Phytogenics in Animal Nutrition: Natural Concepts to Optimize Gut Health and Performance

Phytogenics in Animal Nutrition Natural Concepts to Optimize Gut Health and Performance

i

Phytogenics in Animal Nutrition Natural Concepts to Optimize Gut Health and Performance

Edited by T Steiner

Nottingham University Press Manor Farm, Main Street, Thrumpton Nottingham, NG11 0AX, United Kingdom www.nup.com First published 2009 © Erber AG, Austria All rights reserved. No part of this publication may be reproduced in any material form (including photocopying or storing in any medium by electronic means and whether or not transiently or incidentally to some other use of this publication) without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1988. Applications for the copyright holder’s written permission to reproduce any part of this publication should be addressed to the publishers.

British Library Cataloguing in Publication Data Phytogenics in Animal Nutrition Natural Concepts to Optimize Gut Health and Performance Steiner, T. ISBN: 978-1-904761-71-6

Disclaimer Every reasonable effort has been made to ensure that the material in this book is true, correct, complete and appropriate at the time of writing. Nevertheless the publishers, the editors and the authors do not accept responsibility for any omission or error, or for any injury, damage, loss or financial consequences arising from the use of the book.

Typeset by Nottingham University Press, Nottingham Printed and bound by Martins the Printers, Berwick upon Tweed

Table of contents



Preface

1

Essential Oils: Biochemistry, Production and Utilisation



Á. Máthé

2

Phytogenic Feed Additives to Young Piglets and Poultry: Mechanisms and Application

3

W. Windisch, E. Rohrer and K. Schedle



T.J. Applegate

Influence of Phytogenics on the Immunity of Livestock and Poultry

4

Phytobased Products for the Control of Intestinal Diseases in Chickens in the Post Antibiotic Era



I. Giannenas and I. Kyriazakis



Enhancing Feed Intake by the Sow during Lactation using Biomin® P.E.P.

5 6

vii 1

19

39

61

87

J.C. Laurenz, J.A. Miller, J. Rounsavall, N.C. Burdick and F. Neher

Phytogenic Compounds in Broiler Nutrition

97



K.C. Mountzouris, V. Paraskevas and K. Fegeros

7 8

Essential Oils as Feed Additives in Ruminant Nutrition



P. Encarnação

9

Application and Benefits of Phytogenics in Egg Production



T. Steiner



Conclusion

167



Index

169

111

C. Benchaar, A.N. Hristov and H. Greathead

The Potential of Phytogenic Compounds in Aquaculture

147

157

Preface

Driven by the European ban of antibiotic growth promoters in 2006, phytogenic feed additives have been gaining considerable attention in livestock feeding in the last few years. More and more commercial products are available on the market and it is expected that the use of these additives will increase even more in the future. The term “phytogenics”, also referred to as botanicals or phytobiotics, describes plant-derived compounds incorporated in animal feed to improve productivity of livestock through amelioration of feed properties and promotion of the animal’s production performance. Phytogenics include a broad range of plant materials, most of which have a long history in human nutrition, where they have been used as flavours, food preservatives and medicines since ancient times. These plant materials usually contain a cocktail of numerous different active principles (e.g. eugenol, cinnamaldehyde, carvacrol or thymol), which all play together to determine a specific flavour or scent. Indeed, phytogenics are commonly known for their flavouring properties, thus having impact on the palatability of diets. On the other hand, phytogenics exert a range of distinct biological activities, therefore having the potential to positively affect gut health and increase performance. The in vitro antimicrobial, antiviral, antifungal, antioxidant and other activities of phytogenic compounds are well described and backed up by numerous scientific reports. In the meanwhile, an increasing number of studies addressing the gastrointestinal effects of phytogenics under in vivo conditions, i.e. in animal feeding experiments, are available. The intestinal microflora, gut morphology, gastric emptying, activity of endogenous digestive secretions and, finally, performance parameters are considered to be influenced by dietary phytogenics. A systematic assessment of the potential efficacies of phytogenics has been difficult due to the fact that the majority of in vivo trials were carried out using commercial phytogenic additives, which, in most cases, were blends of several plant extracts, hence representing a mixture of different active ingredients. Only a minor portion of trials used single phytogenic compounds such as pure carvacrol or thymol or a chemically defined essential oil. Based on the studies reported to date, it is the aim of this compendium to summarize the most recent knowledge about the application and benefits of phytogenics in different animal species. In Chapter 1, Ákos Máthé presents a brief overview about definitions and the chemistry of aromatic plants, their extracts and active principles. He highlights the different biological activities as reported in in vitro experiments. Wilhelm Windisch and his co-authors summarise the potential modes of action of phytogenic feed supplements and their effects on animal performance (Chapter 2), while specific attention is paid to the potential impact of phytogenics on immune parameters by Todd Applegate (Chapter 3). In Chapter 4, Ilias Giannenas and Ilias Kyriazakis describe the role of phytogenics in the prevention of intestinal diseases in poultry, such as coccidiosis and necrotic enteritis. The beneficial effect of

viii Preface phytogenics on feed intake and lactation performance of sows is presented by Jamie Laurenz and his co-authors by the example of a feeding trial conducted at Texas A&M University (Chapter 5). In Chapter 6, Kostas Mountzouris and colleagues provide a review of the latest literature pertaining to the benefits of phytogenics specifically in broiler nutrition. Recently, as presented by Chaouki Benchaar and co-authors, the potential to manipulate the ruminal microflora with phytogenics has attracted growing interest. Up-to-date knowledge about this application is summarised in Chapter 7. Due to the increasing importance of aquaculture and the shift from animal protein to plant protein-based diets in feeding programs for fish and shrimp, it is anticipated that phytogenics will also gain increasing attention in aquatic species, as Pedro Encarnação reports in Chapter 8. Finally, this work is completed by the editor by highlighting the application and benefits of phytogenics in the feeding of laying hens (Chapter 9). This compendium represents a review of existing knowledge, as well as a basis for future research and development for scientists and the feed industry in order to develop efficacious phytogenic preparations for animal nutrition.

Tobias Steiner, PhD Editor

A. Máthé 1

1 ESSENTIAL OILS – BIOCHEMISTRY, PRODUCTION AND UTILISATION Ákos Máthé Department of Botany, Faculty of Agriculture and Food Science, University of West Hungary, 9200 Mosonmagyaróvár, Vár 2, Hungary, e-mail: [email protected]

Introduction Essential oils are concentrated hydrophobic liquids containing the volatile aroma compounds of plants. They are also known as volatile or ethereal oils, since they are volatile in steam. They differ in both chemical and physical properties from the so called fixed oils. The essential oils are not simple compounds, but a mixture of various compounds (mainly terpenes and terpene derivatives) (Baer and Demirci, 2007). Consequently, the term “essential oil” corresponds only to the practical technological feature of these compound mixtures, i.e. it generally denotes active principles that become volatile at room temperature and evaporate without residues. Essential oils do not or only poorly dissolve in water and are generally distilled with water steam. Herbs and spices, or plants used in perfumery (cosmetic industry), or even as phytogenic feed additives are chosen mainly because they produce small quantities of characteristic flavours (taste and odour), when added to food or animal feed. To date, it is common knowledge that the chemicals responsible for these distinctive tastes and smells are mainly essential oils.

Occurrence of essential oils in the plant kingdom In the plant kingdom 24 Families are reported to contain more than one, and further 40 Families only one essential oil producing genera (Protzen and Hose, 1993). It is, however, a commonly accepted fact, that practically speaking, nearly all plants might contain certain quantities of essential oils, even if only in minute quantities. Major essential oil containing plant families (in alphabetical order) are: Anacardiaceae, Annonaceae, Apiaceae, Araceae, Aristolochiaceae, Asteraceae, Burseraceae, Calycanthaceae, Cannabinaceae, Asteraceae, Geraniaceae, Gramineae, Hyperaceae, Lamiaceae, Lauraceae, Leguminosae, Magnoliaceae, Myrtaceae, Myoporaceae, Orchidaceae, Pinaceae, Piperaceae, Rosaceae, Rutaceae, Santalaceale, Saururaceae, Solanaceae, Zingiberaceae.

1

2 Essential oils - biochemistry, production and utilisation

Biochemical nature of essential oils Essential oils are versatile and are made up of several chemical constituents with the basic building elements being primarily carbon, hydrogen and oxygen. The aromatic constituents of essential oils are built from hydrocarbon chains (carbon and hydrogen atoms). The basic precursor of many essential oils is a five-carbon molecule called isoprene. Most essential oils are synthesized from isoprene, the building block of terpenoids. The main groups of constituents found in essential oils include: a) alcohols, b) aldehydes, c) esters, d) ethers, e) ketones, f) phenols, g) terpenes. Each of these compounds can be broken down into numerous smaller components (units), e.g. the terpenes into mono-, di- and sesquiterpenes, etc. There are several hundred naturally occurring monoterpenes. These are known to constitute the most common odor-bearing components of essential oils. Essential oils can differ not only in their chemical structures but also in the biosynthetic pathways in which these are synthesized. In a general context, the characteristic main components of essential oils are: •

Monoterpenes: C10 compounds that are mostly synthesized from geranyl pyrophosphate, the ubiquitous C10 intermediate of the isoprenoid pathway (Croteau, 1997). They are colorless, steam distillable, water insoluble liquids with a characteristic aroma, with boiling points ranging from 140 to180°C. These compounds are formed by the head-to-tail, head-to-head or tail-to-tail condensation of two isoprene residues and exhibit every possible mode of ring closure, various degrees of insaturation and substitution of different functional groups. In all, 450 monoterpenes have been discovered (Sticher, 1977) and these can be classified as derivatives of 15 common types of basic and 15 less common types of basic monoterpenes (Devon and Scott, 1972). Based on their chemical structures, monoterpenoids are classified into the following groups: (a) normal monoterpenes, (b) cyclopentanoid monoterpenes and (c) tropolones.

•

Sesquiterpenes: These are C15 compounds with either open chains (e.g. farnesole) or aromatic compounds (e.g. chamazulene). More than 1200 sesquiterpenes are known today. Their structures are based on 30 main skeletal structures (approximately 700 compounds) and 70 less common skeletal structures (approximately 500 compounds) (Daniel, 2008). Sesquiterpenes are steam distillable volatile oils contributing to their flavour. Main groups of sesquiterpenes are: (a) acyclic, (b) monocyclinc and (c) bicyclic

•

Other compounds of non-terpene character (e.g. terpene interemedieries, phenylpropane derivatives, etc.)

The aromatic-ring structure of essential oils is much more complex than that of the simpler, linear carbon-hydrogen structure of fatty oils. Unlike fatty oils, the essential oils

A. Máthé 3

also contain sulfur and nitrogen atoms. In terms of biological activity and effects, each individual chemical constituent has its own characteristic properties. This means that the essential oils (i.e. the mixtures of several chemical components) are of a complex character with several rather diverse effects. Different molecules in the same essential oil can exert different effects, e.g. the azulene in German Chamomile has powerful anti-inflammatory compounds, whereas its bisabolol component has sedative and mood-balancing properties. Other compounds in German Chamomile perform still different functions, such as enhancing the regeneration of tissues. Phenols are, generally, responsible for the antibacterial activity. Carvacrol has anti-inflammatory activity and limonines are antiviral. Based on its chemical composition, a single plant species can have several different chemotypes, i.e. a plant, such as sage grown in the same area, might produce essential oil with a different chemical setup than the sage grown in another location (Máthé et al., 1993). Table 1 shows a survey of the various essential oils species in view of their important chemical constituents, according to Trease and Evans (2002). Table 1. The chemical composition of volatile oils (Trease and Evans, 2002)

Name

Botanical name

Terpenes or sesquiterpenes Turpentine Pinus spp. Juniper Juniperus communis Cade (Juniper Tar Oil) Juniperus oxycedrus Alcohols Coriander Coriandrum sativum Otto of rose Rosa spp. Geranium Pelargonium spp. Indian or Turkish Cymbopogon spp. geranium (Palmarosa) Sandalwood Santalum album Esters and alcohols Lavender Lavandula officinalis Rosemary Rosmarinus officinalis Pumilio pine Pinus mugo var. Pumilio

Important constituents

Terpenes (pinenes, camphene) Terpenes (pinene, camphene); sesquiterpene (cadinene); alcohols Sesquiterpenes (cadinene); phenols (guaiacol, cresol) Linalol (65–80% alcohols); terpenes Geraniol, citronellol (70–75% alcohols); esters Geraniol; citronellol; esters Geraniol (85–90%) Santalols (sesquiterpene alcohols), esters, aldehydes Linalol; linalyl acetate (much); ethylentyl ketone Borneol and linalool (10–18%); bornyl acetate, etc. (2–5%); terpenes; cineole Bornyl acetate (about 10%); terpenes; sesquiterpenes

4 Essential oils - biochemistry, production and utilisation Table 1. Contd.

Name

Botanical name

Peppermint Mentha piperita Aldehydes Cinnamon bark Cinnamomum verum Presl. Cassia Cinnamomum cassia Lemon Lemon grass Cymbopogon spp. terpenes ‘Lemon-scented’ Eucalyptus citriodora eucalyptus Ketones Spearmint Mentha spicata and M. cardiaca Caraway Carum carvi Dill Anethum graveolens Sage Salvia officinalis cineole, etc. Wormwood Artemisia absinthium Phenols Cinnamon leaf Cinnamomum verum Presl. Clove Syzygium aromaticum (L.) Merr & L. M. Perry Thyme Thymus vulgaris Horsemint Monarda punctata Ajowan Trachyspermum ammi Ethers Anise and Star-anise Pimpinella anisum and Illicium verum Fennel Foeniculum vulgare Eucalyptus Eucalyptus globulus Cajuput Melaleuca spp. Camphor Cinnamomum camphora

Important constituents Menthol (about 45%); menthyl acetate (4–9%) Cinnamaic aldehyde (60–75%); eugenol; terpenes Cinnamic aldehyde (80%) Citral (over 3.5%); limonene (about 90%) Citral and citronellal (75–85%); Citronellal (about 70%)

Carvone (55–70%); limonene, esters Carvone (60%); limonene, etc. Carvone (50%); limonene, etc. Thujone (about 50%); camphor; Thujone (up to 35%); thujyl alcohol; azulenes Eugenol (up to 80%) Eugenol (85–90%); acetyl eugenol, methylpentyl ketone, vanillin Thymol (20–30%) Thymol (about 60%) Thymol (4–55%)

Anethole (80–90%); ehavicol methyl ether, etc. Anethole (60%); fenchone (20%) Cineole (over 70%); terpenes, etc. Cineole (50–60%); terpenes, alcohols and esters After removal of the ketone camphor contains safrole; terpenes, etc.

A. Máthé 5 Table 1. Contd.

Name

Botanical name

Parsley Petroselinum sativum Indian dill Peucedanum soia Nutmeg Myristica fragrans Peroxides Chenopodium Chenopodium ambrosioides var. anthelmintica Nonterpenoid and derived from glycosides Mustard Brassica spp. Wintergreen Gaultheria procumbens Bitter almond Prunus communis var. amara

Important constituents Apiole (dimethoxysafrole) Dill-apiole (dimethoxysafrole) Myristicin (methoxysafrole) up to 4%; terpenes (60–85%); alcohols, phenols Ascaridole (60–77%), an unsaturated terpene peroxide

Glucosinolates Methyl salicylate Benzaldehyde and HCN (from amygdalin)

Factors influencing the production of essential oils The plant Organ specific production of essential oils

The content and composition of essential oils depend also on the type of plant organ analysed. The list of essential oil containing organs with some characteristic species is given in Table 2. There is also experimental evidence that both composition and amount of essential oils accumulated in various organs of the same plant could be distinct and different, respectively (Figueiredeo, 1997). In the majority of cases, however, the different organs are of similar character. Secretory structures

In a characteristic of the plant family form, volatile oils are synthesized, accumulated (stored) and released by a variety of specialized secretory structures (Table 3). The most common are: • •

Cavities or ducts: These are clusters of cells just below the epidermis, e.g. skins of citrus fruit, or the leaves of eucalypts or ngaio; Glands or glandular hairs: Originating from epidermal cells, e.g. the glands on lavender florets, or the modified leaf hairs of mint, geranium, and oregano.

6 Essential oils - biochemistry, production and utilisation Table 2. Essential oils derived from various organs of plants

Bark Berries Flowers Leaves Peel Resin Root Rhizome Seeds Wood

Cassia, Cinnamon, Sassafras Allspice, Juniper Cannabis, Chamomile, Clary sage, Clove, Scented geranium, Hops, Hyssop, Jasmine, Lavender, Manuka, Marjoram, Orange, Rose, Ylang-ylang Basil, Bay leaf, Cinnamon, Common sage, Eucalyptus, Lemon grass, Melaleuca, Oregano, Patchouli, Peppermint, Pine, Rosemary, Spearmint, Tea tree, Thyme, Wintergreen Bergamot, Grapefruit, Lemon, Lime, Orange, Tangerine Frankincense, Myrrh Valerian Galangal, Ginger Almond, Anise, Celery, Cumin, Nutmeg oil, Camphor, Cedar, Rosewood, Sandalwood, Agarwood

Table 3. Different types of secretory structures occurring in some plant Families (Figueiredo, 1997; adapted from Fahn, 1988)

Secretory structures

Families

External secretory structures Trichomes Asteraceae, Lamiaceae, Rutaceae, Geraniaceae, Solanaceae and Cannabinaceae Osmophores Piperaceae, Orchidaceae and Araceae Internal secretory structures Idioblasts Lauraceae, Magnoliaceae, Piperaceae, Araceae, Aristolochiaceae, Calycanthaceae and Saururaceae Cavities Rutaceae, Myrtaceae, Myoporaceae, Hypericaceae and Leguminosae Duct Apiaceae, Asteraceae, Pinaceae, Myrtaceae, Hypricaceae, Leguminosae and Anacardiaceae

There is also evidence that in certain species (e.g. Leonotis leonutus, Plectranthus madagascariensis) different types of the same secretory structure exist, heterogeneously distributed over the plant body, secreting also different types of compounds (Ascensao et al., 1997). Phenophase dependent accumulation

The harvest of aromatic plants has always been related to the special phases of development of plants, i.e. to the phenophases. There are numerous examples, how the development of essential oil species can be related to the accumulation of their essential oil content. A

A. Máthé 7

list of species where the time of harvest affected the essential oil yield and composition of species is given by Figueiredo et al. (1997). Some of the well known characteristic species include: Artemisia judiaca, Chrysanthemum balsamita, Citrus bergamia, Cymbopogon spp., Dracocephalum moldavica, Eucalyptus spp., Matricaria recutita, Mentha × piperita, Origanum vulgare, Salvia spp., Satureja hortensis, Thymus spp. and Vanilla planifolia.

Ecological factors Essential oil production is highly influenced by the ecological factors and climatic conditions. The special literature abounds in examples on the influence of soil, nutrients, water, light and temperature on the production and quality of essential oils. Whereas there are several individual exceptions, it seems to be a general rule that an increase in light and temperature beneficially influences (increases) the essential oil production (Figueiredo et al., 2008). Water supply is also essential, although hydric stress has been reported to increase essential oil yield in several species (e.g. Anethum graveolens, Artemisia dracunculus, Satureja douglasii, Mentha × piperita and Ocimum basilicum. According to Simon et al. (1992), in O. basilicum, the increase in hydric stress was coupled with an increase and a change in the essential oil composition. The type and composition of the soil is also regarded as one of the determinant factors. In addition to nutrient supply, soil factors closely related with pH are also important for the growth and production of essential oil species (Figueiredo et al., 1997).

Plant cultivation and processing The origin of plant materials used for the production of essential oils is decisive for the quality of the oil obtained. Formerly plants were mainly collected from their wild populations and were extracted for oils of mostly local use. The demand of essential oil commerce and industries, however, cannot be met by these traditional methods, where also due to the regular and occasionally inexpert collection practices, the natural populations are frequently damaged. As a solution to the above problem, today’s intensive cropping industry, equipped with and using all of the modern agricultural technologies is already capable of securing high yields of high quality oil. As a consequence, essential oil species, like sage (Salvia officinalis) can be grown, even outside the area of their natural occurrence (Máthé et al., 1992). In addition to good agronomic features, improved crop performance producers get control over oil production and processing processes with the ultimate result of improved and stabilized quality and supply.

Isolation methods As the essential oils are contained by special secretory cells and/or tissues of plants, they have to be obtained (isolated) from their location of accumulation prior to utilization.

8 Essential oils - biochemistry, production and utilisation Depending upon the nature of the part (organ) in which they occur, they are obtained from plants in various ways, such as steam-distillation, solvent extraction, absorption, pressure and maceration. The various physical and chemical isolation methods are varied in their rate of efficiency and can also influence both the amount and the quality (including composition) of the essential oil obtained. The main isolation methods are the following: • •

•

Expression: mainly used with citrus fruits, where the essential oils are mechanically cold pressed out from the fruits. Distillation: The most frequently used method, steam distillation is mainly used to obtain essential oils from Labiatae, Apiaceae species, from eucalyptus and bitter orange leaf. Further methods of distillation include: hydro-distillation of flowers (e.g. rose, jasmine or bitter orange), hydrodiffusion (where low pressure steam <0.1 bar replaces the volatiles in plant cells). Extraction: This method is mainly used with flowers that contain too minute amounts of volatile oils to undergo expression, or else, their volatile oil components are easily denaturated in the course of high heat steam distillation. Instead, a solvent such as hexane or supercritical carbon dioxide is used to extract the oils (Boelens, 1997).

Utilisation of essential oils Essential oils are generally high value, low volume commodities. These characteristics make them attractive crops to grow and process nearly all over the world. They are popular also with smallholder farmers and remote communities in the less developed countries of the world, where transport problems would even prevent from marketing high volume cash crops. Essential oils are important commercial items, especially with a main area of utilization in the food industry (55%). Due to their broad range of use and production opportunities, large quantities of essential oils are produced world wide. About 300 different plant species are used for the production of essential oils for the food, flavor and fragrance industry (Boelens, 1997). Table 4 gives a list of global imports and exports, as well as their trade tendencies, according to essential oil sub-categories (SADC, 2006).

Conventional uses Implicitly the biological activities of essential oils have been known and utilized since ancient times (e.g. in food seasoning, medicine, etc.). Spice plants have been popular and widely used throughout the entire history of mankind. Essential oils have also a number of further uses, such as perfumes, bath oils, flavourings, burning (for scent) and in cleaning products. However, most people know of their use in medicine and especially in aromatherapy as well as other forms of alternative medicine. These uses are based on important biological activities, such as antimicrobial activity, anti-oxidant activity, anti-

A. Máthé 9 Table 4. Global imports and exports per essential oils sub-category (SADC, 2006)

2000 2005 Imports (US-$ ‘000) Concentrates 195,850 Concretes and absolutes 522,658 Bergamot 20,612 Other citrus fruit 71,638 Geranium 15,074 Jasmin 8,914 Lavender or lavandin 37,550 Lemon 129,667 Lime 35,686 Mints 97,156 Orange 83,007 Peppermint 137,024 Vetiver 7,189 Resinoids 44,060 Exports (US-$ ‘000) Concentrates 184,428 Concretes and absolutes 420,220 Bergamot 22,833 Other citrus fruit 77,158 Geranium 9,681 Jasmin 5,934 Lavender or lavandin 32,267 Lemon 127,200 Lime 23,244 Mints 97,190 Orange 82,642 Peppermint 147,831 Vetiver 3,992 Resinoids 63,987

Annual growth, 2000–2005 (%)

309,796 680,777 27,694 154,656 13,455 10,005 48,482 188,400 57,188 134,858 158,399 131,347 15,326 42,927

10 5 6 17 -2 2 5 8 10 7 14 -1 16 -1

287,059 595,418 36,791 142,470 10,244 12,488 38,731 182,065 39,482 85,708 155,746 131,354 5,175 87,003

9 7 10 13 1 16 4 7 11 -2 14 -2 5 6

inflammatory activity, antiplasmodial activity, cytotoxic activity, cytotoxicity against human cancer cells, cytotoxicity against human epithelial cells, etc. (Kamatou et al., 2008). Due to their concentrated nature, undiluted or “neat” form essential oils are generally not applied, since in certain cases, they could cause severe irritation or provoke allergic reactions. Instead, before use, essential oils are blended with vegetable-based “carrier” oils. Some essential oils, including many of the citrus peel oils, are so called photosensitizers, i.e. they may increase the skin’s vulnerability to sunlight, making it more likely to burn. Industrial users of essential oils are advised to consult the Material Safety Data Sheets (MSDS) to determine the hazards and handling requirements of particular oils.

10 Essential oils - biochemistry, production and utilisation Non-conventional uses – feed additives Due to their versatility, essential oils can have numerous further, so called non-conventional uses. A comprehensive compilation on these, with a special focus on agriculture, was given by Palevits (1994) (Table 5). Remarkably, this compilation ignored the use of essential oils in animal nutrition (animal feeding). Table 5. Non-conventional uses of volatile oils in agriculture (Palevits, 1994)

1. 2. 3. 4. 5. 6. 7. 8.

Botanical pesticides Botanical Insecticides 2.1. Stored Products Fungicidal effects 3.1. Stored products 3.2. Post-harvest treatments 3.3. Field fungi Herbicidal effects Nematocididal effects Honeybee pathogens Potato sprouting Bovine aromatherapy

Similarly, a search in the veterinary medicinal records of the databases BEAST and AGRICOLA, even at the end of 1990s, revealed an only limited scientific approach to the application of essential oils, or in general, essential-oil plants, in animal feeding (Máthé, 1997). Despite the obvious great importance of animal feeding for agriculture, this class of feed additives has only recently gained increased interest, especially for use in swine and poultry feeding. This tendency, i.e. a significant increase in the number of scientific publications since 2000, was highlighted also by Windisch et al. (2008). The driving factor behind this appears to be the ban on most of the antibiotic feed additives within the European Union in 1999. A complete ban was enforced in 2006. There are ongoing discussions to restrict their use also outside the European Union, since it is speculated that they could generate antibiotic resistance in pathogenic microbiota. The European Food Safety Authority (EFSA) has come up with the regulation of feed additives. According to Regulation (EC) No 1831/2003, only additives that have been through an authorization procedure may be placed on the market. Authorizations are granted for specific animal species, specific conditions of use and for ten years periods. In 2004, EFSA listed the assessment of essential herbs, essential oils and other plant products as “additives” among the items of self-tasking proposals: The ongoing changes in the nutritional habits of the human population, the increased concern for the environment have brought about an upsurge of interest towards the consumption and production of natural foods. To achieve

A. Máthé 11

this goal, similarly to organic agriculture, the food producing ‘animal industry’ will also have to reduce the application of synthetic chemicals and turn towards the more healthy natural ways and means of production for which Phytogenic Feed Additives (PFA) can offer a solution.

Feed additives vs. Phytogenic Feed Additives (PFA) A feed additive - as defined by the European Feed Additive Directive 70/524/EEC - is “any substance, or preparation containing any substance which, when incorporated into a feeding stuff, is likely to affect its characteristics or livestock production”. Phytogenic Feed Additives are of plant origin. In practice, various supplements are used to improve the nutritive balance or performance of the total feed. The role of such feed additives (e.g. protein supplements, vitamins, etc.) is vital from the viewpoint of feed utilization efficiency. The improvement of efficiency is, however, only one aspect of up-to-date animal production. The matters and the methods applied should contribute not only to the production of superior meat quality but they should also be conform with the increasingly severe food safety regulations. From this point of view, feed ingredients of natural origin, especially medicinal and aromatic plants containing biologically active substances seem to open up a favourable prospective. Owing to the manifold and mostly safe effect (GRAS = Generally Recognized as Safe) of their active principles and aromatic components, their use in the form of feed additives seems to be continuously increasing.

Biologically active principles in PFAs Regarding their manifold biologically active properties, essential oil plants, as feed additives, can offer an alternative for most feed additive categories. Some of the species, e.g. garlic, seem to be extremely potent from this point of view (Table 6).

Antioxidant Activity All five groups, i.e. antioxidants, anti-depressants, antivirals, bactericides and sedative, have obvious relevance for animal husbandry. From the nutrition physiological point of view, however, the antioxidants may merit special attention, since undesirable oxidation can produce uninviting changes in colour, flavour, aroma and other quality factors of meat as well as food. In the case of fats and oils a rancid taste and odour may develop which not only impairs the nutritional value of the product but might also form the grounds of toxic effects (Kanner, 1994). Natural antioxidants, as compared to synthetic products have the advantage that they are readily accepted by the consumers since they are considered to be safe and not a ‘chemical’.

12 Essential oils - biochemistry, production and utilisation Essential oil crops have a huge potential as antioxidants, offering an unexploited great choice of species and essential oil components (Katmatou et al., 2008). Some of the characteristic essential oil components with identified potential antioxidant properties are summarized in Table 7. Especially species of the Families Apiaceae and Lamiaceae have been identified to possess significant antioxidative properties (Deans and Waterman, 1993). Table 6. Some important flavoring spices as sources of biologically active compounds (after Duke and Beckstrom-Sternberg, 1994)

Name of the spice Number of identified biologically active compounds Antioxidant Sedative Anti-depressant Anti-viral Bay Black Mustard Black Pepper Cassia Cayenne Clove Coriander Cumin Garlic Ginger Licorice Oregano Poppy-seed Rosemary Saffron Sage Sesame Thyme Turmeric Vanilla

3 4 4 3 9 3 7 5 9 6 10 14 3 12 2 7 7 4 3 7

5 - 7 - 7 - 8 6 5 11 6 - - 6 - - - - - -

- 5 - 4 - - 3 7 6 3 - 12 - 7 5 5 5 6 - 8 - 11 5 - 10 - - 7 3 3 - 3 - 3

Bactericide 0 5 14 3 8 20 II 13 17 20 19 19 6 5 5 8 7

Antifungal Activity Essential oils have been shown to exhibit antifungal activity, even at very low concentrations in the growth medium. As an example, Deans and Svoboda (1990) pbserved that 1–10 µl-1 marjoram oil in the culture broth reduced the growth of the filamentous fungus species Aspergillus flavus, A. niger, A. ochraceus, A. parasiticus and Trichoderma viride by up to 89%. In optimal cases essential oils interfere already with spore germination, whereby the inhibitory effect of the essential oil components has been demonstrated to vary substantially (Table 8).

A. Máthé 13 Table 7. Essential oil components with potential antioxidant properties (Dorman et al., 1995; Burt, 2004)

Oil component

Monarda citriodora

Myristica fragrans

Origanum vulgare

Pelargonium sp.

Thymus vulgaris

α-pinene 3 3 ß-pinene 3 3 α-terpineol 3 3 α-phellandrene 3 α-terpinene 3 γ-terpinene 3 3 ß-caryophyllene 3 3 p-cymene 3 3 3 1,8-cineole 3 terpinene-4-ol 3 3 isoeugenol 3 isomenthone 3 methyl eugenol 3 geranyl acetate 3 geranyl formate 3 citronellic acid 3 borneol camphene carvacrol 3 3 citronellol 3 elemicin 3 eugenol 3 geraniol 3 limonene 3 3 linalool 3 3 3 myrcene 3 neral 3 sabinene 3 3 safrole 3 thymol 3 3

3 3

3 3 3 3 3 3 3

3 3 3

3 3

3

Table 8. Effect of essential oil components on spore germination in various fungi[Minimum Inhibitory Concentration (MIC) (ppm)]

carvacrol p-anis-aldehyde (-)-carvone (E)-anethole

A 250 1000 - -

B 125 1000 1000 -

C

D

E

F

62 250 250 250 250 500 250 250 250 1000

G 125 1000

A=B. cinerea, B=Monilia laxa, C=Mucor piriformis, D=Penicillium digitatum, E= P. expansum. F=P. italicum, G=Rhizopus stolonifer check after 24 h at 20°C, data are the average of five replications.

14 Essential oils - biochemistry, production and utilisation Sedative and Antidepressant Activity The beneficial value of some essential oil plants (e.g. Valeriana officinalis, Melissa officinalis, Lavandula angustifolia, etc.) in the treatment of nervous instabilities and sleep disorders (Weiss and Fintelmann, 1999) has been utilized by phytotherapy for a long time. Although, obviously, PFAs, and among them essential oil plants, are expected to have analogous effects on the animal organism, the ways and means of application need to be cleared in scientific experiments.

Antibacterial and Antiviral Activity Antibacterial activity (Benchaar et al., 2008) seems to be equally determined by both the concentration and the composition of essential oils. Remarkably, according to some authors (Knobloch et al., 1989) the aseptic physiological potency (capacity) of terpenoid compounds is positively related to their water solubility. Although there is less scientific evidence, certain species, e.g. garlic, are generally recognized to possess antibiotic (antiviral) properties. In a feeding experiment with poultry, Achillea millefolium, Hypericum peforatum and Levisticum officinale were efficiently used as a substitute for antibiotics and it was also established that the diet favorably affected the sensory characteristics of meat (Fritz et al., 1993).

Physiological effects of essential oils in animals Nutritional products used as additives/supplements to bulk feedstuffs (e.g. grains, oilseeds, forage, etc.) are meant to improve performance, or in certain cases to cure nutritional deficiency and/or metabolic disorders. To date, mainly synthetics have been used to this end. It has, however, been established that phytogenic feed additives can be used with similar efficacy, since they are capable of influencing important physiological processes in the animal organism (Table 9). Table 9. Physiological effects of essential oils (Günther, 1990)

1. Intensification of the impulses sent by the taste and smelling-nerves in the nasal cavity area towards the central nervous system 2. Increasing the secretion of digestive juices, e.g. saliva, gastric juice, gall, pancrease and intestinal secretion 3. Intensification of the activity of digestive enzymes in the gastro-intestinal area 4. Increasing nutrient absorbtion by activating the transport mechanisms 5. Inhibition of oxidation-processes of intermediary metabolism, e.g. amino acids 6. Inhibiting the growth of bacteria and fungi within the alimentary-tract and stabilization of the microbial flora 7. Inhibition of mould growth on feedstuffs (fungicide effect)

A. Máthé 15

PFAs in animal production technologies Several companies make use of PFAs in their everyday production technologies and there is accumulating scientific evidence demonstrating the benefits of PFAs for animal production (Windisch et al., 2008). It should, however, be mentioned that the use of PFAs is still surrounded by contradictory research reports. As an example, in a recent trial on swine, Muhl and Liebert (2007) have found no impact of an essential oil mix (carvacrol and thymol) on digestion and unspecific immune reaction in piglets. In contrast, Kroismayr et al. (2008) reported that performance was increased when weaned piglets were fed a blend of essential oils (derived from oregano, anis and citrus peels). In a comprehensive review on PFAs, Windisch et al. (2008) have come to the conclusion that the current experience in feeding swine and poultry seems to justify the assumption that PFAs may have the potential to promote production performance and productivity, and thus add to the set of non-antibiotic growth promoters, such as organic acids, probiotics and enzymes. Based on the feed back from production practice, the main advantages of PFA application may be: • • • • • • • • • • •

Reduced risk of enteric imbalances (such as diarrhea), especially of young animals Improved growth performance, including weight gain and feed conversion Stimulated feed intake Increase in egg production Reduction of mortality Better acceptance of feed ingredients with unpleasant taste (such as rapeseed byproducts, protein-concentrates or mineral premixes) Reduced need to apply of chemotherapeuticals Improved product quality (in terms of taste, colour or texture) Improved barn-climate including the reduction of unpleasant odour and toxic gases No withdrawal period in most cases Presumably no harmful residues in animal products

To improve the efficiency and profitability of animal production by means of animal feeding (nutrition) remains to be an economic priority. To achieve these goals, by virtue of their biologically active principles, several essential oil plants can offer a natural and healthy alternative. These plants offer not simply an alternative for synthetically produced feed additives, but owing to their not yet fully discovered synergistic effect of chemical components, they can envision an even more environment-friendly, positive prospective in improving production efficiency and meat quality, including longer shelf-life.

References Ascensao L, Marques N and Pais MS (1997) Peltate glandular trichomes of Leonotis leonurus leaves: ultrastructure and histochemical characterization of secretions. International Journal of Plant Sciences 158: 249–258.

16 Essential oils - biochemistry, production and utilisation Baer HK and Demirci F (2007) Chemistry of Essential Oils. In: Berger RG (Ed): Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Springer Berlin, Heidelberg, New York. Pp. 43–86. Benchaar C, Calsamiglia S, Chaves AV, Fraser GR, Colombatto D, McAllister TA and Beauchemin KA (2008) A review of plant derived essential oils in ruminant nutrition and production. Animal Feed Science and Technology 145: 209–228. Boelens MH (1997) Production of essential oils. In: Franz Ch, Máthé Á and Buchbauer G (Eds) Essential Oils: Basic and Applied Research. Allured Publishing Corporation, Carol Stream, IL, USA. Pp. 283–292. Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods – a review. International Journal of Food Microbiology 94: 223–253. Croteau R (1997) Biochemical and molecular genetic aspects of monoterpene formation. In: Franz Ch, Máthé Á and Buchbauer G (Eds) Essential Oils: Basic and Applied Research. Allured Publishing Corporation, Carol Stream, IL, USA. Pp. 71–80. Daniel M (2008) Medicinal Plants: Chemistry and Properties. Science Publishers, Enfields (NH)-Jersey-Plymouth. Pp. 250. Deans SG and Svoboda KP (1990) The anti-microbial properties of marjoram (Origanum majorana L) volatile oil. Flavour Fragrance Journal 5: 187–190. Deans SG and Waterman PG (1993) Biological activity of volatile oils. In: Hay RKM and Waterman PG (Eds) Volatile Oil Crops. Longman Scientific and Technical, Essex. Pp. 97–111. Devon TK and Scott AI (1972) Handbook of Naturally Occurring Compounds: Vol. II. Terpenes, Academic Press, New York. Dorman HJD, Surai P, Deans SG and Noble RC (1995) Evaluation in vitro of plant essential oils as natural antioxidants. Journal of Essential Oil Research 7: 645–651. Duke J and Beckstrom-Sternberg SM (1994) Acceptable levels of flavoring ingredients. In: Charalambous G (Ed) Developments in Food Science Vol. 34. Spices, Herbs and Edible Fungi. Elsevier Science B.V., Amsterdam, The Netherlands. Pp. 741–758. European Commission (2003) Regulation (EC) No. 1831/2003 of the European Parliament and of the council of 22 September 2003 on additives for use in animal nutrition. Official Journal of the European Union. Fahn A (1988) Secretory tissues in vascular plants. New Phytologist 108: 229–257. Figueiredo AC, Barroso JG, Pedro LG and Scheffer JC (1997) Physiological aspects of essential oil production. In: Franz C, Máthé Á and Buchbauer G. (Eds) Essential Oils: Basic and Applied Research. Proceedings of the 27th International Symposium on Essential Oils. Allured Publishing Corporation, Carol Stream, IL. Pp. 95–107. Figueiredo AC, Barroso JG, Pedro LG, Scheffer JJC (2008) Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour and Fragrance Journal 23: 213–226. Fritz Z, Schleicher A and Kinal S (1993) Effect of substituting milfoil, St. Johns wort and Lovage for antibiotics on chicken performance and meat quality. Journal of Animal and Feed Sciences 2: 189–195.

A. Máthé 17

Günther KD (1990) Gewürzstoffe können die Leistung erhöhen. Kraftfutter 73: 469–474. Kamatou GPP, Makunga NP, Ramogola WPN and Viljoen AM (2008) South African Salvia species: A review of biological activities and phytochemistry. Journal of Ethnopharmacology 119: 664–672. Kanner J (1994) Oxidative processes in meat and meat products: Quality implications. Meat Science 36: 169–189. Knobloch KA, Pauli A, Iberl BH, Weigand H and Weis N (1989) Antibacterial and antifungal properties of essential oil components. Journal of Essential Oil Research 1: 119–128. Kroismayr A, Schedle K, Sehm J, Pfaffl MW, Plitzner C, Foissy H, Ettle T, Mayer H, Schreiner M and Windisch W (2008) Effects of antimicrobial feed additives on gut microbiology and blood parameters of weaned piglets. Die Bodenkultur 59: 111–120. Lawrence BM (1993) A planning scheme to evaluate new aromatic plants for the flavor and fragrance industries. In: Janick J and Simon JE (Eds) New Crops. Wiley, New York. Pp. 620–627. Máthé Á. (2007) Essential oils as phytogenic feed additives. In: Franz Ch, Máthé Á and Buchbauer G (Eds) Essential Oils: Basic and Applied Research. Allured Publishing Corporation, Carol Stream, IL, USA. Pp. 315–325. Máthé Jr I, Miklóssy VV, Máthé Á, Bernáth J, Oláh L, Blunden G and Patel AV (1993) Essential oil content as chemotaxonomic marker for the genus Salvia with reference to its variation in Salvia officinalis L. Acta Horticulturae 330: 123–132. Máthé Jr I, Oláh L, Máthé A, Miklóssy VV, Bernáth J, Bluden G, Patel AV and Máthé I (1992) Changes in the essential oil production of Salvia officinalis under climatic conditions of the temperature belt. Planta Medica 58: A680. Muhl A and Liebert F (2007) No impact of a phytogenic feed additive on digestion and unspecific immune reaction in piglets. Journal of Animal Physiology and Animal Nutrition 91: 426–431. Palevits D (1994) Le point sur les usages non conventionels des huiles essentielles et des extraits de plantes (The non-conventional uses of essential oils and plant extracts). In: 4ème Rencontres Internationales – Nyons. Pp. 26–40. Protzen KD and Hose S (1993) Produktion und Marktbedeutung ätherischer Öle. In: Carle R (Ed) Ätherische Öle: Anspruch und Wirklichkeit. Wissenschaftliche Verlagsgesellschaft, Stuttgart. Pp. 248. Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003. SADC (2006) Trade Information Brief. Essential Oils. Southern African Development Community. Simon JE, Reiss-Bubenheim D, Joly RJ and Charles DJ (1992) Water stress-induced alterations in essential oil content and composition of sweet basil. Journal of Essential Oil Research 4:71–75.

18 Essential oils - biochemistry, production and utilisation Sticher O (1977) Plant mono- di and sesquiterpenoids with pharmacological or therapeutical activity. In: Wagner P and Wolff P (Eds) New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical Activity. Springer Verlag, Berlin. Pp. 137–176. Trease GE and Evans WCh (2002) Pharmacognosy, Harcourt Publishers. UK. Pp. 253–257. Weiss RF and Fintelmann V (1999) Lehrbuch der Phytotherapie. Hippokrates Verlag, Stuttgart. Pp. 485. Windisch W, Schedle K, Plitzner C and Kroismayr A (2008) Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86: E140–E148.

W. Windisch et al. 19

2 PHYTOGENIC FEED ADDITIVES TO YOUNG PIGLETS AND POULTRY: MECHANISMS AND APPLICATION Wilhelm Windisch, Elisabeth Rohrer, Karl Schedle University of Natural Resources and Applied Life Sciences, Vienna, Department of Food Science and Technology, Division of Animal Food and Nutrition, 1180 Vienna, Austria E-mail: [email protected]

Introduction The term phytogenic feed additives (often also called phytobiotics or botanicals) is commonly defined as plant derived feed additives included into livestock diets to improve the animals’ productivity, properties of feed and food quality, as well as promotion of zootechnical performance. Composition of phytogenic feed additives comprises a very wide range of substances with respect to botanical origin, processing, and chemical description including purity. This class of feed additives has recently gained rising interest especially in piglets and poultry for fattening since banning the antibiotic feed additives within the EU in the year 2006. Discussions to restrict their use outside the EU due to speculated risk for generating antibiotic resistance in pathogenic microbiota are still up to date. Pronounced effort has also been made on developing other non-antibiotic growth promoters such as organic acids and pre- or probiotics. Those substances are well established in animal nutrition. In contrast phytogenic substances are a young class of feed additives and the knowledge regarding modes of action and aspects of application are still rather fragmentary in knowledge. Further complications arise because phytogenic feed additives comprise a group of feed additives widely varying with respect to botanical origin, processing, and composition. Most studies comprise blends of various active compounds and report effects on zootechnical performance rather than physiological impacts. This restricts the assessment of phytogenic feed additives to a selected sub-group of well known active substances and some commercial products, ignoring that this is incomplete. In this context the following will provide an overview about recent knowledge on general application in young piglets and growing poultry for fattening and principal modes of action of phytogenic feed additives.

19

20 Phytogenic feed additives to young piglets and poultry

Growth promoting efficacy During recent years, phytogenic feed additives have attracted increasing interest as an alternative growth promoter to replace the use of antibiotic feed additives. In principal, the primary mode of action of growth promoting feed additives arises from beneficially affecting the ecosystem of gastrointestinal microbiota through controlling potential pathogens. This applies especially to critical phases of the animals’ production cycle or hygienic disorders of the environment. Sensitive phases for digestive disorders are characterised at the weaning phase of piglets or the early life span of poultry. Due to a more stabilized intestinal health, animals are less exposed to microbial toxins and other undesired microbial metabolites, such as ammonia and biogenic amines. Consequently, growth promoting feed additives relieve the host animal from immune defence stress during critical situations, raise the intestinal availability of essential nutrients for absorption, and thus, assist the animal to grow better within the framework of its genetic potential (Windisch et al., 2008). Freitag et al. (1998) reviewed the potential of common growth promoting feed additives (antibiotics, organic acids, probiotics) in piglets housed under comparable European conditions. On average, the rise in feed intake, growth rate, and feed conversion ratio induced by these feed additives ranged around 4%, 8% and 4%, respectively. Phytogenic feed additives are often used for improving flavour and palatability of feed, thus enhancing zootechnical performance. Indeed, there are reports on higher feed intake through phytogenic feed additives, especially in piglets (Table 1). However, a rise in feed intake is a common declaration of growth promoting feed additives such as antibiotics, organic acids, and probiotics. In the first instance, it reflects the higher consumption capacity of larger grown animals compared to untreated controls but not necessarily a specific enhancement of voluntary feed consumption due to an improved palatability. The rise in feed intake seems to reflect the growth promoting efficacy in general (enhanced consumption capacity due to better growth) rather than a specific stimulation through improved palatability of the feed. Few experimental assessments of palatability as affected by phytogenic feed additives have been reported so far. This limited number of available studies indicates reductions in voluntary feed intake in piglets through essential oils from fennel and caraway, as well as through thyme and oregano herbs (Schöne et al., 2006; Jugl-Chizzola et al., 2006). This indicates the presence of potentially offensive components in phytogenic feed additives. Therefore, the assumption that herbs, spices and their extracts generally improve feed intake does not seem to be justified in general. In poultry (Table 2), most studies revealed a lack of significant efficacy of phytogenics on feed intake. But growth was often enhanced and especially feed conversion rate was improved in most of the studies. Since poultry is known to adjust feed intake according to the metabolic demand for energy, feed conversion rate is therefore the most sensible parameter reacting on growth promoting feed additives. Indeed, there are reports on negative effects of phytogenic feed additives in piglets and poultry, mainly associated with depressions in feed intake obviously reflecting uncertainties in choosing the proper herbal origin, extraction technique, and the suitable dietary dose. But in total, there is sufficient experimental evidence that phytogenic feed additives are capable of improving zootechnical performance in piglets and poultry.

W. Windisch et al. 21 Table 1. Effect of phytogenic feed additives on zootechnical performance in piglets (data from literature survey)

Phytogenic feed Dietary Treatment effects References additive dose (% difference to untreated control) (g/kg) Feed Body Daily Feed intake weight weight conversion gain rate Plant extracts Fennel 0.1 +3 +6 -3 Caraway 0.1 -2 0 -2 Fennel 0.1 +3 +4 -2 Caraway 0.1 -9 -7 -3 Lemongrass 5ml -3 +2 -5 Clove leafs 5ml -5 0 -5 Peppermint 5ml -4 -3 -2 Oregano 0.1 +3 +2 0 Oregano 0.1 0 +5 -5 Cassia 0.1 +5 +2 +3 Cassia 0.1 -5 0 -5 Clove leafs 0.1 +1 -1 +3 Clove leafs 0.1 +3 +7 -4 Lemongrass 0.1 -2 +2 -4 Pimento 0.1 -8 -4 -5 Tea 0.1 -2 0 -2 Peppermint 0.1 -9 -3 -7 Peppermint 0.1 -6 -5 -1 Oregano 0.5 -3 +7 -9 Oregano 0.5 +12 +23 -9 Essential oil blend 0.04 +4 +6 -2 Plant extracts* 0.3 +26 +33 +4 Plant extracts* 0.3 +26 +33 +4 Plant extracts* 7.5 -17 -10 +8 Plant extracts* 0.15 -7 -11 -3 Plant extracts* 0.3 -6 -7 +2 Essential oil blend 0.2 Essential oil blend 0.1 +3 0 +3 Essential oil blend 0.1 +4 0 +3 Essential oil blend 0.1 +1 -2 +3 Herbs and spices Herbs blend 2.0 +20 +13 +19 -3 Herbs blend 10.0 -14 +4.7 -18 Oregano 2.0 -1 +9 -10

Schöne et al. (2006) Schöne et al. (2006) Schöne et al. (2004) Schöne et al. (2004) Tartrakoon et al. (2003) Tartrakoon et al. (2003) Tartrakoon et al. (2003) Gollnisch et al. (2001) Wald et al. (2001) Gollnisch et al. (2001) Wald et al. (2001) Gollnisch et al. (2001) Wald et al. (2001) Wald et al. (2001) Wald et al. (2001) Wald et al. (2001) Wald et al. (2001) Wald (2002) Günther and Bossow (1998) Kyriakis et al. (1998) Kroismayr et al. (2008) Manzanilla et al. (2006) Nofrarías et al. (2006) Namkung et al. (2004) Manzanilla et al. (2004) Manzanilla et al. (2004) Gollnisch et al. (2001) Gollnisch et al. (2001) Gollnisch et al. (2001)

Kong et al. (2007) Lien et al. (2007) Schaumacher et al. (2002)

22 Phytogenic feed additives to young piglets and poultry Table 1. Contd.

Phytogenic feed Dietary Treatment effects References additive dose (% difference to untreated control) (g/kg) Feed Body Daily Feed intake weight weight conversion gain rate Oregano Thyme Sage Saint-John’s-wort Saint-John’s-wort Coriander Bahzen Thyme Thyme Thyme Chinese rhubarb Chinese rhubarb Chinese rhubarb Yarrow Garlic Garlic Echinacea purpurea Yucca

2.0 2.0 2.0 2.0 2.0 2.0 10 1.0 5.0 10.0 2.5 5.0 10.0 2.0 1.0 1.0

+4 +5 +4 +6 +3 +7 -7 -3 +3 +2 +4 +7 -14 +5 -1 +1 -1 -1 -2 -1 +2 +2 +2 +17 +11 +32 +1 -2 +5 -15 -16 -35 +1 +4 -7 +2 +5 +1

0 -3 -4 -6 +1 -3 -18 -4 +4 -2 -16 +9 +35 -4 -8 +4

18.0 0.125

-2 +1 +4 0

-4 -3

Schaumacher et al. (2002) Schaumacher et al. (2002) Schaumacher et al. (2002) Schaumacher et al. (2002) Schaumacher et al. (2002) Schaumacher et al. (2002) Lien et al. (2007) Hagmüller et al. (2006) Hagmüller et al. (2006) Hagmüller et al. (2006) Straub et al. (2005) Straub et al. (2005) Straub et al. (2005) Schaumacher et al. (2002) Schaumacher et al. (2002) Schaumacher et al. (2002) Maass et al. (2005) Yen et al. (1993)

* = Entire product

Special aspects of using phytogenic substances are their beneficial properties on the intestinal tract. Those effects may improve zootechnical parameters. Table 3 summarizes relevant results of an in vivo comparison between an essential oil blend and an antibiotic feed additive (Kroismayr et al., 2008a, b). In this study, 120 weaning piglets were fed common diets fortified with either no growth promoting feed additives (control), tetracycline (Avilamycin) (40 mg/kg feed), or essential oils from oregano, anise and citrus peels (40 mg/ kg of feed; Biomin® P.E.P., Austria). After 3 weeks of feeding, 12 representative animals per treatment were sacrificed in order to derive samples of gut contents and gut tissues for analysis of indicators of gut functionality. Weight gain, feed intake, and feed conversion ratio of treated animals was numerically improved within a range typical to growth promoting feed additives.

W. Windisch et al. 23 Table 2. Effect of phytogenic feed additives on zootechnical performance in poultry (data from literature survey and Windisch et al., 2008)

Phytogenic feed Dietary Treatment effects References additive dose (% difference to untreated control) (g/kg) Feed Body Daily Feed intake weight weight conversion gain rate A) Broilers Plant extracts Sylimarin 0.04 -6 -2 -4 Sylimarin 0.08 -4 -1 -3 Oregano 0.15 -6 -2 -4 Oregano 0.3 -3 +1 -2 Rosemary 0.15 0 -1 -1 Rosemary 0.3 -2 +1 -4 Thymol 0.1 +1 +1 -1 Cinnamaldehyde 0.1 -2 -3 0 Thymol 0.2 -5 -3 -3 Carvacrol 0.2 +2 +2 -1 Anis 0.15 -1 +1 -1 Cassia 0.1 -4 -3 -1 Lemongrass 0.1 +1 -1 +2 Clove leaf 0.1 -3 -4 +1 Oregano 0.1 -1 +8 -9 Oregano 1.0 +3 +6 -3 Oregano 0.1 -2 -1 -1 Peppermint leafs 0.1 -3 -2 -1 Yucca extract 2.0 -1 +1 -6 Essential oil blend 0.024 -4 0 -4 Essential oil blend 0.048 -5 0 -6 Plant extracts* 0.2 -2 0 -2 Plant extracts* 5.0 +2 +3 -4 Plant extracts* 0.5 0 -2 -2 +2 Plant extracts* 1.0 +2 -1 0 +2 Plant extracts* 0.1 +1 +1 0 Essential oil blend 0.2 0 -17 Essential oil blend 0.075 -7 -3 -4 Essential oil blend 0.15 -7 -1 -1 Essential oil blend 0.036 +3 -8 -5 Essential oil blend 0.048 +2 -8 -4 Essential oil blend 0.024 -2 0 -2 Essential oil blend 0.048 0 +14 -12 Essential oil blend 0.072 -2 +8 -9

Schiavone et al. (2007) Schiavone et al. (2007) Basmacioglu et al. (2004) Basmacioglu et al. (2004) Basmacioglu et al. (2004) Basmacioglu et al. (2004) Lee et al. (2003) Lee et al. (2003) Lee et al. (2003) Lee et al. (2003) Mayland-Quellhorst (2002) Wald (2002) Wald (2002) Wald (2002) Halle et al. (1999) Halle et al. (1999) Wald (2002) Wald (2002) Yeo et al. (1997) Cabuk et al. (2006) Cabuk et al. (2006) Hernandez et al. (2004) Hernandez et al. (2004) Botsoglou et al. (2004b) Botsoglou et al. (2004b) Lee et al. (2003) García et al. 2007) Basmacioglu et al. (2004) Basmacioglu et al. (2004) Alcicek et al. (2004) Alcicek et al. (2004) Alcicek et al. (2003) Alcicek et al. (2003) Alcicek et al. (2003)

24 Phytogenic feed additives to young piglets and poultry Table 2. Contd.

Phytogenic feed Dietary Treatment effects References additive dose (% difference to untreated control) (g/kg) Feed Body Daily Feed intake weight weight conversion gain rate Essential oil blend 1.0 -7 -3 -4 Essential oil blend 1.0 -8 +1 -9 Herbs and spices Oregano 5.0 +5 +7 -2 Thyme 1.0 +1 +2 -1 Garlic 1.0 -5 -5 0 Herb mix 0.25 0 +2 -2 Herb mix 0.5 +5 +2 +3 Herb mix 1.0 +2 +1 +1 Herb mix 2.0 +1 +1 0 B) Turkeys Herbs and spices Oregano 1.25 -5 +2 Oregano 2.5 -6 +1 Oregano 3.75 -9 +1 C) Quails Essential oils Thyme 0.06 0 +6 Black seed 0.06 +1 +2 Herbs and spices Coriander 5.0 +3 +1 +1 Coriander 10.0 +3 +5 -1 Coriander 20.0 +4 +8 -4 Coriander 40.0 +5 +4 +1

Halle et al. (2001) Halle et al. (2001)

Florou-Paneri et al. (2006) Sarica et al. (2005) Sarica et al. (2005) Guo et al. (2004) Guo et al. (2004) Guo et al. (2004) Guo et al. (2004)

Bambidis et al. (2005) Bambidis et al. (2005) Bambidis et al. (2005)

Denli et al. (2004) Denli et al. (2004) Güler et al. (2005) Güler et al. (2005) Güler et al. (2005) Güler et al. (2005)

* = Entire product

Antimicrobial action Phytogenic substances are well known to exert inhibiting effects in vitro against pathogens (Adam et al., 1998; Smith-Palmer et al., 1998; Hammer et al., 1999; Dorman and Deans, 2000; Burt, 2004; Si et al., 2006; Özer et al., 2007). Comparable observations in vivo of herbal essential oils and oleoresins on activity of intestinal microbiota were also found in studies with piglets and broilers (e.g. Jamroz et al., 2003; Manzanilla et al., 2004; Mitsch et al., 2004; Namkung et al., 2004; Jamroz et al., 2005; Castillo et al., 2006). For broilers a specific antimicrobial efficacy of essential oils against Eimeria species after experimental

W. Windisch et al. 25 Table 3. Zootechnical and gastrointestinal effects of essential oils and an antibiotic feed additive in weaning piglets (means expressed as % of control level) (data from Kroismayr et al., 2008a, b; Zitterl-Eglseer et al., 2008)

Feed additive Essential oils1) Antibiotic2) Zootechnical performance (day 0 to 21 post weaning) Feed intake 103.8 Growth rate 105.9 Feed:gain ratio 98.1 Sum of aerobic bacterial counts in chyme fresh matter3) Ileum 85.3 * Caecum 92.9* Colon 102.8 Sum of anaerobic bacterial counts in chyme fresh matter3) Ileum 86.1 * Caecum 93.2 * Colon 98.8 Sum of volatile fatty acids4) in chyme fresh matter Ileum 93.8 Caecum 90.3 Colon 89.7 Sum of biogenic amines5) in chyme fresh matter Ileum 91.1 Caecum 84.3 Colon 94.3 Ammonia contents in chyme fresh matter Ileum 81.0 Caecum 75.9 Colon 103.9 Apparent digestibility Dry matter 101.4 * Crude protein 103.3 * mRNA expression of genes relative to untreated control TNFα in mesenterial lymph nodes 107.2 Caspase 3 in mesenterial lymph nodes 118.1 NFκB in mesenterial lymph nodes 48.0 * Cyclin D1 in jejunum 71.7 Cyclin D1 in ileum 70.7 Cyclin D1 in colon 62.9 * *

101.8 104.1 97.4 83.7 * 104.4 102.8 82.8 * 105.8 99.1 100.0 87.3 89.7 87.9 78.2 83.0 93.1 82.5 87.3 101.2 * 101.8 (*) 124.8 126.6 28.1 * 145.4 (*) 84.1 65.1 *

Statistically significant difference of mean compared to untreated control (p<0.05) Statistical tendency of difference of mean compared to untreated control (p<0.10) 1) Extract from oregano, anise and citrus peels containing mainly carvacrol, thymol, anethol, limonene; dietary dose =40 mg/kg 2) Avilamycin (tetracycline); dietary dose =40 mg/kg 3) Original data expressed as log10 of colony forming units per g of chyme 4) Acetic, propionic, lactic, butyric, valearic, and capric acid 5) Colamin, methylamin, histamin, pyrrolidin, isopropylamin, putrescin, cadaverin, spermidin, and spermin (*)

26 Phytogenic feed additives to young piglets and poultry challenge and against other specific pathogens were observed in vivo (Giannenas et al., 2003; 2004; Jamroz et al., 2003; Mitsch et al., 2004; Jamroz et al., 2005; Hume et al., 2006; Oviedo-Rodon et al., 2006). In the study presented in table 3, both the antibiotic and phytogenic feed additive decreased microbial activity in terminal ileum, caecum, and colon, as was obvious from reduced bacterial colony counts and reduced chyme contents of volatile fatty acids as well as of biogenic amines. The antimicrobial mode of action is considered to arise mainly from the potential of the hydrophobic essential oils to intrude into the bacterial cell membrane, to disintegrate membrane structures and cause ion leakage. These effects are also typical to organic acids, which are known to exert a major part of their zootechnical efficacy mainly through antimicrobial actions in the gastrointestinal tract (for review see e.g. Gabert and Sauer, 1994; Roth and Kirchgessner, 1998). Relief from microbial activity and related by-products is of high relevance especially in the small intestine, as production of volatile fatty acids counteracts stabilization on intestinal pH required for optimum activity of digestive enzymes. In addition, intestinal formation of biogenic amines by microbiota is undesired not only because of toxicity, but also due to the fact that biogenic amines are produced mainly by decarboxylation of limiting essential amino acids (e.g. cadaverine from lysine, scatol from tryptophan). Consequently, relief from microbial fermentation in the small intestine may improve the supply status of limiting essential nutrients (e.g. Roth et al., 1998). In total, there is clear experimental evidence for an overall antimicrobial efficacy of phytogenic feed additives whether arising directly from an antimicrobial action or indirectly from factors supporting the intestinal hygiene.

Effects on gut morphology Morphological changes of gastrointestinal tissues can be induced by differences in gut load of microbial content including their metabolites (Xu et al., 2003). For phytogenic feed additives literature does not draw a consistent picture. There are reports that show increased, unchanged as well as reduced villi length and crypt depth in jejunum, and colon for broiler and piglet treated with phytogenics (Namkung et al., 2004; Demir et al., 2005; Jamroz et al., 2006; Nofrarias et al., 2006; Oetting, 2006). In the piglet study summarized in Table 3 and Figure 1, essential oils tended to reduce jejunum and ileum villi length as well as colon crypt depth numerically. Simultaneously, mRNA expression rates of Cyclin D1, a marker of cell proliferation, was down-regulated in these tissues, which further supports the suggested depression of villi and crypt sizes. Considering the different reactions of gut morphology reported so far, it might be hypothesized that one aspect of phytogenic actions seems irritating intestinal tissues leading to shortage of intestinal surface, while other beneficial effects on gut health (e.g. reduced pathogen pressure) could favour villi length and gut surface. Consequently, the overall impact of phytogenics on gut morphology seems to depend on the balance between tissue irritation and beneficial effects on intestinal hygiene. Also changes in digestive enzyme activities associated with phytogenics might

W. Windisch et al. 27

reflect at least in part an enhanced secretion rate of enzymes due to intestinal stress induced by some compounds of these feed additives. 0.8

Control

Essential oils

Antibiotic

0.7

Length (mm)

0.6 0.5 0.4 0.3 0.2 0.1 0.0 Villi length, jejunum

Villi length, ileum

Crypth depth, colon

Figure 1. Effects of avilamycin and essential oils on gut morphology (data adopted from Kroismayr et al., 2008b)

Relief from intestinal immune defence stress Differential inflammatory responses may reflect, at least in part, functional and anatomical differences between the different sections of the gut (Beagley et al., 1995). As shown in Figure 2, the size of the Peyers’ Patches in the ileum was significantly reduced through both the antibiotic and phytogenic feed additive in the above-mentioned study with weaning piglets. 0.6

Control

Essential oils

Antibiotic

a 0.5

Area (mm2)

0.4

b

b

0.3 0.2 0.1 0.0 Area of Peyers' patches

Figure 2. Effects of avilamycin and essential oils on area of Peyers’ patches (data adopted from Kroismayr et al., 2008b). a,b Significant difference between treatments (P < 0.05)

28 Phytogenic feed additives to young piglets and poultry Manzanilla et al. (2006) and Nofrairas et al. (2006) observed diminished number of intraepithelial lymphocytes in jejunum of piglets treated with antibiotic or phytogenic feed additives. These observations suggest less immune defence activities in the small intestine compared to untreated controls. Simultaneous down-regulation of the transcription factor NFκB at fairly unchanged pro-inflammatory cytokine TNFα and Caspase-3 in mesenterial lymph nodes (table 3) gives further rise to the hypothesis of relief from immune stress through phytogenic feed additives. The immune modulation potential of phytogenic feed additives is reported by several authors in different ways (Nabuurs, 1995; Maass et al., 2005; Dong et al., 2007; Böhmer et al., 2008). Those effects ranged from changes in mRNA concentrations of inflammatory marker genes, over antibody count to different T or B cell densities (Dong et al., 2007; Böhmer et al., 2008; Kroismayr et al., 2008b). Although the precise mechanisms of phytogenics are not clear further investigations are necessary.

Specific impacts on gut functions A wide range of spices, herbs and their extracts are known for their beneficial actions within the digestive tract, such as laxative and spasmolytic effects as well as prevention from flatulence (e.g. Chrubasik et al., 2005). Increased, secretions of saliva, bile, mucus as well as enhanced enzyme activity are considered to further add to those beneficial effects of herbs and spices as has been demonstrated in a rat model (e.g. Platel and Srinivaran, 2000a,b, 2004; Rao et al., 2003). Similarly feed additives for broilers showed enhanced activities of trypsin, amylase in tissue homogenates of pancreas, small intestine, and jejunal chyme content (Lee et al., 2003; Jang et al., 2004). Another example are essential oils, which may promote glucose absorption in rats (Kreydiyyeh et al., 2003). Manzanilla et al. (2004) fed a combination of essential oils and capsaicin to piglets and observed that gastric retention time of ingested feed was slowed down by these additives. Thus, there is evidence that phytogenic feed additives may favourably affect gut functions (passage rate of ingesta, activity of digestive enzymes, and nutrient absorption). Such additives also stimulated intestinal secretion of mucus (Jamroz et al., 2006). This raises the question whether mode of action of phytogenic additives on gut functions might arise at least in part indirectly from an irritant action on exposed tissues leading to higher secretion of mucus (and possibly enzymes). The latter may be considered to result in an impaired adhesion of pathogens and thus to produce an overall benefit to the organism due to relief from pathogen pressure despite the fact that higher secretory activities raise the expenditure of energy and nutrients. Saponins are a special class of phytogenic substances, because they are considered to reduce intestinal NH3 and hence to alleviate an important stress factor to animal health (Franzis et al., 2002). Nazeer et al. (2002) for example reported reduced intestinal and faecal urease activities feeding Yucca schidigera extracts. For such extracts the possibility to contain sub-fractions with partially antagonistic properties on intestinal urease activity and NH3 formation were reported (Killeen et al., 1998; Cho et al., 2006). Hence, further research seems to be required to improve efficacy of such products.

W. Windisch et al. 29

Further considerations to the use of phytogenics Antioxidative effects A high oxidative stability of meat is important when attempting to avoid or delay development of rancid products or warmed-over flavour. In relation to character the process of lipid oxidation, effect of antioxidants is more pronounced by a dietary application than the post mortem addition to meat (Govaris et al., 2004). Significant antioxidative properties are well described for herbs and spices especially in the case of the phenolic compounds derived from Labiatae plant family (e.g. Cuppett and Hall, 1998; Craig, 1999; Nakatani, 2000; Botsoglou et al., 2005; Wei, 2007). The antioxidant property of many phytogenic compounds may be assumed to protect feed lipids from oxidation like antioxidants usually added to diets (e.g. α-tocopheryl acetate, BHT). Although this aspect has not been explicitly investigated for piglet and poultry feeds, there is wide practice of using essential oils successfully, especially from the Labiatae plant family as ‘natural’ antioxidants in human food (Cuppett and Hall, 1998) as well as in feed of companion animals. The potential of herbal phenolic compounds to improve oxidative stability of meat from different species of livestock or eggs, has been demonstrated in a series of studies (Botsoglou et al., 2002; 2003a, b; Papageorgiou et al., 2003; Young et al., 2003; Basmacioglu et al., 2004; Govaris et al., 2004; Botsoglou et al., 2004a, Botsoglou et al., 2005; Giannenas et al., 2005; Florou-Paneri et al., 2006; Janz et al., 2007). Interestingly, at least for essential oregano oils the dietary additions improved tissue retention of α-tocopherol (Botsoglou et al., 2003a,b). Oregano and rosemary in combination tended to be more pronounced than with comparable additions of phenolic compounds from either plant species (Basmacioglu et al., 2004). These results suggest that the protective properties of herbal phenolic compounds against lipid peroxidation might arise at least in part from specific interactions with lipid metabolism rather than from an increment of total dietary antioxidative potential. This hypothesis is supported by observing improved oxidative stability of tissues in oregano fed chicken exposed to transport stress (Young et al., 2003). Thus, the antioxidative properties of phytogenic feed additives may clearly improve quality of animal derived products and presumably the animals’ health too.

Safety aspects As phytogenics are natural substances or derived thereof, there is common sense that safety concerns do not apply per se. Hence application of phytogenics to agricultural livestock has to be safe to the animal, the user, the consumer of the animal product and the environment. In case of the user (e.g. feed manufacturer, farmer), the handling of pure formulations of such feed additives usually needs protective measures as they are potentially irritating and therefore causing allergic contact dermatitis (Burt, 2004). With respect to consumer safety, the ‘natural’ phytogenic feed additives cannot be disburdened globally from check

30 Phytogenic feed additives to young piglets and poultry for possibly undesired residues in products derived from treated animals. For example, the improved oxidative stability of meat from animals fed essential oils might suggest the transition of respective compounds from feed into tissues. Indeed, Zitterl-Eglseer et al. (2007) reported almost complete absorption of carvacrol and thymol in piglets fed an essential oil blend at amounts reflecting their common use as feed additives. Glucuronic and sulphate metabolites of carvacrol and thymol were found in blood plasma and kidney, while respective residues could not be detected in liver, spleen, muscle or in abdominal fat tissues indicating effective excretion via urine. Similarly, a study in humans demonstrated rapid absorption and effective urinary excretion of glucuronic and sulphate metabolites of rosmarinic essential oils (Baba et al., 2005). Although minor residues of phenolic compounds from the Labiatae plant family in animal products do not seem to pose a specific safety risk to the consumer due to the common inclusion of these plants as spices into human food, these examples demonstrated that the fate of phytogenic feed additives may not be limited to the digestive tract.

Conclusions Phytogenic substances are claimed to exert in general growth promoting effects in agricultural livestock, partially associated with an enhanced feed consumption due to an improved palatability of the diet. Additionally antioxidative and antimicrobial actions are in discussion. The potential of phytogenic feed additives to promote zootechnical performance in young piglets and poultry has been clearly demonstrated. In this context, the raise in feed intake observed in piglets seems to arise mainly indirectly from growth promoting efficacy and improved digestive capacity rather than from specific improvement in dietary palatability. Also in regards to antimicrobial action, in vivo evidence is given for a final reduction of intestinal pathogen pressure through phytogenics. But it still remains unclear to which extent this effect originates directly from the antimicrobial action of the active substances as observed in vitro. Nevertheless, when compared with antimicrobial feed additives, phytogenics seem to similarly modulate relevant gastrointestinal parameters, such as microbial colony counts, fermentation products including undesirable or toxic substances, gut tissue morphology and immunological reactions. This might raise the hypothesis that the mode of action of phytogenic feed additives is based primarily on an antimicrobial activity. The pronounced mucus production through phytogenics, however, suggests another mechanism, namely inhibition of adherence of pathogens to the intestinal tissue. The same applies to enhanced activities of digestive enzymes observed in animals treated with phytogenic substances. Both a higher mucus production and activity of digestive enzymes suggests irritation of intestinal tissues as primary mode of action. Unfortunately, respective experimental data is available only from commercial products containing blends of different active substances. Therefore, it is unclear whether irritating actions are a feature of phytogenics in general or of single compounds (e.g. hot and pungent principles). According to the current state of knowledge, phytogenic feed additives (especially commercial products

W. Windisch et al. 31

usually comprising a blend of different active compounds) seem to act through a combination of different modes such as antioxidative and antimicrobial action as well as specific effects on gut tissue (irritation, mucus production, enzyme activity, etc.).

References Adam K, Sivropoulou A, Kokkini S, Lanaras T and Arsenakis M (1998) Antifungal activities of Origanum vulgare subsp. hirtum, Mentha spicata, Lavandula angustifolia, and Salvia fruticosa essential oils against human pathogenic fungi. Journal of Agricultural and Food Chemistry 46: 1739–1745. Alcicek A, Bozkurt M and Cabuk M (2003) The effect of an essential oil combination derived from selected herbs growing wild in Turkey on broiler performance. South African Journal of Animal Science 33: 89–94. Alcicek A, Bozkurt M and Cabuk M (2004) The effect of a mixture of herbal essential oils, an organic acid or a probiotic on broiler performance. South African Journal of Animal Science 34: 217–222. Baba S, Osakabe N, Natsume M, Yasuda A, Muto Y, Hiyoshi K, Takano H, Yoshikawa T and Terao J (2005) Absorption, metabolism, degradation and urinary excretion of rosmarinic acid after intake of Perilla frutescens extract in humans. European Journal of Nutrition 44: 1–9. Bampidis VA, Christodoulou V, Florou-Paneri P, Christaki E, Chatzopoulou PS, Tsiligianni T and Spais AB (2005) Effect of dietary dried oregano leaves on growth performance, carcase characteristics and serum cholesterol of female early maturing turkeys. British Poultry Science 46: 595–601. Basmacioglu H, Tokusoglu O and Ergul M (2004) The effect of oregano and rosemary essential oils or alpha-tocopheryl acetate on performance and lipid oxidation of meat enriched with n-3 PUFA’s in broilers. South African Journal of Animal Science 34: 197–210. Beagley KW, Fujihashi K, Lagoo-Deenadaylan S, Black CA, Murray MA, Sharmanov AT, Yamamoto M, McGhee JR and Elson CO (1995) Differences in intraepithelial lymphocyte T cell subsets isolated from murine small versus large intestine. Journal of Immunology 154: 5611–5619. Botsoglou NA, Florou-Paneri P, Christaki E, Fletouris DJ and Spais AB (2002) Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. British Poultry Science 43: 223–230. Botsoglou NA, Grigoropoulou SH, Botsoglou E, Govaris A and Papageorgiou G (2003a) The effects of dietary oregano essential oil and alpha-tocopheryl acetate on lipid oxidation in raw and cooked turkey during refrigerated storage. Meat Science 65: 1193–1200. Botsoglou NA, Govaris A, Botsoglou EN, Grigoropoulou SH and Papageorgiou G (2003b)

32 Phytogenic feed additives to young piglets and poultry Antioxidant activity of dietary oregano essential oil and alpha-tocopheryl acetate supplementation in long-term frozen stored turkey meat. Journal of Agricultural and Food Chemistry 51: 2930–2936. Botsoglou NA, Florou-Paneri P, Christaki E, Giannenas I and Spais AB (2004a) Performance of rabbits and oxidative stability of muscle tissues as affected by dietary supplementation with oregano essential oil. Archives of Animal Nutrition 58: 209–218. Botsoglou NA, Christaki E, Florou-Paneri P, Giannenas I, Papageorgiou G and Spais AB (2004b) The effect of a mixture of herbal essential oils or alpha-tocopheryl acetate on performance parameters and oxidation of body lipid in broilers. South African Journal of Animal Science 34: 52–61. Botsoglou NA, Florou-Paneri P, Botsoglou E, Dotas V, Giannenas I, Koidis A and Mitrakos P (2005) The effect of feeding rosemary, oregano, saffron and alpha-tocopheryl acetate on hen performance and oxidative stability of eggs. South African Journal of Animal Science 35: 143–151. Böhmer BM, Salisch H, Paulick BR and Roth XF (2009) Echinacea purpurea as a potential immunostimulatory feed additive in laying hens and fattening pigs by intermittent application. Livestock Science 122: 81–85. Burt S (2004) Essential oils: their antibacterial properties and potential applications in food – a review. International Journal of Food Microbiology 94: 223–253. Cabuk M, Bozkurt M, Alcicek A, Akbas Y and Kücükyilmaz K (2006) Effect of a herbal essential oil mixture on growth and internal organ weight of broilers from young and old breeder flocks. South African Journal of Animal Science 36: 135–141. Castillo M, Martin-Orue SM, Roca M, Manzanilla EG, Badiola I, Perez JF and Gasa J (2006) The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs. Journal of Animal Science 84: 2725–2734. Cho JH, Chen YJ, Min BJ, Kim HJ, Kwon OS, Shon KS, Kim IH, Kim S and Asamer A (2006) Effects of essential oils supplementation on growth performance, IgG concentration and fecal noxious gas concentration of weaned pigs. Asian-Australasian Journal of Animal Science 19: 80–85. Chrubasik S, Pittler MH and Roufogalis BD (2005) Zingiberis rhizome: A comprehensive review on the ginger effect and efficacy profiles. Phytomedicine 12: 684–701. Craig WJ (1999) Health promoting properties of common herbs. American Journal of Clinical Nutrition 70(suppl): 491S–499S. Cuppett SL and Hall CA (1998) Antioxidant activity of Labiatae. Advances in Food and Nutrition Research 42: 245–271. Demir E, Sarica S, Özcan MA and Suicmez M (2005) The use of natural feed additives as alternative to an antibiotic growth promoter in boiler diets. Archiv für Geflügelkunde 69: 110–116. Dong XF, Gao WW, Tong JM, Jia HQ, Sa RN and Zhang Q (2007) Effect of polysavone (Alfalfa Extract) on abdominal fat deposition and immunity in broiler chickens. Poultry Science 86: 1995–1959.

W. Windisch et al. 33

Denli M, Okan F and Uluocak AN (2004) Effect of dietary supplementation of herb essential oils on the growth performance, carcass and intestinal characteristics of quail (Coturnix coturnix japonica). South African Journal of Animal Science 34: 174–179. Dorman HJD and Deans SG (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology 88: 308–316. Florou-Paneri P, Giannenas I, Christaki E, Govaris A and Botsoglou N (2006) Performance of chickens and oxidative stability of the produced meat as affected by feed supplementation with oregano, vitamin C, vitamin E and their combinations. Archiv für Geflügelkunde 70: 232–240. Francis G, Kerem Z, Makkar HPS and Becker K (2002) The biological action of saponins in animal systems: a review. British Journal of Nutrition 88: 587–605. Freitag M, Hensche HU, Schulte-Sienbeck H and Reichelt B (1998) Kritische Betrachtung des Einsatzes von Leistungsförderern in der Tierernährung. Forschungsbericht des Fachbereichs Agrarwirtschaft Soest. ISBN 3-00-003331-9. UniversitätGesamthochschule Paderborn, Germany. Gabert VM and Sauer WC (1994) The effect of supplementing diets for weanling pigs with organic acids. Journal of Animal and Feed Sciences 3: 73–87. Garcia V, Catalá-Gregori P, Hernández F, Megias MD and Madrid J (2007) Effect of formic acid and plant extracts on growth, nutrient digestibility, intestine musa morphology, and meat yield of broilers. Journal of Applied Poultry Research 16: 555–562. Giannenas I, Florou-Paneri P, Papazahariadou M, Christaki E, Botsoglou NA and Spais AB (2003) Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition 57: 99–106. Giannenas I, Florou-Paneri P, Papazahariadou M, Botsoglou NA, Christaki E and Spais AB (2004) Effect of diet supplementation with ground oregano on performance of broiler chickens challenged with Eimeria tenella. Archiv für Geflügelkunde 68: 247–252. Giannenas I, Florou-Paneri P, Botsoglou NA, Christaki E and Spais AB (2005) Effect of supplementing feed with oregano and/or alpha-tocopheryl acetate on growth of broiler chickens and oxidative stability of meat. Journal of Animal and Feed Sciences 14: 521–535. Gollnisch K, Wald C and Berk A (2001) Einsatz unterschiedlicher ätherischer Öle in der Ferkelaufzucht. XXXXVI. In: Vortragstagung der Deutschen Gesellschaft für Qualitätsforschung (Pflanzliche Nahrungsmittel) e.V. in Zusammenarbeit mit der Vereinigung für Angewandte Botanik.19. –20. 03. 2001 in Jena/Thüringen, Germany. Pp. 259–262. Govaris A, Botsoglou N, Papageorgiou G, Botsoglou E and Ambrosiadis I (2004) Dietary versus post-mortem use of oregano oil and/or alpha-tocopherol in turkeys to inhibit development of lipid oxidation in meat during refrigerated storage. International Journal of Food Sciences and Nutrition 55: 115–123. Güler T, Ertaş ON, Ciftci M and Dalkılıc B (2005) The effect of coriander seed (Coriandrum

34 Phytogenic feed additives to young piglets and poultry sativum L.) as diet ingredient on the performance of Japanese quail. South African Journal of Animal Science 35: 261–267. Günther KD and Bossow H (1998) The effect of etheric oil from oreganum vulgaris in the feed ration of weaned pigs on their daily feed intake, daily gains and food utilization. In: Proceedings of the 15th IVPS Congress, Birmingham, UK. P. 223. Guo FC, Kwakkel RP, Soede J, Williams BA and Verstegen MWA (2004) Effect of a Chinese herb medicine formulation, as an alternative for antibiotics, on performance of broilers. British Poultry Science 45: 793–797. Hagmüller W, Jugl-Chizzola M, Zitterl-Eglseer K, Gabler C, Spergser J, Chizzola R and Franz C (2006) The use of Thymi herba as feed additive (0.1%, 0.5%, 1.0%) in weanling piglets with assessment of the shedding of haemolysing E. coli and the detection of thymol in the blood plasma. Berliner und Munchener Tierarztliche Wochenschrift 119: 50–54. Halle I, Thoman R and Flachowsky G (1999) Einfluss eines Oreganoöl-Zusatzes zum Futter auf die Zusammensetzung das Chymus sowie die Mikroflora im Darmkanal von Absetzferkeln. In: 7th Symposium Vitamins and Additives in Nutrition of Man and Animal, Jena, Germany. Pp. 469–471. Halle I (2001) Effects of essential oils and herbal mixtures on growth of broiler chicks. In: 8th Symposium Vitamins and Additives in Nutrition of Man and Animal, Jena, Germany. P. 84. Hammer KA, Carson CF and Riley TV (1999) Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 86: 985–990. Hernandez F, Madrid J, Garcia V, Orengo J and Megias MD (2004) Influence of two plant extracts on broilers performance, digestibility, and digestive organ size. Poultry Science 83: 169–174. Hume ME, Clemente-Hernandez S and Oviedo-Rondon EO (2006) Effects of feed additives and mixed Eimeria species infection on intestinal microbial ecology of broilers. Poultry Science 85: 2106–2111. Jamroz D, Orda I, Kamel C, Wiliczkiewicz A, Wertelecki T and Skorupinska I (2003) The influence of phytogenic extracts on performance, nutrient digestibility, carcass characteristics, and gut microbial status in broiler chickens. Journal of Animal and Feed Sciences 12: 583–596. Jamroz D, Wiliczkiewicz A, Wertelecki T, Orda J and Skorupinska J (2005) Use of active substances of plant origin in chicken diets based on maize and locally grown cereals. British Poultry Science 46: 485–493. Jamroz D, Wertelecki T, Houszka M and Kamel C (2006) Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90: 255–268. Jang IS, Ko YH, Yang HY, Ha JS, Kim YI, Kang SY, Yoo DH, Nam DS, Kim DH and Lee CY (2004) Influence of essential oil components on growth performance and the functional activity of the pancreas and small intestine in broiler chickens. Asian-

W. Windisch et al. 35

Australasian Journal of Animal Science 17: 394–400. Janz JAM, Morel PCH, Wilkinson BHP and Purchas RW (2007) Preliminary investigation of the effects of low-level dietary inclusion of fragrant essential oils and oleoresins on pig performance and pork quality. Meat Science 75: 350–355. Jugl-Chizzola M, Spergser J, Schilcher F, Novak J, Bucher A, Gabler C, Hagmuller W and Zitterl-Eglseer K (2005) Effects of Thymus vulgaris L. as feed additive in piglets and against haemolytic E. coli in vitro. Berliner und Münchner Tierärztliche Wochenschrift 118: 495–501. Killeen GF, Connolly CR, Walsh GA, Duffy CF, Headon DR and Power RF (1998) The effects of dietary supplementation with Yucca schidigera extract or Fractions thereof on nitrogen metabolism and gastrointestinal fermentation processes in the rat. Journal of the Science of Food and Agriculture 76: 91–99. Kong XF, Wu GY, Liao YP, Hou ZP, Liu HJ, Yin FG, Li TJ, Huang RL, Zhang M, Deng D, Kang P, Wangb RX, Tang ZY, Yang CB, Deng ZY, Xiong H, Chu W-Y, Ruan Z, Xie MY and Yin YL (2007) Effects of Chinese herbal ultra-fine powder as a dietary additive on growth performance, serum metabolites and intestinal health in earlyweaned piglets. Livestock Science 108: 272–275. Kreydiyyeh SI, Usta J, Knio K, Markossian S and Dagher S (2003) Aniseed oil increases glucose absorption and reduces urine output in the rat. Life Science 74: 663–673. Kroismayr A, Schedle K, Sehm J, Pfaffl MW, Plitzner C, Foissy H, Ettle T, Mayer H, Schreiner M and Windisch W (2008a) Effects of antimicrobial feed additives on gut microbiology and blood parameters of weaned piglets. Bodenkultur 59: 111–120. Kroismayr A, Sehm J, Pfaffl MW, Schedle K, Plitzner C and Windisch W (2008b) Effects of Avilamycin and essential oils on mRNA expression of apoptotic and inflammatory markers and gut morphology of piglets. Czech Journal of Animal Science 53: 377–387. Kyriakis SC, Sarris K, Lekkas S, Tsinas AC, Giannakopoulos CG, Alexopolos C and Saoulidis K (1998) Control of post weaning diarrhoea syndrome of piglets in-feed application of origanum essential oils. In: Proceedings of the 15th IVPS Congress, Birmingham, UK. P. 106. Lee KW, Everts H, Kappert HJ, Frehner M, Losa R and Beynen AC (2003) Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broiler chickens. British Poultry Science 44: 450–457. Lien TF, Horng YM and Wu CP (2007) Feasibility of replacing antibiotic feed promoters with the Chinese traditional herbal medicine Bazhen in weaned piglets. Livestock Production Science 107: 92–102. Maass N, Bauer J, Paulicks BR, Bohmer BM and Roth-Maier DA (2005) Efficiency of Echinacea purpurea on performance and immune status in pigs. Journal of Animal Physiology and Animal Nutrition 89: 244–252. Manzanilla EG, Perez JF, Martin M, Kamel C, Baucells F and Gasa J (2004) Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 82: 3210–3218.

36 Phytogenic feed additives to young piglets and poultry Manzanilla EG, Nofrarias M, Anguita M, Castillo M, Perez JF, Martin-Orue SM, Kamel C and Gasa J (2006) Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 84: 2743–2751. Mayland-Quellhorst D (2002) Untersuchungen zum Einfluss von Anis auf die Mastleistung von Broilern. Master Thesis, Fachhochschule Osnabrück, Germany. Mitsch P, Zitterl-Eglseer K, Kohler B, Gabler C, Losa R, and Zimpernik I (2004) The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poutry Science 83: 669–675. Nabuurs MJA (1995) Microbiological, structural and functional changes of the small intestine of pigs at weaning. Pig News and Information 16: 93–97. Nakatani N (2000) Phenolic antioxidants from herbs and spices. BioFactors 13: 141–146. Namkung H, Li M, Gong J, Yu H, Cottrill M and de Lange CFM (2004) Impact of feeding blends of organic acids and herbal extracts on growth performance, gut microbiota and digestive function in newly weaned pigs. Canadian Journal of Animal Science 84: 697–704. Nazeer MS, Pasha TN, Abbas S and Ali Z (2002) Effect of yucca saponin on urease activity and evelopment of ascites in broiler chicken. International Journal of Poultry Science 1: 174–178. Nofrairas M, Manzanilla EG, Pujols J, Gilbert X, Majo N, Segales J and Gasa J (2006) Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaning pigs. Journal of Animal Science 84: 2735–2742. Oetting LL, Utiyama CE, Giani PA, Ruiz UD and Miyada VS (2006) Effects of herbal extracts and antimicrobials on apparent digestibility, performance, organs morphometry and intestinal histology of weanling pigs. Revista Brasileira de Zootecnia 35: 1389–1397. Oviedo-Rondon EO, Hume ME, Hernandez C and Clemente-Hernandez S (2006) Intestinal microbial ecology of broilers vaccinated and challenged with mixed Eimeria species, and supplemented with essential oil blends. Poultry Science 85: 854–860. Özer H, Sökmen M, Güllüce M, Adigüzel A, Sahin F, Sökmen A, Kilic H and Baris Ö (2007) Chemical composition and antimicrobial and antioxidant activities of the essential oil and methanol extract of Hippomarathum microcarpum (Bieb.) from Turkey. Journal of Agricultural and Food Chemistry 55: 937–942. Papageorgiou G, Botsoglou N, Govaris A, Giannenas I, Iliadis S and Botsoglou E (2003) Effect of dietary oregano oil and alpha-tocopheryl acetate supplementation on ironinduced lipid oxidation of turkey breast, thigh, liver and heart tissues. Journal of Animal Physiology and Animal Nutrition 87: 324–335. Platel K and Srinivasan K (2000a) Influence of dietary spices and their active principles on pancreatic digestive enzymes in albino rats. Food 44: 41–46. Platel K and Srinivasan K (2000b) Stimulatory influence of select spices on bile secretion

W. Windisch et al. 37

in rats. Nutrition Research 20: 1493–1503. Platel K and Srinivasan K (2004) Digestive stimulant action of spices: A myth or reality? Indian Journal of Medical Research 119: 167–179. Rao RR, Platel K and Srinivasan K (2003) In vitro influence of spices and spice-active principles on digestive enzymes of rat pancreas and small intestine. Food 47: 408–412. Roth FX and Kirchgessner M (1998) Organic acids as feed additives for young pigs: nutritional and gastrointestinal effects. Journal of Animal and Feed Sciences 8: 25–33. Roth FX, Windisch W and Kirchgessner M (1998) Effect of potassium diformiate (FormiTM LHS) on nitrogen metabolism and nutrient digestibility in piglets at graded dietary lysine supply. Agribiological Research 51: 167–175. Sarica S, Ciftci A, Demir E, Kilinc K and Yildirim Y (2005) Use of an antibiotic growth promoter and two herbal natural feed additives with and without exogenous enzymes in wheat based broiler diets. South African Journal of Animal Science 35: 61–72. Schiavone A, Righi F, Quarantelli A, Bruni R, Serventi P and Fusari A (2007) Use of Sibyllum marianum fruit extract in broiler chicken nutrition: influence on performance and meat quality. Journal of Animal Physiology and Animal Nutrition 91: 256–267. Schöne F, Vetter A, Hartung H, Bergmann H, Lutz J, Richter G and Müller S (2004) Prüfung der ätherischen Öle aus Fenchel- und Kümmelsaat bei Absetzferkeln. In: 8. Tagung Schweine- und Geflügelernährung, Jena, Germany. Pp. 120–122. Schöne F, Vetter A, Hartung H, Bergmann H, Biertumpfel A, Richter G, Muller S and Breitschuh G (2006) Effects of essential oils from fennel (Foeniculi aetheroleum) and caraway (Carvi aetheroleum) in pigs. Journal of Animal Physiology and Animal Nutrition 90: 500–510. Schuhmacher A, Hofmann M, Boldt E and Gropp JM (2002) Kräuter als alternative Leistungsförderer beim Ferkel. In: Forum angewandte Forschung in der Rinder- und Schweinefütterung, Fulda, Germany. Pp. 85–87. Si W, Gong J, Tsao R, Zhou T, Yu H, Poppe C, Johnson R and Du Z (2006) Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology 100: 296–305. Smith-Palmer A, Stewart J and Fyfe L (1998) Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Food Microbiology 26: 118–122. Straub R, Gebert S, Wenk C and Wanner M (2005) Growth performance, energy, and nitrogen balance of weanling pigs fed a cereal-based diet supplemented with Chinese rhubarb. Livestock Production Science 92: 261–269. Tartrakoon W, Sukkasem K, Ter Meulen U and Vearasilp T (2003) Use of essential oil extracted from citronella, cloves and peppermint as supplement in weaner pig diets. In: Deutscher Tropentag, October 8–10, 2003, University of Göttingen, Germany. Wald C, Kluth H and Rodehutscord M (2001) Effects of different essential oils on the

38 Phytogenic feed additives to young piglets and poultry growth performance of piglets. In: Proceedings of the Society of Nutrition Physiology, Göttingen, Germany. Pp. 156. Wald C (2002) Untersuchungen zur Wirksamkeit verschiedener äterischer Öle im Futter von Aufzuchtferkeln und Broilern. PhD thesis. Universität Halle Wittenberg, Germany. Wei A and Shibamoto T (2007) Antioxidant activities and volatile constituents of various essential oils. Journal of Agricultural and Food Chemistry 55: 737–1742. Windisch W, Schedle K, Plitzner C and Kroismayr A (2008) Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86: E140–E148. Xu ZR, Hu CH, Xia MS, Zahn XA and Wang MQ (2003) Effects of dietary fructooligosaccharide on digestive enzyme activities intestinal microflora and morphology of male broilers. Poultry Science 82: 648–654. Yen JT and Pond WG (1993) Effects of carbadox, copper, or yucca shidigeru extract on growth performance and visceral weight of young pigs. Journal of Animal Science 71: 2140–2146. Yeo J and Kim KI (1997) Effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on growth and intestinal urease activity in broiler chicks. Poultry Science 76: 381–385. Young JF, Stagsted J, Jensen SK, Karlsson AH and Henckel P (2003) Ascorbic acid, alphatocopherol, and oregano supplements reduce stress-induced deterioration of chicken meat quality. Poultry Science 82: 1343–1351. Zitterl-Eglseer K, Wetscherek W, Stoni A, Kroismayr A and Windisch W (2008) Bioverfügbarkeit der ätherischen Öle eines phytobiotischen Futterzusatzes und der Einfluss auf die Leistung bzw. Nährstoffverdaulichkeit bei Absetzferkeln. Bodenkultur 59: 121–129.

T.J. Applegate 39

3 INFLUENCE OF PHYTOGENICS ON THE IMMUNITY OF LIVESTOCK AND POULTRY Todd J. Applegate Department of Animal Science, Purdue University, 915 West State Street, West Lafayette, Indiana 47907-2054 USA, e-mail: [email protected]

Abstract Balancing the response to naïve pathogens and development of an immunological memory is essential for survivability of the animal but comes at a considerable price to productivity. The current breath of literature of plants and their extracts as immuno-modulators in livestock and poultry species is marginal and we currently draw much of our knowledge from that of human pharmaceutical and nutrition research. Where plants and plant-derived substances have been used for feed additives for livestock and poultry, their immuno-modulating targets have principally been against gastro-intestinal pathogens. For example, severity of coccidiosis appears to be alleviated in broiler chickens with compounds such as Artemesia annua, Sophora flavescens, Oregeno and Astragulus membranaceus. In weanling piglets, several compounds have been used against enteropathogenic E. coli to reduce the severity of diarrhea and improve immuno-competency. Future work, however, is needed to fully understand the pharmokinetic dynamics of plant-derived substances in our livestock and poultry species and where targeted applications might be most beneficial.

Introduction Immunity can be defined as the resistance of an organism to infection, disease, or other unwanted biological invasion. This resistance can be categorised into non-specific responses, innate immunity and adaptive immunity. Despite a large number of plants that have been used for centuries, less than 5 percent affect the immune response (Guo et al., 2003). Numerous mixtures of plants and their extracts have recently entered into the commercial livestock and poultry feeding sector. While little in vivo research has been done with livestock and poultry, several have emerged as showing promise in improving productivity either through their antioxidant and antimicrobial properties, improvement in diet palatability, improvement of gut function (digestive secretion, absorptive capabilities, or 39

40 Influence of phytogenics on the immunity of livestock and poultry changes to barrier functionality), suppression of pathogen virulence and/or tissue recovery after damage. The sum of these responses from reported poultry literature have shown either no improvement or up to a six percent improvement in feed-to-gain ratios, whereas literature in swine is less consistent (Windisch et al., 2008). This review will explore how plant-derived products might influence the immune capabilities of livestock and poultry. For recent reviews on immunology in poultry and swine please refer to Klasing (2007), Burkey et al. (2008) and Piriou-Guzylack and Salmon (2008). Immuno-modulation can be defined (Bakuridze et al., 1993) as the change (stimulating or suppressing) of the indicators of cellular, humoral and non-specific defence mechanisms. Immunologic responses can be costly in terms of nutrient allocation, but necessary to rid the body of a pathogen(s) and thus keep the animal alive. Typically, the immune system is held in a homeostatic balance between immuno-stimulation and immuno-suppression. Several plants have been identified for their pharmacologic capabilities. These pharmacologic effects are wide-ranging. For example, Ginsing, with its steroidal saponins, has immunostimulating properties including cytokine production (Il-1, Il-6, IL-12, IL-6, TNF-α and IFN-γ), macrophage activation and lymphocyte activity (Tan and Vanitha, 2004). Conversely, Ginko biloba and its bio-active flavenoids and terpenes can mediate production of pre-inflammatory cytokines (Li, 2000). Practical application to livestock and poultry, however, has been limited to measures of a relatively few immuno-modulating measures, particularly in response to intestinal pathogens. However, considerable targeted uses could be developed. These are not limited to, but could include advancements in vaccine adjuvants or improvements in response in immune compromised animals (e.g. use of phytosterols such as β-sitosterol and β-sitosterol glycosides; Bouic and Lamprecht, 1999).

Cost of immunity No review of livestock and poultry immune response would be complete without fully addressing what productive sacrifices are made due to either a non-specific, innate immune response, or adaptive immune response. Of the three, the adaptive immune response requires the least as far as nutrient allocation, but requires prior exposure to an antigen. However, the non-specific response and innate response are nutrient intensive due to changes in tissue maintenance, secretion (e.g. increases in intestinal mucin) and factors associated with an acute-phase response. The acute phase response can include local inflammation, fever, anorexia, muscle catabolism and production of cytokines and acute phase proteins (Gruys et al., 2005; Klasing, 2007). The severity, duration and recovery of feed intake suppression due to the immunological naivety towards a particular pathogen can be influenced by pathogen load, virulence, animal genotype and feed composition (Sandberg et al., 2006). In some cases, the anorexia experienced during the acute phase response is necessary for some genotypes to immunologically cope with the pathogen. For example, Nestor et al. (1999) compared the response of full-fed, growth-selected (F-line) turkeys to a feed-limited, growth-selected and a random-bred line (RBC) of turkeys when challenged with a high dosage of Pasteurella

T.J. Applegate 41

multocida. Interestingly, the mortality in the full-fed, F-line turkeys was over 80% but that of the feed-limited F-line and RBC line turkeys were only 48 and 43%, respectively. Beyond the feed intake reduction during an acute phase response, productivity is also lost due to the acute phase immune response requiring up to 10 percent of nutrient use which otherwise would have gone towards growth (Klasing, 2007). Other researchers have estimated this nutrient cost to be 1.3 times that of maintenance (Webel et al., 1998) or a daily cost of 0.27 g ideal protein per kg body weight (Sandberg et al., 2007).

Immunity and response of the gastro-intestinal tract The gastro-intestinal tract (GIT) has been a primary focus for action of phytogenic feed additives. As an organ system, the GIT must meet two seemingly incompatible goals: maximize nutrient uptake and minimize antigenic insult while tolerating the presence of indigenous microbiota and other antigens introduced by the presence of feed within the intestinal tract. In doing so, the GIT consumes approximately 20% of dietary energy with a protein turn-over rate of 50 to 75% per day (Cant et al., 1996). Nearly 25% of daily protein synthesis is secreted into the GIT to support digestive and barrier functionality. The GIT is residence to 10 times more bacterial cells than our own cells, which the body then allocates nearly 70% of all its immune cells to protecting the body from foreign cells and substances (Kagnoff, 1993). The barrier of the GIT is both specific and non-specific and is far from remaining “static”. Adaptively, the GIT can: • • • • • •

Change peristaltic rate Change enterocyte turnover (including changes to proliferation rate, migration rate, rate of apoptosis - programmed cell death), Regulate tight junction permeability between enterocytes Change mucin production (amount and compositional changes), Change direction of stem cells towards those with greater immune capacity (both innate and adaptive responses), and Facilitate a commensal microflora through luminal secretions.

From a holistic perspective, there is a paucity of data on the influence of plants and plantderived substances across the range of these physiological and immunological responses.

Non-immune defences of the gastro-intestinal tract The initial, non-specific barrier in the GIT includes that of mucin production and epithelial turnover. As described earlier, these processes are far from static and turnover (however costly to growth) can be increased to increase the rate of antigen shed from the GIT.

42 Influence of phytogenics on the immunity of livestock and poultry Mucins Among the first line of non-immune defences associated with gut barrier function is the mucus layers overlying the epithelial cells. Mucins contain a diverse array of carbohydrate structures which are created and secreted by goblet cells located along the villi within the intestinal epithelium (Forstner and Forstner, 1994). Many functional properties have been ascribed to intestinal mucins, such as lubrication of intestinal surfaces, trapping and neutralizing bacteria, detoxification of heavy metal binding, interactions with the intestinal immune system, acting as a diffusion barrier for nutrients and macromolecules and protecting the underlying epithelial cells (Forstner and Forstner, 1994). Because the mucins contain a diverse array of carbohydrate structures, they provide numerous potential attachment sites for commensal and pathogenic bacteria and also serve as a colonization niche for intestinal bacteria found within the intestine (Sonnenburg et al., 2004). Moreover, the tightly adherent mucus layers bind with mucin binding protein receptors on the underlying epithelial cells, thus preventing bacterial access to some epithelial receptors (Slomiany et al., 2001). However, by aiding potential pathogens in gaining an attachment site, the mucus layer may also allow pathogens to migrate through the mucin towards the epithelium where colonization or release of toxins may occur. Protective factors are in place, however, as both mucin layers are known to accumulate bacteriocidal and bacteriostatic compounds and secretory immunoglobulin A, compounds which are capable of neutralizing or killing the trapped bacteria. Secondly, as the loosely adherent mucus layer is sloughed, it also traps and carries the resident or invading bacteria, thus removing the trapped bacteria from the GIT. Therefore, although mucus is generally seen as an important factor in maintaining a strong intestinal barrier, it is difficult to predict exactly how the mucus layer will inhibit or aid specific pathogens in the invasion process, as many factors influence the outcome of bacterial-mucin interactions, such as mucin composition, quality, quantity, digesta flow and gut motility (Forstner and Forstner, 1994). High levels of threonine in digesta have been attributed to the contribution of mucin and may be responsible for the low apparent digestibility of threonine in many feedstuffs (Lien et al., 1997; Sauer and Ozimek, 1986) or during higher antigenic load within the intestine. For example, Corzo et al. (2007) noted a 2 to 10% increase in threonine requirement when birds were reared in used “dirty” litter versus “clean” litter. This increase in nutrient need can have a large economic impact. Little information is available currently as to how different phytogenic products will affect mucin production. Jamroz et al. (2006) noted that when a product containing 5, 3 and 2 mg/kg of carvacrol, cinnamaldehyde and capsiscum oleoresin was fed to broilers, there was no effect on performance, but goblet cells and mucin at the surface of the villi were increased. Unfortunately, there was no direct quantification of this change and what is not known is whether or not this change had a functional effect on commensal or pathogenic bacteria.

T.J. Applegate 43

Turnover of intestinal epithelia As intestinal cells are derived and migrate along the length of the crypt-villus axis, they differentiate and express functionality, either as a digestive/absorptive enterocyte, a mucus-secreting goblet cell, a peptide hormone producing enteroendocrine cell, or an antimicrobial peptide/protein secreting paneth cell. A typical response to ridding infected enterocytes is to increase migration and apoptosis rates. Epithelial turnover rates can vary from as little as 2 days (Imondi and Bird, 1966) to as much as 5 days (Applegate et al., 1999). If the epithelium turns over more quickly, enterocytes are not allowed to reach a fully differentiated state. Examples of where this may become detrimental the animal is in the case of expression of carbohydrases. Uni et al. (1998) noted that the upper 40% of the villus expresses 30 to 40% more sucrase and maltase activity per enteroctye than the lower 60% of the crypt/villus axis. During enteric stress where enteroctyes are not allowed reach a differentiated state, undigested disaccharides enter into the lower digestive tract causing microbial growth resulting in osmotic and secretory diarrhea (Zijlstra et al., 1997). Additionally, intestinal stressors can also influence rates of enteroctye proliferation and apoptosis (programmed cell death; Potturi et al., 2005). Very few papers, however, have measured the effect that plants and plant-derived substances may have on intestinal enterocyte dynamics. Kroismayr et al. (2006) began to look in this direction by quantitative RT-PCR measures of cyclin D (a marker of cell proliferation) in the mesenteric lymph nodes of pigs. Notably, cyclin D was reduced by feeding of a commercial feed additive containing essential oils of oregano, anis and citrus peel with inulin. Thatte et al. (2000) noted that several plant extracts can either induce apoptosis (e.g. mistletoe, Semicarpus anacardium, bryonolic acid from saffron, allicin from Allium sativum, as well as soybean, garlic, ginger and green tea) or prevent apoptosis (e.g. Panax ginseng) in cell culture studies. The question, however, is as to whether any of these compounds affect intestinal enterocyte cell cycle lifetime and differentiation.

Intestinal tight junctions Additional influences on the intestinal tract include that on paracellular tight junctions. These junctions serve as a regulated barrier between the apical and basolateral ends of the enterocyte. Anderson and Cereijido (2001) report that 90% of substances (ions, nutrients, etc.) are absorbed via this paracellular route and are highly regulated with the tight junctions discriminating against different ions depending on the surrounding pH. Influence of Solanaceae spices (paprika and cayenne pepper) on tight junction functionality has been noted in intestinal cell culture models through increased permeability of ions and macromolecules, thus having plausible pathophysiological importance (Jensen-Jarolim et al., 1998). Similar pharmacological work in rats and humans has shown that the alkaloid, piperine, from different pepper species (Piper nigrum and Piper longum) can mediate absorption of different substances such as curcumin (from Curcuma longa) from the intestine (Shoba et al., 1998). Further research is needed in this area to determine the influence of

44 Influence of phytogenics on the immunity of livestock and poultry different plants and plant-derived substances because enteric pathogens and associated virulence factors (Sears, 2000) and stress-induced production of catecholamines (Söderholm and Perdue, 2001) influence the functionality of intestinal tight junctions.

Immune defences of the gastro-intestinal tract Non-immunologic contributions to gut barrier function are very important, yet the immune system cannot be overlooked as the GIT contains greater than 70% of all immune cells found within the body (Kagnoff, 1993). Thus, the intestinal immune system has been given its own designation as GALT, or gastrointestinal-associated lymphoid tissue and contributes greatly to gut barrier function on a day-to-day basis. The protection that is provided by GALT can be broken down into two categories: innate and adaptive immunity. Innate immunity is defined as a non-specific immune defence and consists of physical barriers such as epithelial cells or secretions such as mucus (as discussed previously), as well as antibacterial peptides such as defensins, lysozymes, or lactoferrin and phagocytes and macrophages which engulf and destroy bacteria. None of these factors specifically target invading pathogens, but instead provide an initial defence against them and enhance the mechanisms of adaptive immunity. Adaptive or acquired immunity is significantly more specific to individual antigens, which are defined as any substance that the body recognises as foreign, and is characterised by the development of T cells, B cells and antibodies that are antigen-specific (Muir, 1998). One of the primary components of the GALT is the lamina propria of the intestine, which is the connective tissue that underlies the epithelium of the gut. Highly vascularised and richly innervated by the enteric nervous system (Gaskins, 1997), the lamina propria contains sizeable populations of immune cells such as the B and T lymphocytes, immunoglobulins, macrophages, mast cells and plasma cells, among others (Kagnoff, 1993). Additionally, intraepithelial lymphocytes (IEL) reside within the epithelial layer of the intestine and produce numerous cytokines that mediate the immune response. Where IEL’s have been measured after feeding weanling piglets with plants or plant-derived substances, measured changes have been noted after feeding carvacrol, cinnamaldehyde and capsicum oleoresin but are specific to region of the intestine measured (Manzanilla et al., 2006; Nofrarias et al., 2006). Other changes to the GALT in pigs include oregano increasing T lymphocytes in mesenteric lymph nodes (Walter and Bilkei, 2004), reduction in peyers patches after feeding of essential oils of oregano, anis and citrus peel (Kroismayr et al., 2006) and reduced B lymphocytes in mesenteric lymph nodes and increased lymphocyte density in the colon (Nofrarias et al., 2006).

Phytogenic feed additives and in vivo anti-microbial effects One of the primary explored functional attributes of plants and plant extracts includes that of anti-microbial activity. As reviewed by Greathead (2003), anti-microbial activity in

T.J. Applegate 45

vivo is due to either their lipophilic nature of essential oils disrupting bacterial membrane structure and integrity, through inactivation of bacterial extracellular enzymes, or through up-regulation of immunological defences. This inherent activity, therefore, has caused research to focus either on pathogenic bacteria (including food-borne pathogens) and that for modification of ruminal microflora. Ruminal function modifications due to the antimicrobial activity of different plant secondary metabolites have been the focus of several recent reviews (Greathead, 2003; Rochfort et al., 2008). Most of research in this area has focused on the effect of forage type, growth stage and on active tannin and saponin content. Benefits of ruminal modification include anti-bloating through proanthocyanidin/tannin content (e.g. birdsfoot tre-foil and dock), anti-methanogen activity of the tannin component (e.g. trefoil, Acacia mearnsii), reductions in ruminal protozoa by saponins and essential oils (e.g. Soapberry tree, Artemisia annua) and nematodes by tannins (e.g. Hedysarum coronarium; Min and Hart, 2003; Rochfort et al., 2008). In non-ruminants, the distribution of indigenous microflora within the GIT is not random, but organised qualitatively and quantitatively along vertical and horizontal regions in the GIT (Berg, 1996). The vertical distribution refers to the distribution of bacteria from the oral cavity to the colon and concentrations of bacteria are vastly different among sections of the GIT. Furthermore, bacteria are distributed horizontally along the GIT and occupy the intestinal lumen, mucus lining, crypt spaces and adhere to the epithelial cells (Rozee et al., 1982). Thus, each segment and horizontal layer of the GIT harbors its own specific bacterial community, a phenomenon which may be attributed to environmental factors in the intestine, such as nutrition, bile salts, oxygen concentration and intestinal pH (van der Wielen et al., 2002). Currently, our knowledge of how and why different bacterial species come to reside in different horizontal layers of the intestinal tract is minimal and most studies have focused on the lower small intestine and hind-gut of poultry and swine.

Anti-coccidial effects Avian coccidiosis is the most costly disease of poultry resulting in an annual global loss between $1 to 3 billion USD in medication prevention, treatment, mortality, malabsorption and productivity losses (Williams, 1999; Shirley et al., 2007). Anti-coccidial effects of individual plants, plant extracts and unidentified commercial plant-derived feed additives have been noted in domestic poultry (Table 1). Many of these studies used proprietary mixtures and therefore repeatability and further investigation of physiologic mechanisms is very limited. Necrotic enteritis in Poultry – Additional intestinal insult, typically caused by Eimeria, can allow for Clostridium perfringens enterotoxins to cause necrotic enteritis. Necrotic enteritis is particularly severe in countries that have stopped using antibiotic growth promoters (van Immerseel et al., 2004). Several researchers have established necrotic enteritis bio-assays, of which commercial essential oil mixtures show promise in reducing enteritis severity (Table 2).

46 Influence of phytogenics on the immunity of livestock and poultry Table 1. Plant-derived compound effects on poultry response after Eimeria (coccidosis) challenge

Compound Raw soybeans

Dietary concentration

Measured response

Reduction of lesions after singular challenges of different Eimeria species Artemesia annua leaves 50 g/kg (not Reduction in lesions after E. acervulina effective at lower & tenella challenge. No effect on E. concentrations) maxima lesion scores. 119 mg/kg Camphor extract from Reduction in lesions after E. acervulina Artemesia annua leaves & tenella challenge. Water soluble extract 6 to 30 g/L of Reduction in lesions, bloody diarrhea & from Sophora flavescens drinking water oocyte shedding after E. tenella challenge Water soluble extracts 6 to 30 g/L of Show promise against Eimeria but not as from Pulsatilla koreana, drinking water efficacious as S. flavscens Sinomenium acutum, Ulmus macrocarpa, or Quisqualis indica Oregeno oil 300 mg/kg Reduction in E. tenella excretion & lesions – intermediate response versus coccidiostat Oregeno Up to 10 g/kg Reduction in mortality & lesion scores after E. tenella challenge Reduction in bloody diarrhea score, litter Commercial extract from 0.5 g/kg oocyst count, with no change in cecal Agrimonia eupatoria, lesion score after E. tenella challenge Echinacea angustifolia, Ribes nigrum, & Cinchona succirubra 1 g/kg Astragulus After E. tenella challenge: IgA & IgM membranaceus extract but not IgG were increased in serum at day 14 & 21, IgA increased in cecae at day 14 but not 7 or 21, splenocyte proliferation increased at 14 & 21 days to E. tenella antigen, & erythrocyte rosette forming cells & erythrocyte antibody complement cells increased at 14 & 21 days 1 g/kg Reduction in lesions & oocyte shedding Astragulus after E. tenella challenge, but not when membranaceus extract given a coccidial vaccine. 10, 30, & 100 g/ Improved recovery after accidental Nigella sativa seeds kg (uncrushed) E. tenella infection. No effect on paramyxovirus Type 2 or 3 titers Commercial essential oil 100 ppm No effect on lesion scores after challenge blend with a coccidial vaccine containing E. acervulina, maxima, & tenella

Reference Mathis et al. (1995) Allen et al. (1997) Allen et al. (1997) Youn & Noh (2001) Youn & Noh (2001)

Giannenas et al. (2003) Giannenas et al. (2004) Christaki et al. (2004)

Guo et al. (2004a)

Guo et al. (2005) El-Sayed et al. (2005) OviedoRondon et al. (2005)

T.J. Applegate 47 Table 2. Plant-derived compound effects on poultry response to a necrotic enteritis challenge

Compound

Dietary concentration

Commercial essential oil mix Commercial essential oil mix Commercial mix containing thymol, carvacrol, eugenol, curcumin, & piperin Commercial oregano 330 & 660 g/ based essential oil mix MT

Measured response

Reference

Reduction in C. perfringens in ileium, rectum, & colon Reduction in C. perfringens from field study Reduction in C. perfringens proliferation

Losa & Kohler (2001) Mitsch et al. (2002) Mitsch et al. (2004)

Reduction in lesion scores in necrotic enteritis model

Saini et al. (2003)

Intestinal microflora and immune responses in poultry Additional studies have noted effects of plants and plant-derived substances on specific immunological and GIT microbiota measures, which are summarised in Table 3. In vitro results have suggested that different plant-derived substances can have an anti-microbial effect on C. perfringens (e.g. curcumin; Bhavanishankar and Sreenivasa Murthy, 1985). However, in vivo results after feeding carvacrol, cinnamaldehyde, capsacin, marjoram, oregano, rosemary, yarrow and/or thyme do not suggest a specific antimicrobial effect on C. perfringens (Jamroz et al., 2005; Cross et al., 2007). Other compounds, including carvacrol, cinnamaldehyde and capsacin reduced coliforms and/or E. coli (Jamroz et al., 2005) whereas marjoram, oregano, rosemary, yarrow or thyme did not (Cross et al., 2007). Additionally, Peganum harmala extract has been shown to reduce the severity and recovery from colibacillosis (Arshad et al., 2008). Beneficial microbiotia including lactobacilli and bifidobacteria have been shown to increase after feeding of Astragalus membranaceus radix extract (Guo et al., 2004b).

Intestinal microflora and immune responses in swine Diarrhea caused by either viral or enterotoxigenic E. coli causes substantial economic impact to the swine industry (Fairbrother et al., 2005). As with poultry and necrotic enteritis, the removal of growth-promoting antibiotics has increased the incidence and severity of postweaning diarrhea in piglets (Fairbrother et al., 2005). Not surprisingly, this production phase has been the focus of much of the research of plants and plant-derived substances for support of growth (Windisch et al., 2008), microbial and immunological characteristics in the intestine (Table 4).

48 Influence of phytogenics on the immunity of livestock and poultry Table 3. Plant-derived compound effects on microflora and immune response of poultry

Compound

Administrative Measured response dose Acemannan from Aloe 500 μg injection Adjuvant-like effect. IFN-γ in vitro, vera intra-muscular monocytes enhanced NO production, but a weaker effect on MHC II cell surface antigen expression, stimulating effect on spontaneous & inducible NO production for splenocytes with a higher capacity to proliferate in response to a T cell-mitogen PHA. Increased in vivo capacity to produce NO, after intravenous LPS injection. Aloe secundiflora var. 200 mg/kg prior Reduction in mortality after to infection; 400 Salmonella gallinarium infection. secundiflora mg/kg postIncrease in antibody titres & serum infection IL-6 concentrations after infection. No difference in Salmonella translocation to spleen or liver between groups. Water soluble extract of 1.5 x 104 µg/mL C. officinalis-treated birds (but not M. Calendula officinalis or (C. officinalis) officinalis) had higher CD3 positive or 3.0 x 104 Melissa officinalis T-cells & lower numbers of neutrophils µg/mL (M. after Salmonella enteriditis footpad officinalis) in injection. drinking water Undefined essential oils 25 or 50 mg/kg No change in ileal digesta Lactobacilli or E. coli 2 g/kg No effect on aerobes or enterococci in Astragalus cecae but an increase in bifidobacteria membranaceus radix & lactobacilli after Mycoplasma extract gallisepticum challenge Commercial 100 mg/kg No effect on C. perfringens. Mixed feed additive effects depending on age but generally containing carvacrol, when were fed wheat-based diets cinnamaldehyde, & observed reductions in E. coli. capsacin 15 mg/kg Feeding reduced Salmonella on neck Oregeno (Origanum skin after slaughter. onites) Oregeno essential oil 2.5 to 6.25 mL/ Increase in minimum inhibitory kg concentrations of amikacin, apramycin and streptomycin of E. coli chicken isolates. Marjoram, oregano, 10 g/kg or 1 g/ No significant changes in cecal or fecal rosemary, yarrow, kg of essential coliforms, lactic acid bacteria, total thyme (fed individually) oil anaerobes, or C. perfringens. Trend towards thyme, marjoram, & rosmary decreasing cecal C. perfringens

Reference Djeraba & Quere (2000)

Waihenya et al. (2002)

Barbour et al. (2004)

Jang et al. (2004) Guo et al. (2004b)

Jamroz et al. (2005)

Aksit et al. (2006) Horosova et al. (2006)

Cross et al. (2007)

T.J. Applegate 49 Table 3. Contd.

Compound

Administrative Measured response dose 25 or 50 mg/kg No effect on ileal-cecal E. coli or lactobacilli.

Commercial feed additive containing undefined essential oils including thymol Ethanol extract from 75 mg/kg body Improved clinical score, reduction Peganum harmala seeds weight in lesion score, & reduced bacterial recovery per gram of tissue & reisolation frequency in intra-peritoneal injection model of colibacillosis. Feeding for 6 weeks altered some serum biochemical measures.

Reference Jang et al. (2007)

Arshad et al. (2008)

Table 4. Plant-derived compound effects on swine

Compound

Dietary concentration 300 mg/kg

Commercial feed additive containing gentian root, juniper oil, thyme oil tannins & sialcic acid 0, 125, 250 or Quillaja saponaria 500 mg/kg extract

Ascophyllum nodosum seaweed extract

5, 10, or 20 g/ kg

Oregeno

1,000 mg/kg

Oregeno

1,000 mg/kg

Measured response

Reference

Increase in leucocytes, neutrophils, & lympcytes in blood from weanling piglets.

Savoini et al. (2002)

No effect on food intake, rectal temperature, serum IgM, blood phagocytic function or circulating acute phase proteins after Salmonella typhimurium challenge No effect on food intake, rectal temperature, serum IgM, blood phagocytic function or circulating acute phase proteins after Salmonella typhimurium challenge. Increased production of PGE2 in vitro from porcine alveolar macrophages Improved post-weaning performance on farms with history of enterotoxogenic & verotoxogenic E. coli. Reduction in sow mortality, lactation culling of sows, increase in farrowing rate & number of liveborn pigs

Turner et al. (2002a)

Turner et al. (2002b)

Ken & Bilkei (2003)

Amrik & Bilkei (2004); Mauch & Bilkei (2004); Allan & Bilkei (2005)

50 Influence of phytogenics on the immunity of livestock and poultry Table 4. Contd.

Compound Horseradish

Dietary concentration 1,000 mg/kg

Measured response

Reduction in peri-parturient disease complex as well as pre- & post-wean mortality Garlic 1,000 ppm No effect on sow peri-parturient disease complex or piglet mortality 5 mg/kg body Trend towards improvements in serum Quillaja sapronaria weight IgA in low-body weight suckling piglets. In weanling pigs, no change in Carvacrol (Origanum 7.5 mg spp.), cinnamaldehyde carvacrol, 4.5 intestinal morphology, increased cinnamaldehyde lactobacilli:enterobacteria with (Cinnamonum spp.) concomitant reduction in total and capsicum oleoresin and 3.0 microbial mass in ileal digesta. Cecum capsicum (Capsicum annum) oleoresin/kg or and colon increases in acetate with reductions in butyrate and valerate. 15.0 mg carvacrol, 9.0 cinnamaldehyde and 6.0 capsicum oleoresin/kg Extract of cinnamon, 7.5 g/kg Post-weaning reduction in fecal thyme & oregeno coliforms but not lactobacilli. No change in serum IgG, IL-1, TNFα or circulating white blood cell populations. Oregeno essential oils 3 g/kg In piglets of smaller-than-average body weight - increase in CD4/CD8 MHC Class II antigen positive & non-T/non-B cells in peripheral blood lymphocytes & CD4/CD8 positive T lymphocytes in blood & mesenteric lymph nodes. 10 g/kg Thymus vulgaris No effect on hemolytic E. coli shed from weanling piglets despite the essential oil having bactericidal activity against 39 haemolytic E. coli isolates in vitro. β-glucan from 500 or 1,000 Increase in lymphocyte proliferation Astragalus mg/kg induced by ConA, IL-2 bioactivity, membranaceus decreased cortisol, & decreased circulating IL-1β & PGE2 at 500 mg/ kg in weanling pigs.

Reference Sika & Bilkei (2003) Sika & Bilkei (2003) Garcia et al. (2004) Manzanilla et al. (2004)

Namkung et al. (2004)

Walter & Bilkei (2004)

Jugl-Chizzola et al. (2005)

Mao et al. (2005)

T.J. Applegate 51 Table 4. Contd.

Compound

Dietary Measured response concentration No effect on performance, white Sows Echinacea purpurea aerial portion (dried – (gestation) 12 blood cell populations, lymphocyte proliferation, colostral IgG, or or 36 g/kg for sows & weanling pigs) & pressed extract Sows (lactation) circulating aminotransferases. Increase in antibody titers to swine erysipelas 5 or 15 g/kg (for grow/finish pigs) Weanling pigs: after second immunization for both sources of Echinacea. 18 g/kg Grow/finish pigs: 15g/kg or 4 to 6 mL/day Reductions in total bacteria in cecum Carvacrol (Origanum 15 mg and distal colon digesta of weanling spp.), cinnamaldehyde carvacrol, 9 cinnamaldehyde pigs but no differences in bacterial (Cinnamonum spp.) and capsicum oleoresin and 6 capsicum enzymatic activity in the hind gut. oleoresin/kg (Capsicum annum) 1, 5 and 10 g/kg No effect on haemolytic E. Coli Thymus vulgaris shedding from weanling pigs. 1,000 g/kg In unchallenged weanling piglets to Commercial feed additive containing 50 days of age – reduction in peyers essential oils of patches as well as NFκB & cyclin D in oregano, anis, & citrus mesenteric lymph nodes peel with chicory (inulin source) In weanling pigs, no change in volatile Carvacrol (Origanum 15 mg fatty acid concentrations or intestinal spp.), cinnamaldehyde carvacrol, 9 cinnamaldehyde villus dimensions but decreased intra(Cinnamonum spp.) and capsicum oleoresin and 6 capsicum epithelial lymphocytes (IEL) in the oleoresin/kg jejunum but increased lymphocyte (Capsicum annum) density in the colon. Commercial feed Exp. 1: 0.5, 1, In unchallenged weanling piglets observed no effect on circulating additive containing & 1.5 g/kg inulin, essential oils Exp. 2: 1 g/kg concentrations of acute phase proteins (haptoglobin or C-reactive protein). (containing thymol & carvacrol) & chesnut meal (tannin source) In weanling pigs, decreased intraCarvacrol (Origanum 15 mg epithelial lymphocytes (IEL) in the spp.), cinnamaldehyde carvacrol, 9 cinnamaldehyde jejunum, reduced blood cytotoxic (Cinnamonum spp.) and capsicum oleoresin and 6 capsicum cells and B lymphocytes in mesenteric lymph nodes but increased lymphocyte oleoresin/kg (Capsicum annum) density in the colon and increased blood monocytes.

Reference Maass et al. (2005)

Castillo et al. (2006)

Hagmüller et al. (2006) Kroismayr et al. (2006)

Manzanilla et al. (2006)

Muhl & Liebert (2007)

Nofrarias et al. (2006)

52 Influence of phytogenics on the immunity of livestock and poultry Table 4. Contd.

Compound Fermentable carbohydrate diet

Dietary concentration 50 g/kg sugar beet pulp, 7.5 g/ kg inulin, 20 g/ kg lactulose, & 50 g/kg wheat starch

Measured response

Reference

In newly weaned piglets, noted Pié et al. increase in IL-6 (but not Il-1β or (2007) TNFα) mRNA in colon & were associated this with changes in fermentation products in digesta (especially branched chain fatty acids & ammonia)

Conclusions and future directions In summary, balancing the response to naïve pathogens and development of an immunological memory is essential for survivability of the animal but maintenance can come at considerable price to productivity in our livestock and poultry species. The current breath of literature of plants as immuno-modulators in livestock and poultry species is marginal and we currently have to draw much of our knowledge from that of human pharmaceutical and nutrition research. Further reviews on cell-culture, rodent and human studies and the impact of plants and plant-derived substances on immunological characteristics include: Bouic and Lamprecht, 1999; Li, 2000; Lovkova et al., 2001; Williams, 2003; Tan and Vanitha, 2004; Spelman et al., 2006. There exists the need to move beyond a perfunctory performance-based, product-driven research to a more mechanistic approach to fully understand where and how plant-derived compounds are working in our domestic livestock and poultry species. The improvements we can make in this direction are both pharmacokinetic and agronomic and include: • • • • •

Site of action Effective dosage(s) In vivo metabolism (by the animal and microflora in the GIT) In vivo digestibility Effect of cultivar type, growing conditions, processing and storage on the active components/secondary plant-metabolites.

Once these needs are met, an improved targeted use of plants and/or plant-derived compounds can be implemented.

References Aksit M, Goksoy E, Kok F, Ozdemir D and Ozdogan M (2006) The impacts of organic acid and essential oil supplementations to diets on the microbiological quality of chicken carcasses. Archiv für Geflugelkunde 70: 168–173.

T.J. Applegate 53

Allan P and Bilkei G (2005) Oregano improves reproductive performance of sows Theriogenology 63: 716–721. Allen PC, Lydon J and Danforth HD (1997) Effects of components of Artemisia annua on coccidian infections in chickens. Poultry Science 76: 1157–1163. Amrik B and Bilkei G (2004) Influence of farm application of oregano on performances of sows. Canadian Veterinary Journal 45: 674–677. Anderson JM and Cereijido M (2001) Introduction: Evolution of Ideas on the Tight Junction. In: Tight Junctions pp 1–18 Eds M Cereijido and J Anderson. 2nd ed. CRC Press, Boca Raton, Florida, USA. Applegate TJ, Dibner JJ, Kitchell ML, Uni Z and Lilburn MS (1999) Effect of turkey (Meleagridis gallopavo) breeder hen age and egg size on poult development. 2. Intestinal villus growth, enterocyte migration and proliferation of the turkey poult. Comparative Biochemistry and Physiology B 124: 381–389. Arshad N, Neubauer C, Hasnain S and Hess M (2008). Peganum harmala can minimize Escherichia coli infection in poultry, but long-term feeding may induce side effects. Poultry Science 87: 240–249. Bakuridze AD, Kurtsikidze S, Pisarev VM, Makharadze RV and Berashvili DT (1993). Immunomodulators of plant origin (Review). Pharmaceutical Chemistry Journal 27: 589–595. Barbour EK, Sagherian VK, Talhouk RS, Harakeh S and Talhouk SN (2004) Cellimmunomodulation against Salmonella enteritidis in herbal extract-treated broilers. Journal of Applied Research in Veterinary Medicine 2: 67–73. Berg RD (1996) The indigenous gastrointestinal microflora. Trends in Microbiology 4: 430–434. Bhavanishankar, TN and Sreenivasa Murthy B (1985) Inhibitory effect of curcumin on intesintal tgas formation by Clostridium perfringens. Nutrition Reports International 32: 1285–1292. Bouic PJD and Lamprecht JH (1999) Plant sterols and sterolins: a review of their immunemodulating properties. Alternative Medicine Reviews 4 170–177. Burkey TE, Skjolaas KA and Minton JE (2008) Board-Invited Review: Porcine mucosal immunity of the gastrointestinal tract. Journal of Animal Science doi:10.2527/ jas.2008–1330. Cant JP, McBride BW and. Croom WJ Jr. (1996) The regulation of intestinal metabolism and its impact on whole animal energetic. Journal of Animal Science 74: 2541–2553. Castillo M, Martin-Orue SM, Roca M, Manzanilla EG, Badiola I, Perez JF and Gasa J (2006) The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs. Journal of Animal Science 84: 2725–2734. Christaki E, Florou-Paneri P, Giannenas I, Papazahariadou M, Botsoglou NA and Spais AB (2004) Effect of a mixture of herbal extracts on broiler chickens infected with Eimeria tenella. Animal Research 53: 137–144. Corzo A, Kidd MT, Dozier WA III, Pharr GT and Koutsos E A (2007) Dietary threonine needs for growth and immunity of broilers raised under different litter conditions.

54 Influence of phytogenics on the immunity of livestock and poultry Journal of Applied Poultry Research 16: 574–582. Cross DE, McDevitt RM, Hillman K and Acamovic T (2007) The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. British Poultry Science 48: 496–506. Djeraba A and Quere P (2000) In vivo macrophage activation in chickens with Acemannan, a complex carbohydrate extracted from Aloe vera. International Journal of Immunopharmacology 22: 365–372. El-Sayed A, Jager J, Bonner BM, Redmann T and Kaleta EF (2005) The seeds of Nigella sativa as a feed additive to male layer-type chicks: lack of hepato- and nephrotoxicity and failure of immuno-modulation following vaccinations with paramyxovirus types 2 and 3 and only minor efficacy on spontaneous Eimeria tenella coccidiosis. Archiv für Geflugelkunde 69: 27–34. Fairbrother JM, Nadeau E and Gyles CL (2005) Escerichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Animal Health Research Reviews 6: 17–39. Forstner JF and Forstner GG (1994) Gastrointestinal mucus. Pp 1255–1283 in Physiology of the Gastrointestinal Tract LR Johnson, ed. Raven Press, New York, New York, USA. Garcia MR, Lopez P, Williams RH, Lukefahr SD and Laurenz JC (2004) Effect of Quillaja saponaria extract on passive immunization in a pig model. Journal of Animal and Veterinary Advances 3: 538–544. Gaskins HR (1997) Immunological aspects of host/microbiota interactions at the intestinal epithelium pp 537–587. In: Gastrointestinal microbiology No. 2. RI Mackie, BA White and RE Isaacson (Eds) Chapman and Hall, New York, New York, USA. Giannenas I, Florou-Paneri P, Papazahariadou M, Christaki E, Botsoglou NA and Spais AB (2003) Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition 57: 99–106. Giannenas I, Florou-Paneri P, Papazahariadou M, Botsoglou, Christaki E and Spais AB (2004) Effect of diet supplementation with ground oregano on performance of broiler chickens challenged with Eimeria tenella. Archiv für Geflugelkunde 68: 247–252. Greathead H (2003) Plants and plant extracts for improving animal productivity. Proceedings of the Nutrition Society 62: 279–290. Gruys E, Toussaint MJM, Niewold TA and Koopmans SJ (2005) Acute phase reaction and acute phase proteins. Journal of Zheijiang University Science 6B: 1045–1056. Guo FC, Savelkoul HFJ, Kwakkel RP, Williams BA and Verstegen MWA (2003) Immunoactive, medicinal properties of mushroom and herb polysaccharides and their potential use in chicken diets. World’s Poultry Science Journal 59: 427–440. Guo FC, Kwakkel RP, Williams BA, Parmentier HK, Li WK, Yang ZQ and Verstegen MWA (2004a) Effects of mushroom and herb polysaccharides on cellular and humoral immune responses of Eimeria tenella-infected chickens. Poultry Science 83: 1124–1132.

T.J. Applegate 55

Guo FC, Williams BA, Kwakkel RP, Li HS, Li XP, Luo JY, Li WK and Verstegen MWA (2004b) Effects of mushroom and herb polysaccharides, as alternatives for an antibiotic, on the cecal microbial ecosystem in broiler chickens. Poultry Science 83: 175–182. Guo FC, Kwakkel RP, Williams BA, Suo X, Li WK and Verstegen MWA (2005) Coccidiosis immunization: Effects of mushroom and herb polysaccharides on immune responses of chickens infected with Eimeria tenella. Avian Disease 49: 70–73. Hagmüller W, Jugl-Chizzola M, Zitterl-Eglseer K, Gabler C, Spergser J, Chizzola R and Franz C (2006) The use of Thymi herba as feed additive (0.1%, 0.5%, 1.0%) in weanling piglets with assessment of the shedding of haemolysing E. coli and the detection of thymol in the blood plasma. Berliner und Munchener Tierarztliche Wochenschrift 119: 50–54. Horosova K, Bujnakova D and Kmet V (2006) Effect of oregano essential oil on chicken lactobacilli and E. coli Folia Microbiologica 51: 278–280. Imondi AR and Bird FH (1966) The turnover of intestinal epithelium in the chick. Poultry Science 45: 142–147. Jamroz D, Wiliczkiewicz A, Wertelecki T, Orda J and Skorupinska J (2005) Use of active substances of plant origin in chicken diets based on maize and locally grown cereals. British Poultry Science 46: 485–493 Jamroz D, Wertelecki T, Houszka M and Kamel C (2006) Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90: 255–268. Jang IS, Ko YH, Yang HY, Ha JS, Kim JY, Kim JY, Kang SY, Yoo DH, Nam DS, Kim DH, and Lee CY (2004) Influence of essential oil components on growth performance and the functional activity of the pancreas and small intestine in broiler chickens. Asian-Australian Journal of Animal Science 17: 394–400. Jang IS, Ko YH, Kang SY and Lee CY (2007) Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Animal Feed Science and Technology 134: 304–315. Jensen-Jarolim E, Gajdzik L, Haber I, Kraft D, Scheiner O, and Graf J (1998) Hot spices influence permeability of human intestinal epithelial monolayers. Journal of Nutrition 128: 577–581. Jugl-Chizzola M, Spergser J, Schilcher F, Novak J, Bucher A, Gabler C, Hagmuller W, and Zitterl-Eglseer K (2005) Effects of Thymus vulgaris L. as feed additive in piglets and against haemolytic E. coli in vitro. Berliner und Munchener Tierarztliche Wochenschrift 118: 495–501. Kagnoff MF (1993) Immunology of the intestinal tract. Gastroenterology 105 1275– 1280. Ken C and Bilkei G (2003) Effects of vaccination and of a phytogenic feed additive on postweaning mortality due to Escherichia coli and on piglet performance. Veterinary Record 153: 302–303.

56 Influence of phytogenics on the immunity of livestock and poultry Klasing KC (2007) Nutrition and the immune system. British Poultry Science 48: 525–537. Kroismayr A, Sehm J, Plitzner C and Windisch, W (2006) Effect of an essential oil blend (oregano, anis, citrus peels) or Avilamycin on growth performance, microbiological and histological parameter and mRNA expression of inflammatory and apototic genes in the gut of weaned piglets. Proceedings of the Society of Nutrition and Physiology: Gesellschaft für Ernährungsphysiologie 15: 47. Lovkova MY, Buzuk GN, Sokolova SM and Kliment’eva NI (2001) Chemical features of medicinal plants. Applied Biochemistry and Microbiology 37: 229–237. Li XY (2000) Immunomodulating components from Chinese medicines. Pharmaceutical Biology 38: 33–40. Lien KA, Sauer WA and Fenton M (1997) Mucin output in ileal digesta of pigs fed a protein-free diet. Zeitschrift für Ernahrungswissenschaft 36: 182–190. Losa R and Kohler B (2001) Prevention of colonization of Clostridium perfringens in broilers intestine by essential oils. Pp. 133–134 in: 13th European Symposium on Poultry Nutrition. WPSA Blankenberge, Belgium. Mao XF, Piao XS, Lai CH, Li DF, Xing JJ and Shi BL (2005) Effects of ß-glucan obtained from the Chinese herb Astragalus membranaceus and lipopolysaccharide challenge on performance, immunological, adrenal, and somatotropic responses of weanling pigs. Journal of Animal Science 83: 2775–2782. Maass N, Bauer J, Paulicks BR, Böhmer BM and Roth-Maier DA (2005) Efficiency of Echinacea purpurea on performance and immune status in pigs. Journal Animal Physiology and Animal Nutrition 89: 244–252. Manzanilla EG, Perez JF, Martin M, Kamel C, Baucellis F, and Gasa J (2004) Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 82: 3210–3218. Manzanilla EG, Nofrarıas M, Anguita M, Castillo M, Perez JF, Martın-Orue SM, Kamel C and Gasa J (2006) Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 84: 2743–2751. Mathis GF, Dale NM and Fuller AL (1995) Effect of dietary raw soybeans on coccidiosis in chickens. Poultry Science 74: 800–804. Mauch C and Bilkei G (2004) Strategic application of oregano feed supplements reduces sow mortality and improves reproductive performance - a case study. Journal of Veterinary Pharmacology and Therapeutics 27: 61­–63. Min BR and Hart SP (2003) Tannins for suppression of internal parasites. Journal of Animal Science 81: E102–E109. Mitsch P, Kohler B, Gabler C, Losa R and Zitterl-Eglseer K (2002) CRINA poultry reduces colonization and proliferation of Clostridium perfringens in the intestine and faeces of broiler chickens. In: XI European Poultry Conference, Bremen, Germany. Mitsch P, Zitterl-Eglseer K, Kohler B, Gabler C, Losa R, and Zimpernik I (2004) The effect of two different blends of essential oil components on the proliferation of Clostridium

T.J. Applegate 57

perfringens in the intestines of broiler chickens. Poutry Science 83: 669–675. Muhl A and Liebert F (2007) No impact of a phytogenic feed additive on digestion and unspecific immune reaction in piglets. Journal of Animal Physiology and Animal Nutrition 91: 426–431. Muir WI (1998) Avian intestinal immunity: Basic mechanisms and vaccine design. Poultry and Avian Biology Reviews 9: 87–106. Namkung H, Li M, Gong J, Yu H, Cottrill M and de Lange CFM (2004) Impact of feeding blends of organic acids and herbal extracts on growth performance, gut microbiota and digestive function in newly weaned pigs. Canadian Journal of Animal Science 84: 697–704. Nestor KE, Lilburn MS, Saif YM, Anderson JW, Patterson RA, Li Z and Nixon JE (1999) Influence of body weight restriction in a body-weight-selected line of turkeys on response to challenge with Pasteurella multocida. Poultry Science 78: 1263–1267. Nofrarias ME, Manzanilla G, Pujols J, Gilbert X, Majo N, Segales J and Gasa J (2006). Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaning pigs. Journal of Animal Science 84: 2735–2742. Oviedo-Rondon EO, Clemente-Hernandez S, Williams P and Losa R (2005) Responses of coccidia-vaccinatedbroilers to essential oil blends supplementation up to forty-nine days of age. Journal of Applied Poultry Research 14: 657–664. Pié S, Awati A, Vida S, Falluel I, Williams BS and Oswald IP (2007) Effects of added fermentable carbohydrates in the diet on intestinal proinflammatory cytokine-specific mRNA content in weaning piglets. Journal of Animal Science 85: 673–683. Piriou-Guzylack L and Salmon H (2008) Membrane markers of the immune cells in swine: an update. Veterinary Research 39: 54 DOI: 10.1051/vetres: 2008030. Potturi LPV, Patterson JA and Applegate TJ (2005) The effects of delayed placement on villus characteristics and barrier functions of the small intestine of newly hatched turkeys. Poultry Science 84: 816–824. Rochfort S, Parker AJ and Dunshea FR (2008) Plant bioactives for ruminant health and productivity. Phytochemistry 69: 299–322. Rozee KR, Cooper D, Lam K and Costerson JW (1982) Microbial flora of the mouse ileum mucous layer and epithelial surface Applied and Environmental Microbiology 43 1451–1463. Sandberg FB, Emmans GC and Kyriazakis I (2006) A model for predicting feed intake of growing animals during exposure to pathogens. Journal of Animal Science 84: 1552–1566. Sandberg FB, Emmans GC and Kyriazakis I (2007) The effects of pathogen challenges on the performance of naïve and immune animals: the problem of prediction. Animal 1: 67–86. Saini R, Davis S and Dudley-Cash W (2003) Oregano essential oil reduces necrotic enteritis in broilers. Pp 95–97 in: Proceedings of the 52nd Western Poulry Disease Conference, Sacramento, CA. Vet Extension, Univ. Calif., Davis.

58 Influence of phytogenics on the immunity of livestock and poultry Sauer WC and Ozimek L (1986) Digestibility of amino acids in swine: results and their practical applications. A Review. Livestock Production Science 15: 376–388. Savoini G, Bontempo V, Cheli F, Baldi A, Sala V, Mancin G, Agazzi A and Del’Orto V (2002) Alternative antimicrobials in the nutrition of postweaning piglets. Veterinary Record 151: 577–580. Sears CL (2000) Molecular physiology and pathophysiology of tight junctions V. Assault of the tight junction by enteric pathogens. American Journal of Physiology Gastrointestinal and Liver Physiology 279: G1129–G1134. Shirley MW, Smith AL and Blake DP (2007) Challenges in the successful control of the avian coccidia. Vaccine 25: 5540–5547. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, and Srinivas PSSR (1998) Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Medica 64: 353–356. Sika J and Bilkei G (2003) Effect of garlic (Allium savitum), horseradish (Aromatica rusticana) and enrofloxacin in the prevention of periparturient disorders and pre- and post-weaning mortality in swine. Pig Journal 51: 83–91. Slomiany A, Grabska M and Slomainy BL (2001) Essential components of antimicrobial gastrointestinal epithelial barrier: specific interaction of mucin with an integral apical membrane protein of gastric mucosa. Molecular Medicine 7: 1–10. Spelman K, Burns JJ, Nichols D, Winters N, Ottersberg S and Tenborg M (2006) Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Alternative Medicine Review 11: 128–150. Söderholm JD and Perdue MH (2001) Stress and the gastrointestinal tract II. Stress and intestinal barrier function. American Journal of Physiology - Gastrointestinal and Liver Physiology 280: G7–G13. Sonnenburg JL,. Angenent LT and Gordon JI (2004) Getting a grip on things: how do communities of bacterial symbionts become established in our intestine? Nature Immunology 5: 569–573. Tan BKH and Vanitha J (2004) Immunomodulatory and antimicrobial effects of some traditional Chinese medicinal herbs: a review. Current Medicinal Chemistry 11: 1423–1530. Thatte U, Bagadey S and Dahanukar S (2000) Modulation of programmed cell death by medicinal plants. Cellular and Molecular Biology 46: 199–214. Turner JL, Dritz SS, Higgins JJ, Herkelman KL and Minton JE (2002a) Effects of a Quillaja saponaria extract on growth performance and immune function of weanling pigs challenged with Salmonella typhimurium. Journal of Animal Science 80: 1939–1946. Turner JL, Dritz SS, Higgins JJ and. Minton JE (2002b) Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium. Journal of Animal Science 80: 1947–1953. Uni Z, Platin R and Sklan D (1998) Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. Journal of Comparative Physiology

T.J. Applegate 59

B 168: 241–247. Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F and Ducatelle R (2004) Costridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathology 33: 537–549. Van der Wielen PWJJ, Keuzenkamp DA, Lipman LJA, van Knapen F and Biesterveld S (2002) Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microbial Ecology 44: 286–293. Waihenya RK, Mtambo MM, Nkwengulila G and Minga UM (2002) Efficacy of crude extract of Aloe securndiflora against Salmonella gallinarum in experimentally infected free-range chickens in Tanzania. Journal of Ethnopharmacology 79: 317–323. Walter BM and Bilkei G (2004) Immunostimulatory effect of dietary oregano etheric oils on lymphocytes from growth-retarded, low-weight growing-finishing pigs and productivity. Tijdschrift voor Diergeneeskunde 129: 178–181. Webel D, Johnson RW and Baker DH (1998) Lipopolysaccharide-induced reductions in food intake do not decrease the efficiency of lysine and threonine utilization for protein accretion in chickens. Journal of Nutrition 128: 1760–1766. Williams JE (2003) Portal to the interior: viral pathogenesis and natural compounds that restore mucosal immunity and modulate inflammation. Alternative Medicine Reviews 8: 395–409. Williams RB (1999) A compartmentalized model for the estimation of the cost of coccidiosis to the world’s chicken production industry. International Journal of Parasitology 29: 1209–1229. Windisch W, Schedle K, Plitzner C and Kroismayr A (2008) Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86: E140–E148. Youn HJ and Noh JW (2001) Screening of the anticoccidial effects of herb extracts against Eimeria tenella. Veterinary Parasitology 96: 257–263. Zijlstra RT, Donovan SM, Odle J, Gelberg HB, Petschow BW and Gaskins HR (1997) Protein-energy malnutrition delays small-intestinal recovery in neonatal pigs infected with rotavirus. Journal of Nutrition 127: 1118–1127.

I. Giannenas and I. Kyriazakis 61

4 PHYTOBASED PRODUCTS FOR THE CONTROL OF INTESTINAL DISEASES IN CHICKENS IN THE POST ANTIBIOTIC ERA Ilias Giannenas and Ilias Kyriazakis Laboratory of Animal Nutrition & Husbandry, Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece, e-mail: [email protected]

Abstract The use of in-feed antibiotics and chemical anticoccidial drugs has been the main strategy for the control of bacterial and parasitic intestinal infections in broiler chickens. Increasing public concerns about arising antibiotic resistance in bacteria and EU legislation has forced the poultry industry to explore new approaches to the methods of their control. Among these alternatives aromatic plants (herbs), herbal extracts and essential oils may have a predominant place, since they are known to exhibit antimicrobial and antiparasitic activity; we call all these phytobased products. The aim of this chapter is the evaluation of the use of phytobased products on broiler chicken health and performance; we focus on their effects on two major poultry diseases: necrotic enteritis and coccidiosis. The knowledge on the effects of consumption of aromatic plants or their extracts in poultry nutrition is based mainly on ethnoveterinary sources, rather than rigorous scientific evidence. Successful application of aromatic plants or their extracts in chicken diets requires a detailed understanding of their modes of action. Administration of such products to broilers is characterised by an enormous variability in response, even when the same herb derived products are used on the same pathogen. As performance and health responses to phytobased products is so variable, the major challenge is the standardization of the biological multi-component composition derived from herbal sources, which will make botanical products as effective as synthetically made antimicrobials and antiparasitic drugs. New technologies are required to modernize traditional herb usage into mainstream phytobased products or supplements. Further in vivo studies are necessary to account for the inconsistent effectiveness, overcome the drawbacks and maximise the benefits from the use of phytobased products for both chicken health and performance and consumer health.

61

62 Control of intestinal diseases in poultry

Introduction The use of antibiotics as growth promoters has been curtailed by legislation in recent years in EU countries. The reason for this ban has been concerns over the widespread development of resistant bacterial strains that has arisen over a short period of time, and the potential of transfer of this resistance to other strains of bacteria. There is concern that if pathogenic bacteria gain resistance to antibiotics, infections, including human ones, may become untreatable. Antibiotics given in small amounts halt the growth of bacteria, prevent outbreaks of diseases and enhance the rate of growth in intensively reared animals. Consequently, considerable effort has been directed towards developing alternative products to antibiotics to maintain intestinal health and improve animal performance. Aromatic plant extracts and plant based products offer an opportunity in this regard, as many plants produce secondary metabolites which have considerable antimicrobial or antiparasitic properties against a wide range of pathogens. These active components, generally recognised as being safe for human and animal consumption in the USA (FDA, 2004), have prompted scientists to examine their potential to improve production efficiency and health in poultry. Aromatic plants have been used since ancient times for their preservative and medicinal properties, and to impart aroma and flavour to food. Hippocrates, the ‘father of medicine’, used plant extracts and prescribed perfume fumigations. For centuries, aromatic plants, also known as herbs and spices, their essential oils and herbal extracts have been used as natural pharmaceuticals in traditional medicine and veterinary medicine. However, their use has not been based on rigorous scientific investigation, but has stemmed from ethnoveterinary or even folkloric sources (Chang, 2000; Athanasiadou and Kyriazakis, 2004). By the middle of the 20th century, the role of essential oils had been reduced almost entirely to use in perfumes, cosmetics and food flavourings. This was because control of pathogens was based mainly on synthetic drugs. However, it must be noted that almost 25% of active medical compounds currently prescribed in the USA and UK have actually been isolated from higher plants (Anthony et al., 2005). Today, more than 85,000 plant species have been documented for medicinal use globally. The World Health Organization (WHO) estimates that almost 75% of the world’s population has therapeutic experience with herbal remedies. The ban on the use of antimicrobial growth promoters within the EU (Barton, 1999) and the demand by consumers for safe products has renewed the interest in aromatic plants and their extracts mainly as a source of alternative therapeutics or natural antioxidants. In some extensive reviews about the use of aromatic plants, their extracts or essential oils in animal nutrition, their mode of action has been examined and experimental evidence on their consequences has been presented (Lee et al., 2004; Windisch et al., 2008). However, these papers have highlighted the paucity of knowledge of both the modes of action and aspects of efficacy of herbal extracts, essential oils or even botanical products. Most studies investigate blends of various active compounds and report effects on production performance rather than physiological impacts. Also, results are often contradictory both in terms of health and performance, as botanical feed additives may vary widely with respect to botanical origin, processing and composition.

I. Giannenas and I. Kyriazakis 63

This chapter discusses recent developments in the use of phytobased products to potentially benefit poultry health and production. We focus on the effects of aromatic plants or their extracts on intestinal diseases and particularly on necrotic enteritis and coccidiosis, as they both have the most significant economic impacts on poultry production, and there is an urgent need to develop and use effective alternative ways of their control. The consequences of inclusion of aromatic plants, herbal extracts and essential oils are extensively reviewed, alongside their potential mechanisms of action. It has proved difficult to obtain experimental evidence for all of the purported effects of phytobased products and possible reasons for their varying consequences on poultry health and performance are discussed. In the last part, we describe challenges and opportunities in the development of phytobased products that could take into account recent progress in production of aromatic plants, quality control and analysis of their composition in biological active compounds. Development of phytobased dietary supplements will be based on the scientific evidence of biological activity of their functional compounds. This approach is a necessity for establishing the true value of phytobased products in poultry systems of production.

Main threats to broiler chickens in the post antibiotic era Intestinal diseases, antibacterial growth promoters and alternatives Intestinal diseases are an important concern of the modern broiler poultry industry, because of lost productivity, increased mortality, reduced welfare and the associated contamination of poultry products with pathogenic bacteria and/or their toxins (Dahiya et al., 2006). For the past 60 years, antibiotics have been supplemented to poultry feed to improve growth performance and efficiency and protect animals from adverse effects of pathogenic and nonpathogenic enteric microorganisms. There are three main categories of intestinal diseases that affect health and performance of chickens: coccidiosis, necrotic enteritis and infectious diseases caused by gram-negative bacteria. Coccidiostatic or anticoccidial drugs (ionophore antibiotics or chemoprophylactics) and growth promoters are mainly protecting chickens from the first two categories. The net effect of using antibiotic growth promoters in the poultry industry was estimated to be a 3-5% improvement in growth and feed conversion (Thomke and Elwinger, 1998). However, it has been argued that continued, unregulated excessive use of antibiotic growth promoters in animal feeds imposes a selection pressure for bacteria that are resistant to antibiotics (Wegener et al., 1998). Hence, the use of feed antibiotics as growth promoters has come under increasing scrutiny by scientists, consumers and government regulators, because of this potential development of antibiotic resistant human pathogenic bacteria after prolonged use (Bedford, 2000). Antibiotics are mostly used for therapeutic purposes in humans, secondly for therapeutic purposes in animals, while medicated animal feeds are their third use. With the ban on subtherapeutic or prophylactic antibiotic usage in European Union countries in 2006, and the

64 Control of intestinal diseases in poultry potential for a ban in North America, there is an increasing interest in finding alternatives to antibiotics for poultry production. Unless alternative feed additives are introduced and routinely used, this ban will have a strong economic impact on the poultry industry and on the health and welfare of the birds. There is, therefore, a necessity for intensive research into the identification and evaluation of alternatives to traditional antibiotics and ionophore anticoccidials that would satisfy consumer perceptions and maintain enhanced productivity rates and poultry health. Amongst alternatives, aromatic plants and their extracts may have a central role to offset any possible adverse effects on production. In recent years, there has been an increased interest in finding alternative management or dietary strategies to control the incidence and severity of intestinal diseases. Cost effective approaches, particularly for the control of necrotic enteritis and coccidiosis, are being sought. These approaches should fulfil both demands of consumers for healthy products and farmers for reproducible effectiveness in biological model systems. In this line, various phytobased feed additives may have a predominant role to play.

Chicken coccidiosis Intestinal coccidiosis, caused by the intracellular growth and replication of protozoa belonging to the genus Eimeria (phylum Apicomplexa), is a major cause of concern and economic loss to the poultry industry worldwide. Seven species of Eimeria are generally accepted to be the causative agents of chicken coccidiosis, namely E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella (Shirley and Millard, 1986). The species E. acervulina, E. maxima and E. tenella are considered to be of the greatest importance to the poultry industry from the point of view of their ubiquity in broiler flocks, pathogenicity and/or immunological features (Crouch et al., 2003). Coccidiosis remains one of the most important diseases in the poultry industry and results in the annual loss of millions of US-$ to the poultry industry (Naidoo et al., 2008). Because of the structure of their breeding programmes, the greatest number by far of chickens being reared in the world at any time is standard broilers. In the United Kingdom the total cost of coccidiosis in chickens was estimated to be US-$ 77 million in 1995 and resulted from a combination of in-feed medication costs, veterinary costs and production losses (98.1% was associated with the 625 million broilers produced, the major percentage, 80.6%, due to the poor performance and 17.5% due to costs of prophylaxis and treatment) (Williams, 1999). At present, commercial poultry farmers worldwide spend approximately US-$ 0.02 per bird annually on the use of in-feed prophylactic anticoccidials in order to limit mortalities and enhance broiler growth and production (Naidoo et al., 2008). The detrimental effects of the disease are a worsened feed conversion ratio and body weight gain rate, and an increased mortality (McDougald, 1998). Commercially reared broilers are extremely vulnerable to the disease due to the intensity of farming, the use of deep litter and the avoidance of use of breeding broilers in cages. The anticoccidials added in feed include the polyether (ionophore) group of chemotherapeutics, sulphonamides,

I. Giannenas and I. Kyriazakis 65

pyrimidine derivatives, triazinetriones and the benzenacetonitriles (Naidoo et al., 2008). The strategy of dietary administration of coccidiostatic drugs, over the last four decades, has, in part, provided the basis for the rapid growth of the poultry industry and the increased availability of high quality chicken meat products to the consumer. However, there are considerable concerns about residues of coccidiostatic drugs in poultry meat products (McEvoy, 2001; Young and Craig, 2001) and resistance of coccidia has been developed to all of the anticoccidial drugs introduced so far (Chapman, 1986; 2000). The current trend is to use fewer chemical coccidiostats but to prevent reduced performance and economic losses, as well. As a consequence, natural solutions with proven anticoccidial activity would be welcomed by the poultry industry.

Necrotic enteritis Necrotic enteritis (NE) of domestic broiler chickens is a world wide disease. It is a potentially fatal disease and flock mortality rates may reach up to 1% per day with total mortalities reaching 30% (Helmboldt and Bryant, 1971). The causative agent of NE, Clostridium perfringens, types A (Songer and Meer, 1996) or C (Shane et al., 1984; Engstrom et al., 2003), is a nearly ubiquitous gram positive, spore forming, extremely prolific, toxigenic, anaerobic bacterium found in soil, dust, feces, feed, poultry litter and intestinal contents. The disease usually occurs in broiler chickens 2–6 weeks after hatching and is characterized by sudden onset of diarrhea and mucosal necrosis caused by the overgrowth of C. perfringens in the small intestine (Fukata et al., 1991). Necrotic enteritis is estimated to cost the poultry industry worldwide as much as US $0.05 per bird, with a total global loss of nearly US $2 billion per annum (Van der Sluis, 2000). However, this might be an underestimate, given the difficulty in diagnosing mild or subclinical forms of NE. The subclinical form of NE may be the most economically important, because of impaired feed conversion, reduced live weight at slaughter and increased condemnation percentage associated with C. perfringens infection (Lovland and Kaldhusdal, 2001). Outbreaks of NE are sporadic and may result in high mortality and severe economic losses. C. perfringens is frequently found in the intestinal tract of healthy poultry, usually at low levels (<104 cfu/g of digesta) and is spread in poultry production units and processing plants through faeces and intestinal rupture. Although C. perfringens is the main etiological agent of NE, other cofactors are usually required to precipitate an outbreak of NE (Dahiya et al., 2006). The physical and chemical composition of broiler diets has been reported to have a marked effect on the intestinal microflora of chickens, and it has been shown to have an important impact on the incidence of NE in broiler chickens (Riddell and Kong, 1992; Drew et al., 2004; Dahiya et al., 2006). Dietary cereal grains rich in nonstarch polysaccharides and proteins of animal origin encourage the development of NE (Riddell and Kong, 1992; Annett et al., 2005; Wilkie et al., 2005). Drew et al. (2004) observed a significant increase in C. perfringens counts in birds fed a 40% crude protein diet containing

66 Control of intestinal diseases in poultry fish meal; however, C. perfringens counts were low in the ileum of birds fed soy protein concentrate-based diets at various proteinaceous levels. A significant positive correlation between the glycine content of diets and digesta to C. perfringens populations in the ileum and cecum of broilers has been reported (Dahiya et al., 2005, 2007; Wilkie et al., 2005). Some in vitro studies have shown an association between certain amino acids and C. perfringens growth, α-toxin production, or both (Muhammed et al., 1975; Nakamura et al., 1978; Stevens and Rood, 2000). Stevens and Rood (2000) reported that glycine containing peptides accelerated C. perfringens growth and α-toxin production in vitro. Similarly, Muhammed et al. (1975) documented that methionine was stimulatory for the growth of C. perfringens in vitro, while, other studies demonstrated an antibacterial effect of high concentrations of DL- methionine against C. perfringens (Wilkie, 2006). The incidence of NE is expected to increase due to the ban of the use of antimicrobial growth promoters and alternative control of the disease is already being sought. Natural substances with antimicrobial properties can be a part of this control strategy; however, their effectiveness has not been extensively tested by in vivo trials.

Aromatic plants (herbs), herbal extracts and essential oils Definition and biological effects Aromatic plants grow in a wide range of environments. The wealth of information available from traditional medicine from many parts of the world about their purported properties has made them the focus of attention by the scientific community (Athanasiadou et al., 2007). Animals and ruminants in particular often consume aromatic plants when grazing, but such plants may also be incorporated in animal feeds after drying and grinding, with minor preparation. In an attempt to utilise the information available from ethnoveterinary sources on the medicinal activity of such plants, there is a current trend to investigate their properties under controlled experimental conditions. The variety of methodologies used for this purpose include the provision of fresh or conserved whole plants, specific plant parts or their extracts to animals. The antimicrobial properties of aromatic plants are partially attributed to essential oils. Herbal extracts from such plants can be prepared by different extraction methods (e.g. supercritical fluid and subcritical water extractions), with various solvents, such as ethanol, methanol, toluene or other organic solvents. Essential oils can also be extracted by several techniques from different parts of the aromatic plant, including mainly water or steam distillation. Although the term ‘essential oil’ is a poorly defined concept from medieval pharmacy and the term ‘volatile oil’ has been proposed instead (Hay and Waterman, 1993), the term ‘essential oil’ applies more often (Lee et al., 2004). Apart from the volatile compounds found in the essential oils, biological activity is also exhibited by another class of various volatile and non-volatile glycosidically bound constituents, after release by enzymatic or acid hydrolysis. The latter can also be considered as potential precursor

I. Giannenas and I. Kyriazakis 67

substances in plant material (Milos et al., 2000). Most of the essential oil compounds which are obtained by steam-distillation are phenolic compounds. In nature, phenolic and polyphenolic compounds are found in herbs, spices and their extracts. There is a central role of polyphenolic compounds in plants since they are needed for pigmentation, growth, reproduction, resistance to pathogens and fungi and many other functions (Skrubis, 1972). One of the most important groups of polyphenols is flavonoids. These can be divided into the following subgroups: flavones/flavonones, anthocyanins and cathehins/flavonols. In plants, flavonoids usually form complexes with various sugars which are called glycocides. Polyphenols have been found to possess antimicrobial and antifungal activity (Sivropoulou et al., 1996) and also have effective antiparasitic activity against gastrointestinal parasites (Guarrera, 1999). A number of studies provide strong evidence that condensed tannins, another class of phenolic compounds, have direct anthelminthic effects towards gastrointestinal nematodes of ruminants (Athanasiadou et al., 2001). Similar activity has been also exhibited by saponins (Ibrahim, 1992). The composition of individual compounds of essential oils is variable. For example, the concentrations of two predominant components of thyme essential oils, i.e. thymol and carvacrol have been reported to range from as low as 3% to as high as 60% of total essential oils (Lawrence and Reynolds, 1984). Similar variation can be found also in the essential oil of oregano, which is obtained by steam-distillation of Origanum vulgare spp. hirtum plants, and comprises of more than 30 ingredients, most of which are phenolic compounds with varying activities (Economou et al., 1991; Sivropoulou et al., 1996; Adam et al., 1998). The major components carvacrol and thymol comprise about 78-82% of the total oil (Adam et al., 1998). The concentration of other main constituents such as the two monoterpene hydrocarbons, γ-terpinene and p-cymene, that often constitute about 5% and 7% of the total oil, respectively, also vary and the effect of oregano essential oil is often also variable because it depends on all these constituents working together (Adam et al., 1998).

Mode of action of aromatic plants and their extracts or phytobased products Aromatic plants and their extracts have been found to exhibit various biological activities such as antimicrobial, antiparasitic, antiviral and antioxidative in poultry (Kamel, 2001; Youn and Noh, 2001; Williams and Losa, 2001; Botsoglou et al., 2002; Papageorgiou et al., 2003; Giannenas et al., 2003; 2004; 2005; Lee et al., 2004; Wallace, 2005). They are also said to stimulate the endocrine and immune system (Lee et al., 2004). Various botanical ingredients have been shown to facilitate beneficial effects on gut environment and microflora (Besra et al., 2002; Rao and Nigam, 1970). Essential oils are known to stimulate digestive enzymes and may affect lipid metabolism and fat digestibility (Platel and Srinivasan, 1996). The exact antimicrobial mechanism of essential oils is poorly understood. It has been suggested that their lipophilic property (Conner, 1993) and chemical structure (Farag et al., 1989) may play a role. Helander et al. (1998) investigated how two isomeric phenols,

68 Control of intestinal diseases in poultry carvacrol and thymol, and the phenylpropanoid cinnamaldehyde, exert their antibacterial effects on Escherichia coli O157 and Salmonella typhimurium. These researchers found that both carvacrol and thymol acted in a similar fashion, disintegrating the outer membrane of bacteria and leading to the release of membrane-associated material from the cells to the external medium. On the other hand, cinnamaldehyde exhibited antibacterial activity, but failed to affect the outer membrane, indicating that the different molecules have different antibacterial effects. Cinnamaldehyde was inhibitory at similar concentrations as thymol and carvacrol for the growth of the enteric bacteria and exhibited strong antimicrobial activity, yet it exhibited neither outer membrane disintegrating activity nor depletion of intracellular ATP. Cinnamaldehyde inhibited enterobacterial growth by access to the periplasm and to the deeper parts of the cell; the outer membrane proteins have been shown to allow the penetration of lipophilic probes at significant rates (Helander et al., 1998). The reason why exposure to cinnamaldehyde does not result in the disintegration of outer membrane, whereas carvacrol and thymol cause significant liberation of membrane components, might be related to the phenolic character of carvacrol and thymol; phenols are known for their membrane-disturbing activities (Sikkema et al., 1994). Phenolic compounds are known to possess antimicrobial effects as they target the bacterial cell wall, thus affecting cell wall structure. They interact with the cytoplasmic membrane by changing its permeability for cations, like H+ and K+ (Sikkema et al., 1994). The dissipation of ion gradients leads to impairment of essential processes in the cell, allowing leakage of cellular constituents, resulting in water unbalance, collapse of the membrane potential and inhibition of ATP synthesis, and finally cell death (Ultee et al., 1999; 2002). It was thus suggested that terpenoids and phenylpropanoids can penetrate the membrane of the bacteria and reach the inner part of the cell because of their lipophilicity (Helander et al., 1998), but it has also been proposed that structural properties, such as the presence of the functional hydroxyl groups (Farag et al., 1989), and aromaticity (Bowles and Miller, 1993) are also responsible for the antibacterial activity. It is thought that membrane perforation or binding is one principle mode of action (Shapiro and Guggenheim, 1995; Stiles et al., 1995), leading to an increase of permeability and leakage of vital intracellular constituents (Juven et al., 1994), resulting in impairment of bacterial enzyme systems (Farag et al., 1989). Further clarification of the antibacterial activity of essential oil components on various bacteria is needed in order these substances could be used as effective antimicrobial products.

Antimicrobial effects of herbs, herbal extracts and essential oils In vitro antimicrobial activities of herbal essential oils

In numerous in vitro studies essential oils have been shown to have antimicrobial activity (Hammer et al., 1999) and due to this property, they have gained much attention in

I. Giannenas and I. Kyriazakis 69

investigations on their potential as alternatives to antibiotics for therapeutic purposes. For example, Lee and Ahn (1998) found that cinnamaldehyde, derived from the cinnamon essential oil, strongly inhibits C. perfrigens and Bacteroides fragilis, and moderately inhibits Bifidobacterium longum and Lactobacillus acidophilus isolated from human faeces. The selective inhibition by cinnamaldehyde of pathogenic, intestinal bacteria may have a pharmacological role in balancing the intestinal microbiota. The wide range of in vitro antimicrobial activities of essential oils derived from cinnamon, thyme and oregano have been reported (Deans and Ritchie, 1987; Sivropoulou et al., 1996; Adam et al., 1998; Farag et al., 1989; Dorman and Deans, 2000), supporting their possible use as antimicrobial agents. The essential oils and their pure components such as carvacrol, thymol, eugenol, cinnamaldehyde and ionone are known for their antimicrobial activity against selected microorganisms such as species of Escherichia, Staphylococcus, Pseudomonas, Salmonella and Streptococcus (Lee et al., 2004). The minimum inhibitory concentrations of the pure compounds vary between experiments. It is considered beneficial to keep the effective antimicrobial concentration of essential oils as low as possible due to their characteristic flavours. This problem can be overcome, as suggested by Moleyar and Narasimham (1992), by using synergistic properties of different oils, thus improving the antimicrobial activity in spite of low dosages. This synergism was highlighted in studies of Didry et al. (1994) and Montes-Belmont and Carvajal (1998). In vivo studies

On the basis of their in vitro antimicrobial activity, it is logical to expect that essential oils would have potential as prophylactic and therapeutic agents in animal production. It would be expected that the intake of essential oils would affect the gastrointestinal microflora composition and population. Recently, Waldenstedt (2003) investigated the possibility of rearing broilers without growth promoters and coccidiostats by incorporating oregano essential oil in their diet. In this experiment, however, oregano oil was not examined as an anticoccidial compound, but mainly as an antibacterial agent against intestinal colonization by C. perfringens, where it decreased caecal numbers of C. perfringens at 31 days but not at 52 days. Another field study conducted by Köhler (1997) with a commercial preparation of essential oils showed a reduction of colony forming units of C. perfringens as compared to the positive control diet containing zinc bacitracin. This preparation was also reported to slightly lower ileal ATP concentrations (Veldman and Enting, 1996), which is an indicator of microbial activity in broilers (Smits et al., 1998). Similarly, a blend of capsicum, cinnamaldehyde and carvacrol lowered the number of E. coli and C. perfringens in ceacum (Jamroz and Kamel, 2002). Mitsch et al. (2004) showed that an essential oil mixture containing thymol, carvacrol and eugenol could control the proliferation of C. perfrigens in the broiler intestines and could potentially reduce the effects of NE outbreak associated with coccidiosis. Although previous reports indicated a considerable in vivo antimicrobial activity on various bacteria of chicken microflora, effects of essential oils or herbal extracts on growth performance of chickens was variable. There is a significant number of studies using

70 Control of intestinal diseases in poultry different essential oils or essential oil combinations that have contradictory outcomes. In several research studies, it was shown that oregano derived essential oils added in the drinking water of chickens (Hertrampf, 2001) or supplemented as ground plant (Giannenas et al., 2005) had beneficial growth-promoting effects. In addition, Alcicek et al. (2003) found that dietary supplementation of an essential oil combination from 6 herbs including oregano, laurel, sage, myrtle, fennel and citrus improved chicken performance. In contrast, Lee et al. (2003) found that dietary supplementation of broiler chickens with a mixture of essential oils did not improve growth performance, whilst Botsoglou et al. (2002) and Papageorgiou et al. (2003) reported that dietary supplementation of essential oils derived from oregano at levels of 50 or 100 mg/kg in chickens, and at levels of 100 or 200 mg/kg in turkeys, respectively, had no beneficial effect on growth performance. The highly variable effects of aromatic plants and their extracts on chickens growth performance could possibly be explained, on the one hand, by their various effects not only on gut microflora, but also on animal metabolism, and, on the other hand, by the variable chemical composition of these products. A detailed understanding of the bioactive effects of feeding aromatic plants, herbal products and blends of phytochemical substances or plant secondary metabolites as to reduce the incidence of diseases in chickens remains a challenge.

Anticoccidial activity of herbs, herbal extracts and essential oils Several natural products have been tested for their potential to provide protection against or modulate the effects of coccidial infections. Allen et al. (1997) reported that dried leaves of Artemisia annua could protect chickens against caecal lesions due to E. tenella infection. Pure components of A. annua, i.e. artemisinin, 1,8-cineole and camphor, were fed to day-old chicks until 3 weeks of age. During the second week of age, half of the birds were inoculated with E. acervulina and E. tenella. Some prophylactic action against the coccidia challenge was shown in treated chicks, especially in those fed artemisinin. Evans et al. (2001) investigated whether a mixture of essential oils from clove, thyme, peppermint and lemon (at level of 0.1%) could have effects on coccidia oocyte output and the number of C. perfringens in broiler chicks when artificially inoculated. There was no positive control included. Chicks fed the diets containing an essential oil blend showed a reduced oocyte excretion when compared to those fed the non-supplemented diet. Youn and Noh (2001) showed that Sophora flavescens extracts were more effective than Artemisia annua against E. tenella infection in chickens. Earlier, Akhtar and Rifaat (1987) examined the anticoccidial effect of extracts of Melia azedarach Linn. (Bakain) in naturally infected chickens and found a modest effectiveness. A study by Williams (1997) showed that phenols used as disinfectants exhibit oocysticide activity against E. tenella both in vivo and in vitro. In nature, rich sources of phenolic compounds are found in several aromatic plants; those of the Labiatae family, among which

I. Giannenas and I. Kyriazakis 71

oregano is of particular interest (Vekiari et al., 1993). Ibrir et al. (2001) found that thymol and carvacrol improved growth performance of broiler chickens previously infected with oocysts of E. acervulina. Giannenas et al. (2003) reported that the essential oil of oregano exhibited coccidiostatic action against E. tenella when incorporated into chicken diets at the level of 0.3 g/kg. In another experiment broiler chickens, challenged at 14 days of age by oral administration with a 2-ml suspension of 5×104 sporulated oocysts of E. tenella, were given diets supplemented with ground oregano herb at levels of 2.5, 5.0, 7.5 and 10 g/kg feed (Giannenas et al., 2004). It was found that supplementation could reduce the adverse effects of E. tenella infection, as judged by the significantly increased body weights and improved feed conversion efficiencies compared to the challenged control group. Also, the recorded values of the severity of bloody diarrhoea, mortality, caecal lesion scores and oocyst output suggested that the most effective diets against the infection E. tenella were the ones supplementated with oregano at 5.0 and 7.5 g/kg of feed. These results support the hypothesis that oregano could be administered to broiler chickens as an alternative to ionophore antibiotics for protection against coccidial infection. In another study the effect of dietary supplementation with Olympus tea (Sideritis scardica) on the performance of broiler chickens challenged with 6×104 sporulated oocysts of E. tenella at 14 days of age was examined. Data based on evaluation of performance parameters along with the extent of bloody diarrhoea, mortality rate, caecal lesion score and oocyst output suggested that treatment with Olympus tea at the supplementation level of 10 g/kg diet, could alleviate the impact of parasite infection. However, the exerted coccidiostatic effect against E. tenella was considerably lower than that exhibited by lasalocid (Florou-Paneri et al., 2004). Similar results were also noted by Christaki et al. (2004) while studying the effect of dietary supplementation with a commercial preparation of herbal extracts from the plants Agrimonia eupatoria, Echinacea angustifolia, Ribes nigrum and Cinchona succirubra. Experimental diets were prepared by incorporating this commercial mixture of herbal extracts at levels of 0.5 and 1.0 g/kg of feed. Chickens were experimentally infected with 6×104 sporulated oocysts of E. tenella at 14 days of age, and results showed that the mixture of herbal extracts did have a beneficial effect on post infection performance compared to the challenged birds; however, lasalocid exerted a significantly stronger coccidiostatic effect against E. tenella than the tested commercial mixture of herbal extracts. Muriel et al. (2005) reported the positive effect of dietary supplementation with both a saponin-based herbal and a tannin-based extract, after experimental infection with 4×104 sporulated oocysts of E. tenella and 4×106 sporulated oocysts of E. acervulina at 8 days of age. Also, Oviedo-Rondon et al. (2006) observed an interesting effect of dietary supplementation with a commercial preparation of herbal extracts (thymol, eugenol, curcumin and piperin from the plants Thymus vulgaris, Syzygium aromaticum, Cinnaminum zeylanicum and Pipper nigrum, respectively). Chickens were experimentally infected with sporulated oocysts of E. tenella, E. acervulina and E. maxima at 19 days of age and half of the challenged birds were also vaccinated with a coccidian vaccine at day of hatch.

72 Control of intestinal diseases in poultry Results showed that the mixture of essential oils had a beneficial effect on post infection performance compared to challenged birds that did not receive anticoccidial substances. In contrast, in the same study the supplementation of these specific blends of essential oils to coccidian vaccinated broilers did not show any beneficial effects. In another similar study (Toyomizu et al., 2003), two experiments were conducted to determine the effects on chicken performance of anacardic acid and cashew nut shell oil as feed supplements during an experimental coccidial infection with E. tenella oocysts. The study showed that anacardic acid and cashew nut shell oil had no positive effect on oocyst output or bird performance but provided only a slight reducing effect on the severity of caecal lesions in chickens during an experimental coccidial infection. Recently, Naidoo et al. (2008) screened four plant extracts for their anticoccidial activity in vivo with toltrazuril, which is an authorised anticoccidial substance, as the positive control. In this study, it was shown that dietary supplementation with Tulbaghia violacea displayed a beneficial effect on chickens, both improving feed conversion ratio and rate of oocyst shedding. Vitis vinifera and Artemisia afra, although resulted in feed conversion ratios similar to toltrazuril and higher than the untreated control, did not reduce oocyst production in the birds. On the contrary, Combretum woodii proved to be extremely toxic to the birds. These researchers noted that it is important the active plant constituents to be sufficiently lipid soluble to penetrate intracellular and be effector on the coccidian parasite (Naidoo et al., 2008). In conclusion, the results of the above studies showed that dietary supplementation with plant extracts may exert a positive effect on experimentally coccidia challenged broiler chickens. However, the efficacy of plant extracts was significantly lower than that exhibited by anticoccidial drugs (Christaki et al., 2004; Giannenas et al., 2003, 2004; Florou-Paneri et al., 2004; Naidoo et al., 2008). In some studies plant extracts exerted significant effects on reducing oocyst excretion and diminishing caecal lesion scores (Youn and Noh, 2001; Giannenas et al., 2003, 2004), while in other studies there was only a positive effect on growth performance of birds compared to challenged birds that did not receive any authorised anticoccidial treatment (Toyomizu et al., 2003; Muriel et al., 2005; Naidoo et al., 2008). This evidence indicates that the investigation of herbal materials as anticoccidial remedies may hold promise as an alternative in the control of coccidiosis. However, further research is needed to elucidate anticoccidial mechanisms of activity and find plant substances that will provide fully anticoccidial protection in intensively reared broiler chickens.

A prebiotic effect of aromatic plants Another approach which may be employed to manipulate the broiler gut ecosystem is the supplementation of diets with prebiotics. A prebiotic is a feed ingredient that beneficially affects the host by selectively stimulating the growth or activity of beneficial bacterial species already resident in the intestinal tract (Gibson and Roberfroid, 1995). Prebiotics are predominantly polysaccharides including fructo-, inulin-, trans-galacto-, gluco-, lactulose-,

I. Giannenas and I. Kyriazakis 73

lactitol-, malto-, xylo-, stachyose-, raffinose- and sucrose-oligosaccharides (Collins and Gibson, 1999; Orzechowski et al., 2002). Oligosaccharides represent a structurally diverse class of macromolecules of a relatively widespread occurrence in nature, found also in herbs, spices and aromatic plants. Other less investigated but potentially promising biological activities of these biopolymers are their immunomodulation effects. Allen et al. (2000) reported that dietary dried leaves of Echinacea purpurea could increase the development of immunity following live coccidian vaccination and subsequent challenge of broiler chickens with multiple coccidian species. Prebiotics are known for their ability to increase the endogenous growth of intestinal lactobacilli and bifidobacteria populations, which have been recognized beneficial to health (Blay et al., 1999). In vivo studies in rats have also shown that fructo-oligosaccharides increase the proportion of butyrate, which in turn, stimulates water and sodium absorption and modulates intestinal motility, and also increase Ca, Mg, and Fe absorption and enhances bone calcium stores (Ohta et al., 1998; Orzechowski et al., 2002). Whether herbs have a prebiotic effect during NE or coccidiosis, has yet to be addressed.

Challenges in the transforming of aromatic plants to phytobased products Challenges to enhance effectiveness of aromatic plants or their extracts Unlike pharmaceutical products based on a single chemical entity that targets specific body functions in target cells, tissues, or organs, most of the herbal remedies seem to lack scientific basis and their effects fall more into the realm of folklore. In order for botanical products to maintain the high productivity rates achieved by antimicrobial drugs and become acceptable by the mainstream poultry industry market, solid scientific evidence is needed to support their functionality claims. In Table 1 certain differences between antibiotics and phytobased products in terms of origin, ways of activity and effectiveness are presented. Table 1. Critical differences between antibiotics and phytobased products

Antibiotics Origin Synthetic Effectiveness Consistent Action Fast Reliable mode of action Both in vitro and in vivo Activity Specific Approach Molecular target Indication Acute and chronic Safety Toxic in certain dose *GRAS, generally regarded as safe

Phytobased products Natural Variable Gradual In vitro, not always in vivo Multifactorial Holistic Chronic GRAS*

74 Control of intestinal diseases in poultry The market for phytogenic performance promoters has increased tremendously since the 1990s, coinciding with the constraints of antibiotic growth promoters (Steiner, 2006). In 1996 the market for essential oils was valued at 90 t with revenues of €413 (Greathead, 2003). Since then the market has experienced rapid growth, and in 2006 it was over 600 t with revenues of €1.5 million. Today, 61% (non-EU) and 70% (EU) of the poultry producers are using botanical feed additives in broiler feeds (Anonymous, 2008). Worldwide sales of aromatic plants, crude extracts and finished products for both human and animal consumption amounts to 60 billion US $ per annum (Voigt, 2006). With an annual growth rate of 5-15%, as projected by the World Bank, great expansion on a global scale is expected in the first half of this century (Liu and Wang, 2008). Prior to considering incorporating aromatic plants in animal feeds in any form, the scientific community should provide strong evidence of their benefits and address the issue of inconsistency across the studies in the literature. It is believed that in order to minimise these inconsistencies and maximise the repeatability of the results, there is an urgent requirement for the development and utilisation of a standardised methodology for the evaluation of their activity. Currently, there is no system available to sufficiently characterise aromatic plants in terms of their biological properties, quantify the latter and finally standardise their use as antimicrobial, antiparasitic, antiprotozoal or antioxidant agents. Such a system could be used as a common currency from scientists around the world to describe the potential effects of bioactive plants. Phytobased products can consist of an extremely heterogeneous group of substances originating from leaves, stems, roots, tubers, or fruits of herbs, spices or aromatic plants. They may also contain extracts or essential oils of single or a mixture of aromatic plants. The use of feed additives is usually subject to restrictive regulations. In general, they are considered as products applied by the keeper to healthy animals for a nutritional purpose on a permanent basis (i.e. during the entire production period of the respective species and category), in contrast to veterinary drugs (applied for prophylaxis and therapy of diagnosed health problems under veterinarian control for a limited time period, partially associated with a waiting period). In the European Union, for example, feed additives need to demonstrate the identity and traceability of the entire commercial product, the efficacy of the claimed nutritional effects, including the absence of possible interactions with other feed additives, and the safety to the animal (e.g. tolerance), the user (e.g. farmer, worker in feed mills), the consumer of animal-derived products and to the environment (for further details, refer to European Commission, Regulations EC, No 1831/2003 and 1334/2003). Problems with feed additive legislation may, therefore, apply to phytobased feed additives that claim to affect the health or modulate metabolism (e.g. through a phytohormonal mode of action). For these reasons, industrial production of phytobased feed additives in poultry diets should present solid scientific evidence if they claim to have antimicrobial or anticoccidial activity and/or growth-promoting efficacy. The main problem in fact is that the effectiveness of aromatic plants and their extracts on animal performance is neither consistent among different trials nor with the expectations arising from traditional use. One explanation for this inconsistency is that the majority of

I. Giannenas and I. Kyriazakis 75

ethno-pharmacology reports originate from ruminants, (Athanasiadou and Kyriazakis, 2004). Consequently, when biological activity of such plants is tested in rodent models or other non-ruminant animals, part of the reported variation may be due to the physiological difference between these two classes of animals (Githiori et al., 2003). Another explanation, especially among different trials is the fact that there is a great variation in the protocols of collection and storage of the plant material prior to its use. Differences in the preservation methods may affect the plant properties (Skrubis, 1972). Additionally, seasonal and environmental variability may have an impact on the synthetic pathways of these metabolites, which can potentially affect their physical and chemical properties (Cardellina, 2002). Variation in composition will result in variable biological effects of essential oils or herbal extracts (Schilcher, 1985; Janssen et al., 1987; Deans and Waterman, 1993). The chemical properties and biological activities of these compounds and their combinations should be extensively examined, as changes in plant availability in nutrients and metabolites, affect the reproducibility of their biological activity. Variable sources of biomass, unknown active ingredients, difficulties in quality control, lack of safety evaluation, unclear mechanism of action, etc., all constitute major challenges in terms of scientific standardization to adhere to the industry norm (Wang and Ren, 2002). Furthermore, the bioavailability of the active compound in the host is not always directly related to the concentration in the plant. A major advantage of aromatic plants and their extracts is that they are generally considered to be safe products. If they were unsafe, the use of herbs, spices and their extracts in human food would have to be discontinued (Jones, 2002). Despite this popularity of aromatic plants and their extracts, if they were to be used as alternative feed additives in large scale in animal feeds, they would need to be researched in terms of their mechanisms of toxicity and side effects. Fallacies and hyperboles associated with herbal products include: i) herbs, being natural are implicitly safe, ii) they do not have side effects, iii) efficacy can be obtained over a wide range of dosage, and iv) they are a panacea (Chang, 2000). Such claims run counter to fundamental pharmacological tenets and would lessen the value of aromatic herbs in the overall strategy to prevent or treat disease. Research on aromatic plants and their extracts on both efficiency and safety can only be viewed as essential in establishing their true value as phytobased feed additives. Safety issues may also arise from the potential residues of different solvents used for the production of herbal extracts and found in botanical products.

Production of phytobased products The first step for the production of phytobased products is the authentification and identification of active compounds in aromatic plants as raw materials or biomass (Figure 1). Subsequently, this information could be used to characterise unknown plants by estimating their content in the active compound. This would enable comparisons to be made on the same basis across studies and will also explain the variability of results. As the process of

76 Control of intestinal diseases in poultry isolating the active compounds is long, complex, and often requires specialised equipment, bibliographic evidence on the active compounds and subsequent determination of their concentration in the plant would also be useful. Admittedly, this process can be complicated in cases where the biological activities of a plant are attributed to more than one compounds. The extraction procedure is critical to the overall effectiveness of herbal products because excessive fractionation and isolation may result in loss of activity. Chromatographic techniques represent an ideal tool for comprehensive chemical characterization of plant substances and their quality control. Nevertheless, there is no definitive methodology for assuring that herbal content will not vary from one production run to another (Wang and Ren, 2002; Balunas and Kinghorn, 2005). Cultivation of aromatic plants

Production and authentification of raw materials

Chemical standardization

Process chemistry: scale up production

In vivo studies

In vitro studies

GCP trials of candidate products

Quality control

GMP production and marketing

Figure 1. Schematic production of standardised phytobased products, (GCP, good clinical practice; GMP, good manufacturing practice), modified from Liu and Wang (2008)

Usage of botanical products will be related to metabolic processes of an integrated biological system under specific environmental conditions. Host metabolic enzymes and intestinal bacteria may be responsible for the metabolism of botanical products (Lee et al., 2004). Systematic research on metabolic pathways of the various components of essential oils

I. Giannenas and I. Kyriazakis 77

or herbal extracts will not only assist in identifying the bioactive constituents but also improve our knowledge on efficacy, safety and complexity of a given combination of these substances. Chemical analysis will indicate the presence of a particular molecule and can be used to identify active components, contaminants or other chemicals in a botanical preparation (Fan et al., 2006). This approach will serve the demand of standardization and quality control for the production of botanical products. In order to standardise the effects of phytobased products, encapsulation techniques of biological active compounds can provide an advantageous way for the production of these products. Liposomes or coated extracts can be particularly useful in formulating neutraceuticals as they may increase availability and absorption of active substances. The encapsulation form of active substances may control release rate of materials or provide protection for them by reducing oxidation or volatilization or even interactions with other ingredients in the finished product (Keller, 2001; Gortzi et al., 2006). Liposomes have also been shown to increase “bioactivity or activity at special sites” of ingredients and might also provide processing and formulation advantages, primarily by allowing easier incorporation of oil soluble materials into water based products (Storm and Crommelin, 1998). Encapsulation may also be used to incorporate mixtures of essential oils along with organic acids and other alternatives to antibiotic growth promoters. There exists a large gap between traditional herbal practices and contemporary medical science in philosophy, theories and applications. Thus, the opening of opportunities for phytobased products in western countries must bridge the two. The intuitive approach of the ethnoveterinary knowledge can contribute to modern animal feeding. In vivo studies are necessary to exploit the interactions of different components (e.g. molecular pathways and regulatory networks) within the organism and elucidate relevant physiological functions and behaviour. This, to certain extent, resembles the general principles of the holistic approach, which focuses on the analysis of entire body functions and bird health and performance rather than focusing on disease treatment (Yuan and Lin, 2000). Thus, a herbal mixture may display diverse activities by interacting with more than one molecular target. A relationship between modern medicine, traditional knowledge and the robust use of technologies in the context of biological systems is required in order plant oils or extracts to be as effective as commercially available synthetic products.

Conclusions The hypothesis of rearing broilers without antibiotics or coccidiostats is not new (Ekstrand et al., 1994). The ban on the routine feeding of antibiotics in Europe and the potential for a reduction or discontinuing of this use in North America brought about an increased interest in finding alternatives to antibiotics to be used in poultry diets to maintain health and high productivity rates. A critical examination of bioactive plant products has to cover analytical defects, bioavailability and molecular functionality together with clinical trials and feeding experiments. The actual knowledge of the impact of these substances on animal and human

78 Control of intestinal diseases in poultry health, as well as product development and quality assurance management, is possible only through a multidisciplinary approach. Phytobased products should be developed in cooperation of veterinarians, pharmacologists, phytochemists and the respective industrial partners. The consumer as end user expects safe and healthy products; therefore the substitution of conventional medication by natural products generally recognized as safe should be proved. The main challenge for the poultry industry is to ensure that claims made for phytobased products are justified by reliable data. This research field is not only of strategic and applied interest, but also of fundamental interest, since it will expand our knowledge on the interactions between animals and active substances of plant origin. Action is required to achieve reproducible evidence on their biological activity and thus appreciation by the worldwide scientific community. Emphasis should be given on incorporating additional measurements in ongoing research, with performance, immunity and behavioural observations when considering the potential of such products. Additional large scale in vivo studies are needed to investigate the activity of different active substances, their incorporation levels in feeds and their efficacy against intestinal diseases. Plants, their extracts and as a consequence phytobased products could exert specific effects on intestinal microflora, particularly in bacteria or protozoa and also on animal performance. These products may have important potential as alternative growth promoters and could be beneficial to both chicken performance and health.

References Adam K Sivropoulou A Kokkini S Lanaras T, Arsenakis M (1998) Antifungal activities of Origanum vulgare susp. hirtum, Mentha spicata, Lavandula angustifolia, and Salvia fruticosa essential oils against human pathogenic fungi. Journal of Agricultural Food Chemistry 46: 1739–1745. Akhtar MS, Rifaat S (1987) Anticoccidial screening of Melia azedarach Linn. (Bakain) in naturally infected chickens. Pakistan Journal Agricultural Science 21: 95–99. Alcicek A, Bozkurt M, Cabuk M (2003) The effect of an essential oil combination derived from selected herbs growing wild in Turkey on broiler performance. South African Journal Animal Science 33: 89–94. Allen PC, Danforth HD, Skinner HG (2000) Dietary echinacea supplementation and development of immunity to coccidia challenge. Proceedings of the XXI Worlds Poultry Congress, Montreal, Canada. Allen PC, Lydon J, Danforth HD (1997) Effects of components of Artemisia annua on coccidia infections in chickens. Poultry Science 76: 1156–1163. Annett CB, Viste JR, Dahiya J, Hoehler D, Wilkie D, Van Kessel A, Drew M (2005) Severely impaired production performance in broiler flocks with high incidence of Clostridium perfrigens associated hepapitis. Avian Pathology 30: 73–81. Anonymous (2008) Plant extracts popular in poultry. World Poultry, 24(3): 9.

I. Giannenas and I. Kyriazakis 79

Anthony J-P, Fyfe L, Smith H (2005) Plant active components – a resourse for antiparasitic agents? TRENDS in Parasitology 21: 462–467. Athanasiadou S, Githiori J., Kyriazakis I (2007) Medicinal plants for helminth parasite control: facts and fiction. Animal 1:9 1392–1400. Athanasiadou S, Kyriazakis I (2004) Plant secondary metabolites: antiparasitc effects and their role in ruminant production systems. Proceedings of the Nutrition Society 63 631–639. Athanasiadou S, Kyriazakis I, Jackson F, Coop R L (2001) Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies. Veterinary Parasitology 30: 205–219. Balunas MJ, Kinghorn, AD (2005) Drug discovery from medicinal plants. Life Sciences 78: 431–441. Barton MD (1999) The down-side of antibiotic use in pig production: The effect of antibiotic resistance of enteric bacteria. In manipulating Pig Production VII (ed PD Carnwell). Australasian Pig Science Association, Victoria, Australia. Bedford M (2000) Removal of antibiotic growth promoters from poultry diets: implications and strategies to minimise subsequent problems. World Poultry Science Journal 56: 347–365. Besra S, Gomes A, Chaudhury L, Vedasiromoni J, Ganguly D (2002) Antidiarrhoeal activity of seed extract of Albizzia lebeck Benth. Phytotherapy Research 16: 529–533. Blay G, Michel C, Blottiere H, Cherbut C, (1999) Prolonged intake of fructo-oligosaccharides induces a short term elevation of lactic acid-producing bacteria and a persistent increase in cecal butyrate in rats. Journal of Nutrition 129: 2231–2235. Botsoglou N, Florou-Paneri P, Christaki E, Fletouris D, Spais AB (2002) Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. British Poultry Science 43: 223–230. Bowles B, Miller A (1993) Antibotulinal properties of selected aromatic and aliphatic aldehydes. Journal of Food Protection 56: 788–794. Cardellina JH (2002) Challenges and opportunities confronting the botanical dietary supplement industry. Journal of Natural Products 65: 1073–1084. Chang J (2000) Medicinal herbs: drugs or dietary supplements? Biochemistry Pharmacology 59 211–219. Chapman HD (1986) Isolates of Eimeria tenella: studies on resistance to ionophorous anticoccidial drugs. Research in Veterinary Science 41: 281–282. Chapman HD (2000) Long term anticoccidial strategy: chemotherapy plus vaccination. Feed International 21(3): 18–19. Christaki E, Florou-Paneri P, Giannenas I, Papazahariadou M, Botsoglou N, Spais AB (2004) Effect of a mixture of herbal extracts on broiler chickens infected with Eimeria tenella. Animal Research 53: 137–144. Collins M, Gibson G (1999) Probiotics, prebiotics and synbiotics: approaches for modulating the microbial ecology of the gut. American Journal of Clinical Nutrition 69: 1052S–1057S. Conner DE (1993) Naturally occurring compounds. In: Antimicrobials in Foods, Davidson

80 Control of intestinal diseases in poultry PM and AL Branen, eds. Dekker, New York. Crouch CF, Andrews SJ, Ward RG, Francis MJ (2003) Protective efficacy of a live attenuated anti-coccidial vaccine administered to 1-day-old chickens. Avian Pathology 32: 297–304. Dahiya J, Hoehler D, Wilkie D, Van Kessel A, Drew M (2005) Dietary glycine concentration affects intestinal Clostridium perfrigens and lactobacilli populations in broiler chickens. Poultry Science 84: 1875–1885. Dahiya J, Wilkie D, Van Kessel A, Drew M (2006) Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era. Animal Feed Science and Technology 129: 60–88. Dahiya, J, Hoehler D, Van Kessel A, Drew M (2007) Dietary encapsulated glycine influences Clostridium perfringens and lactobacilli growth in gastrointestinal tract of broiler chickens. Journal of Nutrition 137: 1408–1414. Deans SG, Ritchie G (1987) Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5: 165–180. Deans SG, Waterman PG (1993) Biological activity of volatile oils. In: Volatile oil crops, Hay, RKM, and PG Waterman, Eds. Longman Scientific and Technical, Essex. Didry N, Dubreuil L, Pinkas M (1994) Activity of thymol, carvacrol, cinnamaldehyde and eugenol on oral bacteria. Pharmaceutica Acta Helvetiae 69: 25–28. Dorman HJD, Deans SG (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal Applied Microbiology 88: 308–316. Drew M, Syed N, Goldabe B, Laarveld B, Van Kessel A (2004) Effects of dietary protein source and level on intestinal populations of Clostridium perfrigens in broiler chickens. Poultry Science 83: 414–420. Economou KD, Oreopoulou V, Thomopoulos CD (1991) Antioxidant properties of some plant extracts of the Labiatae family. Journal American Oil Chemists Society 68: 109–113. Ekstrand C, Algers B, Thebo P, Hooshmand-Rad P (1994) Rearing broilers without coccidiostats. Proceedings of the Eighth International Congress on Animal Hygiene, Minnesota, USA.. Engstrom BE, Fermer C, Lindberg A, Saarinen, E Baverud V, Gunnarsson A (2003) Molecular typing of isolates of Clostridium perfrigens from healthy and diseased poultry. Veterinary Microbiology 94: 225–235. Evans J W, Plunkett M, Banfield M (2001) Effect of an essential oil blend on coccidiosis in broiler chicks. Poultry Science, 80 (suppl. 1): 258 (Abstract). Fan XH, Cheng YY, Ye ZL, Lin RC, Qian ZZ (2006) Multiple chromatographic fingerprinting and its application to the quality control of herbal medicines. Analytica Chimica Acta 555: 217–224. Farag RS, Daw Z, Hewed F, El-Baroty G (1989) Antimicrobial activity of some Egyptian spice essential oils. Journal of Food Protection 52: 665–667. FDA 2004 U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research. Guidance for Industry Botanical Drug

I. Giannenas and I. Kyriazakis 81

Products. (http://www.fda.gov/cder/guidance). Florou-Paneri P, Christaki E, Giannenas I, Papazahariadou M, Botsoglou N, Spais AB (2004) Effect of dietary Olympus tea (Sideritis scardica) supplementation on performance of chickens challenged with Eimeria tenella. Journal of Animal Feed Sciences 13: 301–311 Fukata T, Hadate Y, Baba E, Arakawa A (1991) Influence of bacteria on Clostridium perfrigens infections in young chickens. Avian Diseases 35: 224–227. Giannenas I, Florou-Paneri P, Botsoglou N, Christaki E, Spais AB (2005) Effect of supplementing feed with oregano and/or a-tocopheryl acetate on growth of broiler chickens and oxidative stability of meat. Journal of Animal and Feed Sciences 14: 521–535. Giannenas I, Florou-Paneri P, Papazahariadou M, Botsoglou N, Christaki E, Spais AB (2004) Effect of diet supplementation with ground oregano on performance of broiler chickens challenged with Eimeria tenella. Archiv fur Geflugelkunde 68: 247–252. Giannenas I, Florou-Paneri P, Papazahariadou M, Christaki E, Botsoglou N, Spais AB (2003) Dietary oregano essential oil supplementation on performance of broilers challenged with Eimeria tenella. Archives Animal Nutrition 57: 99–106. Gibson G R, Roberfroid B M (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal Nutrition 125: 1401–1412. Githiori JB, Hoglund J, Waller PJ and Baker RL (2003) Evaluation of anthelmintic properties of some plants used as livestock dewormers against Haemonchus contortus infections in sheep. Parasitology 129: 245–253. Gortzi O, Lalas S, Chinou I, Tsaknis J (2006). Reevaluation of antimicrobial and antioxidant activity of Thymus spp. extracts before and after encapsulation in liposomes. Journal of Food Protection 69: 2998–3005. Greathead H (2003) Plants and plant extracts for improving animal productivity. Proceedings of the Nutrition Society 62: 279–290. Guarrera PM (1999) Traditional antihelmintic, antiparasitic and repellent uses of plants in Central Italy. Journal of Ethnopharmacology 68: 183–192. Hammer KA, Carson CF, Riley TV (1999) Antimicrobial activity of essential oils and other plant extracts. Journal Applied Microbiology 86: 985–990. Hay RKM, Waterman PG (1993) Volatile oil crops: their biology, biochemistry and production. Longman Scientific and Technical, Essex. Helander IM, Alakomi HL, Latva-Kala K, Mattila – Sandholm T, Pol M, Smid EJ, Gorris LG, Von Wright A (1998) Characterization of the action of selected essential oil components on gram negative bacteria. Journal Agricultural Food Chemistry 46: 3590–3595. Helmboldt CF, Bryant ES (1971) The pathology of necrotic enteritis in domestic fowl. Avian Diseases 15: 775–780. Hertrampf JW (2001) Alternative antibacterial performance promoters. Poultry International 40: 50–52. Ibrahim AM (1992) Anthelmintic activity of some Sudanese anthelmintic plants.

82 Control of intestinal diseases in poultry Phytotherapy Research 6: 155–157. Ibrir F, Greathead HMR, Forbes JM (2001) The effect of thymol/carvacrol on the performance of broiler chickens infected with Eimeria acervulina. Proceedings of workshop on alternatives to feed antibiotics and anticoccidials in the Pig and Poultry Meat Production, Oslo, Norway. Jamroz D, Kamel C (2002) Plant extracts enhance broiler performance. Journal Animal Science 80(Suppl. 1): 41 (Abstract). Janssen AM, Scheffer J, Svendsen AB (1987) Antimicrobial activities of essential oils. Pharmaceutisch Weekblad (Sci) 9: 193–199. Jones G (2002) Phytobiotic solutions. Pig Progress 18: 2–3. Juven BJ, Kanner J, Schved F, Weisslowicz H (1994) Factors that interact with the antibacterial action of thyme essential oil and its active constituents. Journal Applied Bacteriology 76: 626–631. Kamel C (2001) Tracing modes of action and the roles of plant extracts in non-ruminants. In: Recent advances in animal nutrition. Garnsworthy, P. C., and J. Wiseman, eds. Nottingham University Press, Nottingham. Pp. 135–150. Keller BC (2001) Liposomes in Nutrition. Trends in Food Science and Technology 12: 25–31. Köhler B (1997) Effects on gut microflora. Akzo Nobel. Lawrence B, Reynolds R (1984) Progress in essential oils. Perfumer and Flavorist 9: 23–31. Lee HS, Ahn YJ (1998) Growth-inhibiting effects of Cinnamomum cassia bark-derived materials on human intestinal bacteria. Journal Agricultural Food Chemistry 46: 8–12. Lee K-W, Everts H, Beynen AC (2004) Essential oils in broiler Nutrition. International Journal Poultry Science 3: 738–752. Lee K-W, Everts H, Kappert HJ, Frehner M, Losa R, Beynen AC (2003) Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broilers. British Poultry Science 44: 450–457. Liu Y, Wang, MW (2008) Botanical drugs: Challenges and opportunities Contribution to Linnaeous Memorial Symposium 2007. Life Sciences 82: 445–449. Lovland A, Kaldhusdal M (2001) Severely impaired production performance in broiler flocks with high incidence of Clostridium perfrigens associated hepapitis. Avian pathology 30: 73–81. McDougald LR (1998) Intestinal protozoa important to poultry. Poultry Science 77: 1156–1158 McEvoy J (2001) Safe limits for veterinary drug residues: what do they mean? Northern Ireland Veterinary Today 37–40. Milos M, Mastelic J, Jerkovic I (2000) Chemical composition and antioxidant effect of glycosidically bound volatile compounds from oregano (Origanum vulgare L. ssp. hirtum). Food Chemistry 71: 79–83. Mitsch P, Zitterl-Eglseer K, Kohler B, Gabler C, Losa R, Zimpernik I (2004) The effect of

I. Giannenas and I. Kyriazakis 83

two different blends of essential oil components on the proliferation of Clostridium perfrigens in the intestine of broiler chickens. Poultry Science 83: 669–675. Moleyar V, Narasimham P (1992) Antibacterial activity of essential oil components. International Journal of Food Microbiology 16: 337–342. Montes-Belmont R, Carvajal M (1998) Control of Aspergillus flavus in maize with plant essential oils and their components. Journal Food Protection 61: 616–619. Muhammed SI, Morrison SM, Boyd WL (1975) Nutritional requirements for growth and sporulation of Clostridium perfringens. Journal of Applied Bacteriology 38: 245–253. Muriel N, Genevieve F, Thierry P, Francois R (2005) Efficacy study of two plant-extract formulas EMX1 and EMX2 in the prevention of E. acervulina and E. tenella coccidiosis in label chickens, Journees de la Recherche Avicole 384–388. Naidoo V, Mc Gaw LJ, Bisschop SPR, Duncan N, Eloff JN (2008) The value of plant extracts with antioxidant activity in attenuating coccidiosis in broiler chickens. Veterinary Parasitology 153: 214–219. Nakamura M, Cook J, Cross W (1978) Lecithinase production by Clostridium perfringens in chemically defined media. Applied Microbiology 16: 1420–1421. Ohta A, Ohtsuki M, Hosono A, Adachi T, Hara H, Sakata T (1998) Dietary fructooligosaccharides prevent osteopenia after gastrectomy in rats. Journal of Nutrition 128: 106–110. Orzechowski A, Ostaseweski P, Jank M, Berwid SJ (2002) Bioactive substances of plant origin in food-impact on genomics. Reproduction, Nutrition and Development 42: 461–477. Oviedo-Rondon E, Clemente-Hernandez S, Salvador F, Williams P, Losa R, Austin S (2006) Essential oils on mixed coccidian vaccination and infection in broilers. International Journal of Poultry Science 5: 723–730. Papageorgiou G, Botsoglou N, Govaris A, Giannenas I, Iliadis S, Botsoglou E (2003) Effect of dietary oregano oil and α-tocopheryl acetate supplementation on iron-induced lipid oxidation of turkey breast, thigh, liver and heart tissues. Journal Animal Physiology and Animal Nutrition 87: 324–335. Platel K, Srinivasan K (1996) Influence of dietary spices and their active principles on digestive enzymes of small intestinal mucosa in rats. International Journal of Food Science and Nutrition 47: 55–59. Rao B, Nigam S (1970) The in vitro antimicrobial efficiency of essential oils. Indian Journal of Medical Research 58: 627–633. Riddell C, Kong XM (1992) The influence of diet on necrotic enteritis in broiler chickens. Avian Diseases 36: 499–503. Schilcher H (1985) Effects and side-effects of essential oils. In: Proceedings of the 15th international symposium on essential oils. Noordwijkerhout. The Netherlands. Pp. 217–231. Shane SM, Koetting DG, Harrington KS (1984) The occurrence of Clostridium perfrigens in the intestine of chicks. Avian Diseases 28: 1120–1124.

84 Control of intestinal diseases in poultry Shapiro S, Guggenheim B (1995) The action of thymol on oral bacteria. Oral Microbiology and Immunology 10: 241–246. Shirley MW, Millard BJ (1986) Studies on the immunogenicity of 7 attenuated lines of Eimeria given as a mixture to chickens. Avian Pathology 15: 629–638. Sikkema J, De Bont JAM, Poolman B (1994) Interactions of cyclic hydrocarbons biological membranes. Journal of Biological Chemistry 11: 8022–8028. Sivropoulou A, Papanikolaou E, Nikolaou C, Kokkini S, Lanaras T, Arsenakis M (1996) Antimicrobial and cytotoxic activities of Origanum Essential oils. Journal of Agricultural and Food Chemistry 44: 1202–1205. Skrubis GΒ (1972) Seven wild aromatic plants growing in Greece and their essential oils. The flavour industry 3: 556–571 Smits CHM, Veldman A, Verkade HJ, Beynen AC (1998) The inhibitory effect of carboxymethylcellulose with high viscosity on lipid absorption in broiler chickens coinsides with reduced bile salt concentration and raised microbial numbers in the small intestine. Poultry Science 77: 1534–1539. Songer JG, Meer RR (1996) Genotyping of Clostridium perfringens by polymerase chain reaction is a useful adjunct to diagnosis of clostridial enteric disease in animals. Anaerobe 2: 197–203. Steiner, T. (2006) Managing Gut Health – Natural Growth Promoters as a key to Animal Performance. Nottingham University Press, Nottingham, United Kingdom. Stevens DL, Rood JI (2000) Histotoxic clostridia. Gram-Positive Pathogens. ASM Press, Washington, DC. Pp. 563–572. Stiles JC, Sparks W, Ronzio RA (1995) The inhibition of Candida albicans by oregano. Journal of Applied Nutrition 47: 96–102. Storm G, Crommelin DJA (1998) Liposomes: quo vadis? Pharmaceutical Science Technology 1: 19–31. Thomke S, Elwinger K (1998) Growth promotants in feeding pigs and poultry. III. Alternatives to antibiotic growth promotants. Annales de Zootechnie 47: 245–271. Toyomizu M, Nakai Y, Nakatsu T, Aki Y (2003) Inhibitory effect of dietary anacardic acid supplementation on cecal lesion formation following chicken coccidial infection. Animal Science Journal 74: 105–109. Ultee A, Bennik, HJ, Moezelaar R (2002) The phenolic hydroxyl group of carvacrol is essential for action against the food borne pathogen Bacillus cereus. Applied Environmental Microbiology 3: 1561–1568. Ultee A, Kets EPW, Smid EJ (1999) Mechanisms of action of carvacrol on the food borne pathogen Bacillus cereus. Applied Environmental Microbiology 65: 4606–4610. Van der Sluis W (2000) Clostridial enteritis is an often underestimated problem. World Poultry 16: 42–43. Vekiari SA, Oreopoulou V, Tzia C, Thomopoulos CD (1993) Oregano flavonoids as lipid antioxidants. Journal of American Oil Chemists Society 70: 483–487. Veldman A, Enting H (1996) Effects of Crina HC 737 in feed on broiler performance and digestive physiology and microbiology. CLO- Institute for Animal Nutrition “De

I. Giannenas and I. Kyriazakis 85

Schothorst”. Voigt K (2006) Our look at global burgeoning Asian herbal industry. The wall Street Journal, June 11. Waldenstedt L (2003) Effect of vaccination against against coccidiosis in combination with an antibacterial oregano (Origanum vulgare) compound in organic broiler production. Acta Agricultural Scandinavica 53: 101 – 109. Wallace RJ (2005) Antimicrobial properties of plant secondary metabolites. Proceedings of the Nutrition Society 63: 621–629. Wang ZG, Ren J (2002) Current status and future direction of Chinese herbal medicine. Trends in Pharmacological Sciences 23: 347–348. Wegener HC, Aarestrup FM, Jensen LB, Hammerum AM, Bager F (1998) The association between the use of antimicrobial growth promoters and development of resistance in pathogenic bacteria towards growth promoting and therapeutic antimicrobials. Animal Feed Science and Technology 7: 7–14. Wilkie DC (2006) Non-antibiotic approaches to control pathogens in the gastrointestinal tract of broiler chickens. PhD Thesis. University Saskatchewan, Saskatoon, Saskatchewan, Canada. Wilkie DC, Van Kessel AG, White L, Laarveld B, Drew MD (2005) Dietary amino acids affect intestinal Clostridium perfringens populations in broiler chickens. Canadian Journal Animal Science 85: 185–193. Williams P, Losa R (2001) The use of essential oils and their compounds in poultry nutrition. World Poultry 17: 14–15. Williams RB (1997) Laboratory tests of phenolic disinfectants as oocysticides against the chicken coccidium Eimeria tenella. Veterinary Record 141: 447–448. Williams RB (1999) Anticoccidial vaccines: the story so far. World Poultry, Special supplement Coccidiosis 3: 23–25. Windisch WM, Schedle K, Plitzner C, Kroismayr A (2008) Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86(E Suppl.): E140–E148. World Health Organization (2000) Report on the inter-regional workshop on intellectual property rights in the context of traditional medicine, Bangkok, Thailand. Dec 6–8 (http://www.who/int/medicines/lebrary/trm/who.edu.trm-2001-I/whoedutrm-2001-1.pdf). Youn HJ, Noh JW (2001) Screening of the anticoccidial effects of herb extracts against Eimeria tenella. Veterinary Parasitology 96: 257–263. Young R, Craig A (2001) The use and misuse of antibiotics in UK Agriculture- Part 3. Residues of dangerous drugs in intensively produced chicken meat and eggs. Bristol, UK: The Soil Association. Yuan R, Lin Y (2000) Traditional Chinese medicine: an approach to scientific proof and clinical validation. Pharmacology & Therapeutics 86: 191–198.

J.A. Miller et al. 87

5 ENHANCING FEED INTAKE BY THE SOW DURING LACTATION USING BIOMIN® P.E.P. Jodi A. Miller1, Jamie C. Laurenz2*, Jeremy W. Rounsavall3, Nicole C. Burdick4 and Fergus J. Neher5 1 505 E Exchange Pkwy. #7108, Allen, TX 75002, United States; 2Eastern New Mexico University, Station 2, 1500 S Ave. K, Portales NM 88130, United States; 3Agrilife Extension, Office of Nueces County, 710 East Main Street, Suite 1, Robstown, TX 78380, United States; 4Texas A&M University, 401 Kleberg, College Station, Kingsville, TX 77843, United States; 5BIOMIN Deutschland GmbH, Vennweg 77, 48282 Emsdetten, Germany *Corresponding author

Abstract Crossbred sows (Yorkshire × Landrace; n = 47) were selected approximately 14 d prior to farrowing and assigned by weight and parity to one of two treatments. All sows were fed a standard lactation diet containing either 0 (Control; n = 24) or 2 kg/t of the phytogenic feed additive Biomin® P.E.P. (PEP; n = 23) from 10 days prior to the expected farrowing date through weaning. At approximately 110 days of gestation sows were moved to individual farrowing crates and fed the same treatment diets twice daily with enough feed supplied at each feeding to ensure feed refusal (i.e., sows were in essence fed ab libitum). After farrowing (d = 1), feed intake was recorded twice daily for each sow through weaning. Sows and piglets were weighed at weekly intervals. By design, sows in the two treatment groups did not differ (P > 0.70) in initial body weight or parity. Sows in both treatment groups experienced low feed intake immediately post-farrowing and feed intake increased (P < 0.01) as lactation progressed. However, sows supplemented with PEP had a greater (P < 0.01) average daily feed intake then control sows (5.4 ± 0.1 vs. 6.2 ± 0.1 kg/d for Control vs. PEP sows, respectively). Consistent with their enhanced feed intake, sows supplemented with PEP lost substantially less weight (P < 0.05) during the first week of lactation (8.1± 1.4 vs. 3.5 ± 1.6 kg for Control vs. PEP sows, respectively), had increased milk production (P < 0.05) and weaned heavier (P < 0.01) pigs. Collectively, these results demonstrate that supplementation of diets for sows with PEP improves feed intake during the early lactation period and enhances sow and piglet performance. 87

88 Enhancing feed intake in sows

Introduction Selection protocols emphasizing increased litter size and enhanced lean growth have substantially increased productivity in the swine industry. As pig growth rate during the pre-weaning period is dependent upon the quantity and quality of milk production, these improvements have also increased the demand for milk production by the sow (Auldist et al., 1998; Revell et al., 1998). As reviewed by Edwards (2002), milk production by the sow is dependent upon a complex interaction between the level of maternal body energy reserves and current feed intake. In this regard, it is known that inadequate sow feed intake during gestation results in reduced pig birth weights, decreased mammary cell number and a decreased potential for milk production during lactation (Pluske et al., 1995). However, it is also recognized that there is an inverse relationship between the level of feed intake during gestation and subsequent intake during lactation (reviewed by Eissen et al., 2000). Hence, over-conditioning sows in preparation for the high-energy demands of lactation has a negative effect on subsequent sow productivity. Similarly, inadequate energy and protein intake during early lactation, and the ensuing negative energy balance in the sow, results in lowered milk production in later lactation (Mullan and Williams, 1989). This is particularly problematic, as numerous studies have reported that the voluntary intake by the sow during lactation (particularly early lactation) is frequently inadequate to meet the increasing nutrient demands for maintenance, milk production and future reproduction (Noblet et al., 1990; Dormad, 1991; Koketsu et al., 1996a; Koketsu et al., 1996b; Kim and Easter, 2001). Furthermore, environmental influences (e.g. heat stress) can exacerbate these problems by further inhibiting voluntary intake (Eissen et al., 2000). This study investigated the effects of a feed supplement formulated with natural products to improve feed palatability and digestion, on sow feed intake during lactation, and subsequent effects on sow weight change, milk production and pig performance during the pre-weaning period.

Materials and methods Animals, experimental design and feeding Crossbred sows (Yorkshire × Landrace; n = 47) were selected approximately 14 days prior to their expected farrowing dates and assigned by weight and parity to one of two treatments. Treatments included sows that received a standard corn-soybean based lactation diet (Control; n = 24) or sows receiving the same standard lactation diet containing 2 kg/ton of a phytogenic feed additive based on a blend of essential oils originating from oregano, anis and citrus peel, and fructooligosaccharides (Biomin® P.E.P. 1000) (PEP; n = 23). Treatments were initiated 10 days prior to farrowing and continued throughout the lactation period. At 110-112 days of gestation sows were moved to individual farrowing crates and fed the same treatment diets twice daily with enough feed supplied at each

J.A. Miller et al. 89

feeding to ensure feed refusal (i.e., sows were in essence fed ab libitum). After farrowing (day =1), the number of pigs born alive and born dead was recorded, and pigs were weighed and tattooed for permanent identification. The following measurements were then obtained during the subsequent lactation period: 1) Feed amount and refusal was recorded at each feeding and individual daily sow intake (kg/d) determined; 2) Sow weights were obtained at day 1, 7, 14 and 21 of the lactation period and weekly weight change was calculated; and 3) Pig weights were obtained at days 1, 7, 14 and 21 days of age, and total litter weights and average daily gain (ADG; g/d) per pig calculated. In addition, individual sow milk production and average milk intake (g/d) per pig were calculated using the equations generated by Noblet and Etienne (1989) using sows of similar breeding. For early lactation (day 1–7), milk production by the sow (kg/d) was calculated as the sum of individual pig milk intakes (g/d = 2.64 × ADG + 67; R2 = 0.96). Similarly, milk production (kg/d) by the sow during the entire 21-day lactation period was calculated as the sum of individual pig milk intake (g/d = 2.50 × ADG + 80.2 × initial body weight +7; R2 = 0.91).

Statistical analysis All data were subjected to an analysis of variance (ANOVA) for repeated measures. Partitioned sources of variation included treatment, farrowing group and their interactions. Treatment comparisons were made using Fisher’s Protected Least Significant Difference with P < 0.05 considered significant.

Results and discussion Summary of sow performance By design, sows did not differ in initial weight (241.3 ± 10.1 and 236.4 ± 7.8 kg for Control and PEP sows, respectively; P = 0.70) or parity (3.0 ± 0.4 and 3.2 ± 0.3 for Control and PEP sows, respectively; Table 1). Supplementation of sows with PEP did not affect (P > 0.05) the number of pigs born alive (NBA), adjusted NBA, number born dead, number weaned per litter, pre-weaning mortality rate, nor pig birth weight. However, the average weight at weaning (day 21) of pigs from sows supplemented with PEP was 10.2% higher (P < 0.01) then pigs from Control sows (5.82 ± 0.15 vs. 6.42 ± 0.22 kg for pigs from Control vs. PEP sows, respectively). As a result, the 21-day litter weights of sows supplemented with PEP were 15.8% greater (P < 0.01) then those of Control sows (47.8 ± 3.4 vs. 55.4 ± 2.4 kg for Control vs. PEP sows, respectively). The difference in litter weights were not the result of differences in parity or number born alive, as adjusted 21-day litter weights were 12.3% higher (P < 0.01) in PEP sows (51.9 ± 2.8 vs. 58.3 ± 2.0 kg for Control vs. PEP sows, respectively).

90 Enhancing feed intake in sows Table 1. The effect of supplementation of sows with a phytogenic feed additive (PEP) during late gestation through lactation on sow and pig performance

Measure

Control Sows

PEP Sows

PEP Effect (P-value)

Number of Sows 24 23 Parity 3.0 ± 0.4 3.2 ± 0.3 0.74 Initial Sow Weight (kg) 241.3 ± 10.1 236.4 ± 7.8 0.70 Born alive (NBA)/litter 9.4 ± 0.5 9.7 ± 0.3 0.56 Adjusted NBAa 10.1 ± 0.4 10.2 ± 0.3 0.72 Born dead/litter 1.1 ± 0.2 1.3 ± 0.2 0.58 Weaned/litter 8.1 ± 0.5 8.6 ± 0.3 0.38 Preweaning Mortality (%) 13.7 ± 2.7 10.8 ± 2.5 0.45 Average Pig Birth Weight (kg; n = 449 pigs) 1.34± 0.03 1.39± 0.05 0.41 Average Pig 21-day Weight (kg; n=393 pigs) 5.82± 0.15 6.42± 0.22 0.01 21-day Litter Weight (kg; n=47 litters) 47.8 ± 3.4 55.4 ± 2.4 0.04 Adjusted 21-day Litter Weight (kg; n=47 litters) 51.9 ± 2.8 58.3 ± 2.0 0.05 a Adjusted values calculated using the procedures of the National Swine Improvement Federation (NSIF)

Sow feed intake Consistent with previous reports (Koketsu et al., 1996a; Koketsu et al., 1996b; Revell et al., 1998) for both groups of sows, feed intake immediately following farrowing was low and subsequently increased (P < 0.01) during lactation (Figure 1). 10

Control PEP

Daily Feed Intake (kg/d)

8

6

4

2

0 0

7

14

21

Day of Lactation

Figure 1. Effect of supplementation with a phytogenic feed additive (PEP) on daily feed intake (kg/d) of sows during lactation. Feed intake was influenced by time (P < 0.01) and PEP supplementation (P < 0.01).

J.A. Miller et al. 91

Overall, sows supplemented with PEP had a greater (P < 0.01) average daily feed intake then control sows (5.4 ± 0.1 vs. 6.2 ± 0.1 kg/d for Control vs. PEP sows, respectively). The treatment-related effect on intake was not the result of differences in sow weights, as daily intake expressed as a percentage of sow body weight was 17% higher (P < 0.01) in PEP sows (2.3 ± 0.1 vs. 2.7 ± 0.1 % for Control vs. PEP sows, respectively). The ability of PEP to enhance daily feed intake in sows was most apparent during early lactation, with PEP sows consuming 24.9% more feed per day (P < 0.01) during the first week of lactation (4.0 ± 0.3 and 5.0 ± 0.2 kg/d for Control vs. PEP sows, respectively; Figure 2). PEP sows also tended to consume more (P = 0.07) feed per day during the second week of lactation (5.9 ± 0.5 and 6.8 ± 0.3 kg/d for Control vs. PEP sows, respectively). Similar findings were reported by Cabrera et al. (2008) who obtained 5.9% higher feed intake in sows fed supplemental oregano in comparison with control sows fed commercial gestation and lactation diets. 10

Control PEP

Average Intake (Kg/day)

8

6

+15.1% P = 0.07

+8.3% P = 0.14

2

3

+24.9% P < 0.01

4

2

0 1

Week of Lactation

Figure 2. Effect of supplementation with a phytogenic feed additive (PEP) on average daily feed intake (kg/d) by week of lactation. Within week of lactation, the % difference in feed intake between treatments and corresponding P-value are provided.

Sow weight change By design, sows did not differ in initial weight (241.3 ± 10.1 and 236.4 ± 7.8 kg for Control and PEP sows, respectively; P = 0.70). All sows lost weight during the lactation period, with the majority of weight loss occurring in the first 2 weeks of lactation. This is consistent with Revell and Williams (1993) whom suggest that sows lose body reserves during the first 2 to 3 weeks of lactation to support milk production. Consistent with their enhanced feed intake, sows supplemented with PEP lost substantially less weight (P < 0.05) during

92 Enhancing feed intake in sows the first week of lactation (8.1± 1.4 vs. 3.5 ± 1.6 kg for Control vs. PEP sows, respectively; Figure 3). Furthermore, sows supplemented with PEP tended (P = 0.07) to lose less weight than Control sows over the entire lactation period (13.3 ± 2.9 vs. 7.2 ± 2.2 kg for Control and PEP sows, respectively; P = 0.07). 5.0

Control PEP Week 1

Week 2

Week 3

Overall

Weight Change (Kg)

0.0

-5.0

-10.0

P = 0.75

P = 0.34

P = 0.04

-15.0 P = 0.07 -20.0 Week of Lactation

Figure 3. The effect of a phytogenic feed additive on sow weekly weight change. Within week, the P-value for the effect of PEP supplementation is provided.

It can be speculated that an improved body condition at the end of lactation might positively affect the subsequent weaning-to-estrus interval. As reported by Kis and Bilkei (2003), supplementation of sow diets with oregano resulted in a shorter weaning-to-estrus interval and higher farrowing rate in a trial with Landrace × Duroc sows carried out in a commercial farrowing unit in Hungary.

Sow milk production and pig milk intake As expected, daily milk production by the sow was lower during early lactation (day 1-7), and increased (P < 0.01) during the second and third week of lactation. Consistent with the fact that milk production in early lactation is more reflective of body energy reserves prior to farrowing (Pluske et al., 1995), the increased feed intake seen in PEP supplemented sows was not associated (P = 0.28) with a corresponding increase in milk production during early lactation (4.5± 0.3 vs. 4.8 ± 0.2 kg/d for Control vs. PEP sows, respectively; Figure 4).

J.A. Miller et al. 93 10

+19.8% P = 0.04

Control PEP

Milk Production (Kg/d)

8

6

P = .28

4

2

0 Day 1-7

Day 1-21 Day of Lactation

Figure 4. Effect of supplementation with a phytogenic feed additive (PEP) on milk production (kg/d) by the sow during early lactation (day 1-7) and during the entire lactation period (day 1-21). Within time period, the percentage difference between treatments and P-value for the effect of PEP supplementation is provided.

However, milk production by sows supplemented with PEP increased to a greater extent in later lactation, such that over the entire lactation period PEP sows produced 19.8% more (P < 0.05) milk per day then Control sows (6.88 ± 0.55 vs. 8.24 ± 0.52 kg/d for Control vs. PEP sows, respectively). The treatment-related effect on sow milk production was not the result of differences in the number of pigs per litter, as average milk intake per pig (g/d) was 14.5% higher in pigs from sows supplemented with PEP (834 ± 34 vs. 955 ± 52 g/d for pigs from Control vs. PEP sows, respectively). These results in combination with the treatment-related differences seen in sow weight change highlight the importance of adequate energy and protein intake during early lactation in supporting milk production in later lactation (Mullan and Williams, 1989).

Piglet performance Piglet weights did not differ (P = 0.41) at birth (1.34± 0.03 vs. 1.39 ± 0.05 kg for pigs from Control vs. PEP sows, respectively; Figure 5) and, as expected, piglets gained (P < 0.01) weight throughout the lactation period. However, piglets from sows supplemented with PEP grew at a more rapid rate during week 2 and 3 of the lactation period and were 10.2% heavier (P < 0.01) by day 21 (5.8 ± 0.2 vs. 6.4 ± 0.2 kg for pigs from Control vs. PEP sows, respectively).

94 Enhancing feed intake in sows 7

+10.2% P < 0.01

Control PEP

Average Pig Weight (Kg)

6 +5.5% P = 0.19

5 4 P = 0.54 3 2

P = 0.41

1 0 1

7

14

21

Day of Age

Figure 5. Effect of supplementation of sows with a phytogenic feed additive (PEP) on average pig weight (kg). Pig weights were affected by time (P < 0.01). Within day, the percentage difference between treatments and P-value for the effect of PEP supplementation are provided.

In agreement with these results, piglets from Control and PEP supplemented sows had similar (P = 0.85) ADG during week 1 of lactation (186 ± 8 and 189 ± 9 g/d for pigs from Control and PEP sows, respectively; Figure 6). However, pigs from sows supplemented with the phytogenic feed additive tended (P = 0.09) to have increased ADG during week 2 of the lactation (218 ± 12 vs. 241 ± 13 g/d for pigs from Control vs. PEP sows, respectively), and had a 22.2% increase in ADG as compared to pigs from Control sows during week 3 of lactation (236 ± 10 vs. 289 ± 16 g/d for pigs from Control vs. PEP sows, respectively). As a result, sows supplemented with PEP had litter weights 15.8% heavier (P < 0.05) than Control sows at day 21 of the lactation period. Increased piglet weight gain was also reported by Murphy et al. (2007) in a trial with 120 Large White × Landrace sows when control lactation diets containing 100 ppm monensin sodium were supplemented with the same phytogenic additive as investigated herein. Amrik and Bilkei (2004) examined the effect of phytogenic supplementation in a commercial sows herd in Hungary and reported reduced mortality, lower annual culling rate and increased subsequent farrowing rate in sows fed oregano. Similar results were reported by Allan and Bilkei (2005) in a large-scale trial with a commercial sow herd.

J.A. Miller et al. 95 350

+22.2% P < 0.01

Control PEP

300

+10.6% P = 0.09

+16.6% P < 0.01

250 ADG (g/day)

P = 0.85 200

150

100

50

0 Week 1

Week 2

Week 3

Overall

Week of Lactation

Figure 6. Effect of supplementation of sows with a phytogenic feed additive (PEP) on pig average daily gain (ADG; g/d). Within week, the percentage difference between treatments and P-value for the effect of PEP supplementation are provided.

Conclusions Supplementation of sows with the phytogenic feed additive Biomin® P.E.P. increased daily feed intake by the sow, particularly during early lactation. The increase in feed intake was associated with less weight loss in the sow and improved milk production throughout the lactation period. As would be expected from these results, pigs from sows supplemented with the phytogenic feed additive had increased growth rates and these sows had greater 21-day litter weights. Hence, supplementation of sow lactation diets with Biomin® P.E.P. is a viable mechanism for improving piglet production.

References Allan P and Bilkei G (2005) Oregano improves reproductive performance of sows. Theriogenology 63: 716–721. Amrik B and Bilkei G (2004) Influence of farm application of oregano on performance of sows. Canadian Veterinary Journal-Revue Veterinaire Canadienne 45: 674–677. Auldist DE, Morrish L, Eason P and King RH (1998) The influence of litter size on milk production of the sows. Animal Science 67: 333–337.

96 Enhancing feed intake in sows Cabrera R; Jordan N, Wilson M, Hedges J, Knott J, Fent R, Widmer S, Tsinas A and Mellencamp MA (2008) Oregano essential oil in sow diets improves sows and piglet performance. AASV Swine Information. American Association of Swine Veterinarians. Internet: http://www.aasp.org/cdrom/ (accessed 02.03.2009). Dormad JY (1991) Effect of feeding level in the gilt during pregnancy on voluntary feed intake during lactation and changes in body composition during gestation and lactation. Livestock Production Science 27: 309–319. Kim SW and Easter RA (2001) Nutrient mobilization from body tissues as influenced by litter size in lactating sows. Journal of Animal Science 79: 2179–2186. Koketsu Y, Dial GD, Pettigrew JE, March WE and King VL (1996a) Characterization of feed intake patterns during lactation in commercial swine herds. Journal of Animal Science 74: 1202–1210. Koketsu Y, Dial GD, Pettigrew JE and King VL (1996b) Feed intake patterns during lactation and subsequent reproductive performance of sows. Journal of Animal Science 74: 2875–2884. Edwards SA (2002) Perinatal mortality in the pig: environmental or physiological solutions? Livestock Production Science 78: 3–12. Eissen JJ, Kanis E and Kemp B (2000) Sow factors affecting voluntary feed intake during lactation. Livestock Production Science 64: 147–165. Kis RK and Bilkei G (2003) Effect of phytogenic feed additive on weaning-to-estrus interval and farrowing rate in sows. Journal of Swine Health and Production 11: 296–299. Mullan BP and Williams IH (1989) The effect of body reserves at farrowing on the reproductive performance of first litter sows. Animal Production 36: 530–531. Murphy A, Moore D, Henman DJ (2007) Lactation performance of sows after enhancing their gut microflora through a dietary nutraceutical. In: Manipulating Pig Production XI. JE Paterson and JE Barker (Eds.). Proceedings of the Eleventh Biennial Conference of the Australasian Pig Science Association. Noblet J and Etienne M (1989) Estimation of sow milk nutrient output. Journal of Animal Science 67: 3352–3359. Noblet J, Dourmad JY and Etienne M (1990) Energy utilization in pregnant and lactating sows. Journal of Animal Science 68: 562–572. Revell DK and Williams IH (1993) Physiological control and manipulation of voluntary food intake. In: Manipulating Pig Production IV, ASPA, Werribee. Butterham ES (Ed.) Pp 55–80. Revell DK, Williams IH, Mullan BP, Lanford JL and Smits RJ (1998) Body composition at farrowing and nutrition during lactation affects performance of first-litter sows. II. Milk composition, milk yield and piglet growth. Journal of Animal Science 76: 1738–1744.

K.C. Mountzouris et al. 97

6 PHYTOGENIC COMPOUNDS IN BROILER NUTRITION Konstantinos C. Mountzouris, Vassilis Paraskevas and Konstantinos Fegeros Department of Nutritional Physiology and Feeding, Agricultural University of Athens, Iera Odos 75, 118 55 Athens Greece, e-mail: [email protected]

Abstract During the last decade, phytogenic compounds have attracted a lot of attention for their potential role as alternatives to antibiotic growth promoters in animal nutrition. The aim of this work is to review current scientific literature on the use of phytogenics in broiler nutrition. The efficacy of phytogenic applications in broiler nutrition depends on many factors such as composition and feed inclusion level of phytogenic preparations, bird genetics, overall diet composition and overall farm management. It is very difficult to compare different studies using phytogenics since, due to the large variation in composition, the potential biological effects of phytogenic compounds may differ. Nevertheless, a good deal of research data supports a potential role of phytogenics as natural non antibiotic growth promoters in broiler nutrition. However, the mechanisms behind growth promotion are still far from being elucidated, as data on phytogenic effects on nutrient digestibility, gut function and the immune system are still weak. In addition, despite some limited evidence that phytogenic intake could depress pathogen growth in the gut, an understanding of their effects on the complex gut ecosystem is still far from being clear. Whereas there is lack of studies describing the effects of phytogenic dietary intake on carcass meat safety, the beneficial effect of phytogenics on carcass meat quality is very well documented. Finally, in terms of this review, safety issues and further considerations on the efficient applications of phytogenic compounds in broiler nutrition are discussed.

Introduction: Phytogenics as alternatives to antibiotic growth promoters The use of antibiotics in animal nutrition, as antimicrobial growth promoters (AGP), has been without doubt beneficial for the improvement of zootechnical performance parameters and prevention of diseases. However, bio-security threats for human and animal health, arising from the escalating resistance of pathogens to antibiotics and the accumulation of antibiotic 97

98 Phytogenics in broiler nutrition residues in animal products and the environment (Burton, 2000), call for a worldwide removal AGP from animal diets. The European Union has pioneered the complete ban of all AGP since January 2006, while according to regulation EC 1831/2003 on feed additives, coccidiostats and histomonostats should be also phased out by the end of 2012. As a result, the demand for alternative products to antibiotics that can be used as prophylactic and as growth promoting agents is very high. In addition, there is a growing demand by consumers, policy makers and authorities that the food industry promptly addresses hot issues dealing with food safety, environmental pollution and animal welfare. All the above, combined with pessimistic forecasts for elevated feedingstuff prices, makes evident that in the post AGP era contemporary animal husbandry needs to evolve and adopt nutritional intervention strategies capable of optimising livestock performance and health in a cost-effective, safe and environmentally friendly manner. In this sense, the highly intensive broiler production sector of the poultry industry takes a lot of interest to optimise performance and minimise economic losses as a result of AGP removal, whilst ensuring the safety of broiler meat via the control and/or elimination of foodborne pathogens. Ongoing research highlights the beneficial potential of various microbes and bioactive compounds in enhancing animal performance and health. Examples include probiotics, prebiotics, enzymes, organic acids (acidifiers) and phytogenic compounds. The term phytogenic compounds refers to the utilised parts (e.g. seeds, fruits, roots, bark and leafs) of various aromatic herbs and spices (e.g. oregano, thyme, rosemary, coriander, cinnamon, anise, garlic, capsicum, mustard and pepper) as well as to their respective plant extracts in the form of essential oils (EO) and oleoresins (Kamel, 2000; Windisch et al., 2008). Another separate category of plant extracts mainly from fruits includes a group of natural polyphenols soluble in water called flavonoids, which have antioxidant, antimicrobial, anti-inflammatory antioestrogenic and antiproliferative activities (Lopez-Bote, 2004). Many of the purported beneficial properties of phytogenic compounds derive from their content in bioactive molecules (e.g. carvacrol, thymol, cineole, linalool, anethole, allicin, capsaicin, allyl isothiocyanate, piperine). Among the most well documented biological activities of these phytomolecules are their antibacterial and antioxidant ones (Lambert et al., 2001; Ruberto et al., 2002; Burt, 2004; Windisch et al., 2008). In addition, antiviral, antimycotic, antitoxigenic, antiparasitic and insecticidal properties have also been reported (Burt, 2004). Currently, there is a higher interest to use phytogenic EO in animal nutrition, human foods, cosmetics and pharmaceuticals, since they have a much higher biological activity compared to the raw material they were extracted from. Examples of aromatic plants and their composition in major active components are shown in Table 1. As described in Chapter 1, EO are very complex mixtures of plant bioactive compounds with variable chemical composition and concentration. EO consist basically of two classes of compounds, the terpenes and the phenylpropenes. Terpenes can be subdivided into mono-, sesqui- and di-terpenes, depending on the number of a 5-carbon building block (i.e. isoprene) units (i.e. 2, 3 and 4 respectively). Examples of important monoterpenes include carvacrol and thymol. On the other hand, phenylpropenes consist of a 6-carbon aromatic ring with a 3-carbon side chain. Important phenylpropenes include trans-cinnamaldehyde, eugenol, capsaicin and piperine (Lee et al., 2004a).

K.C. Mountzouris et al. 99 Table 1. Examples of aromatic plants and their composition in major bioactive components (data from Burt et al., 2004)

Major essential oil components Linalool

Composition (% of total volatiles) 70%

Trans-cinnamaldeheyde

65%

carvacrol thymol γ-terpinene p-cymene

trace–80% trace–64% 2–52% trace–52%

α-pinene camfor 1,8- cineole

2–25% 0–17% 2–14% 3–89%

Sage Salvia officinalis

camfor α-pinene β-pinene 1,8- cineole a-tujone

6–15% 4–5% 2–10% 6–14% 20–42%

Thyme Thymus vulgaris

thymol carvacrol γ-terpinene p-cymene

10–64% 2–11% 2–31% 10–56%

Plant common and Latin name from which the essential oil is extracted Coriander (seeds) Coriandrum sativum Cinnamon Cinnamomum zeylandicum Oregano Origanum vulgare

Rosemary Rosmarinus officinalis

bornyl acetate

The content and composition of EO depend on the interaction of many factors that deal mainly with the plant raw material and the EO production process. For example, factors such as the plant species and growth stage, the environment (e.g. harvest season, climate, stress conditions), the agricultural practices (e.g. plant density per cultivated area, fertilisation, irrigation level) and the geography will affect the EO content and composition of the plant raw material (Kokkini et al., 1997; Mert et al., 2002; Daferera et al., 2003). As a result, the plant raw materials for EO production vary considerably and the same applies to the resulting EO products (Russo et al., 1998). In addition, the process methods applied in EO production (e.g. hydrodistillation, solvent extraction) can also have a significant effect on the amount and composition of the extracted oil (Tarantilis and Polissiou, 1997). In recent years, phytogenic compounds have attracted a lot of attention for their potential role as alternatives to antibiotic growth promoters in animal nutrition. According to EC regulation 1831/2003 on feed additives in animal nutrition, phytogenic compounds have been categorised as “sensory additives” and in particular as flavouring compounds, i.e. substances the inclusion of which in feedingstuffs increases feed smell and palatability.

100 Phytogenics in broiler nutrition During the last decade, phytogenic compounds have started to become popular as feed additives in broiler nutrition. The aim of this work is to review current scientific literature on the use of phytogenics in broiler nutrition. Their effects on broiler performance, nutrient digestibility, gut microbiology, carcass meat safety and quality properties as measures of their efficacy in broiler nutrition will be examined and assessed. Finally, further considerations for efficient applications of phytogenic compounds in broiler nutrition will be discussed.

Commonly researched phytogenics A representative part of the current literature on phytogenic applications in broiler nutrition shows that oregano, thyme, rosemary, sage, anise, cinnamon and pepper are listed among the most commonly researched phytogenic compounds in broiler nutrition, not only in terms of aromatic plants or their respective EO extracts (Botsoglou et al., 2002; Giannenas et al., 2003; Horosova et al., 2006; Cross et al., 2007), but also as blended combinations of multiple phytogenic compounds (Lee et al., 2003, 2004a, 2004b and 2004c; Hernandez et al., 2004; Mitsch et al., 2004; Jamroz et al., 2005). The thematic areas and some representative references reflecting current research on the efficacy of phytogenic applications in broiler nutrition is given in Table 2.

Table 2. The thematic areas and some representative references reflecting current research on the efficacy of phytogenic applications in broiler nutrition

Phytogenic effects on

Relevant references

Feed palatability and quality

Lambert et al. (2001); Soliman and Badeaa (2002); Burt (2004); Windisch et al. (2008)

Growth parameters

Botsoglou et al. (2002); Giannenas et al. (2003); Lee et al. (2003, 2004b, 2004c); Hernandez et al. (2004); Ciftci et al. (2005); Jamroz et al. (2005); Spernakova et al. (2007); Cross et al. (2007); Soltan et al. (2008)

Gut function and nutrient digestibility

Lee et al. (2003, 2004b, 2004c); Hernandez et al. (2004); Jamroz et al. (2005); Jamroz et al. (2006); Cross et al. (2007)

Gut microflora

Lambert et al. (2001); Giannenas et al. (2003); Mitsch et al. (2004); Burt (2004); Chorianopoulos et al. (2004); Penalver et al. (2005); Jamroz et al. (2005); Si et al. (2006); Horosova et al. (2006)

Immune function

Soltan et al. (2008); Windisch et al. (2008)

Carcass meat safety and quality

Lopez-Bote et al. (1998); Botsoglou et al. (2002); Young et al. (2003); Govaris et al. (2005); Gulmez et al. (2006); Spernakova et al. (2007); Govaris et al. (2007)

K.C. Mountzouris et al. 101

Feed inclusion levels of phytogenics A wide range of phytogenic feed inclusion levels have been reported. Depending on whether aromatic plants or their respective EO are used, up to ten fold differences in feed inclusion levels have been found (Cross et al., 2007). In particular, when aromatic plant parts were used, feed inclusion levels ranged from (0.01–30 g/kg diet). Examples include oregano addition at 30 g/kg feed (Young et al., 2003) or 10 g/kg feed (Cross et al., 2007), rosemary at 5–10 g/kg feed (Govaris et al., 2007; Cross et al., 2007), marjoram, rosemary and yarrow at 10 g/kg feed (Cross et al., 2007), rosemary powder at 0.5 g/kg feed (Spernakova et al., 2007) and anise seeds at 0.25–1.5 g/kg diet (Soltan et al., 2008). Lower feed inclusion levels than the above have been reported for EO. Examples include: rosemary and sage extracts at 500 mg/kg of feed (Lopez Bote et al., 1998), oregano EO at 50–100 mg/kg of feed (Botsoglou et al., 2002; Govaris et al., 2005) or 300 mg/kg feed (Giannenas et al., 2003), thymol and cinnamaldehyde at 100 mg/kg feed (Lee et al., 2003), anise oil at 100–400 mg/ kg feed (Ciftci et al., 2005) and essential oils from other herbs such as thyme, marjoram, rosemary and yarrow at 1000 mg/kg feed (Cross et al., 2007). It should be noted that the above inclusion levels should be considered as indicative only, since actual plant or EO composition in active components may vary a lot between different studies.

Feed palatability and quality While there is not enough evidence to adequately support palatability enhancement from phytogenic inclusion (Windisch et al., 2008), their use might improve feed quality via their antioxidative and antibacterial properties (attributed to phenolic compounds such as the rosmarinic acid, carvacrol and thymol) and their potential to depress the growth of mycotoxigenic fungi (Lambert et al., 2001; Soliman and Badeaa, 2002; Burt, 2004). Antioxidant properties could be of great value when the feed contains a high proportion of poly-unsaturated versus saturated fatty acids. The antimycotic properties of EO could be important in preventing mycotoxin production in stored wheat grains. Thyme, anise and cinnamon EO were found to be highly inhibitory against the growth of toxigenic fungi, such as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium moniliforme, and the production of their mycotoxins (Soliman and Badeaa, 2002).

Effects of phytogenics in broilers Effects on growth parameters Broiler body weights (BW), body weight growth (BWG), feed intake (FI) and feed conversion ratio (FCR) are among the growth parameters studied. The use of oregano EO at 50 and 100 mg/kg wheat-soybean meal (SBM) basal diet fed to Cobb broilers had

102 Phytogenics in broiler nutrition no effect on overall BW and FCR that did not differ from the un-supplemented control treatment and from a treatment supplemented with 200 mg α-tocopheryl acetate (Botsoglou et al., 2002). No significant effects on FI, BWG and FCR were found, when phytogenic compounds (i.e. thymol, cinnamaldehyde and commercial preparation) were added at 100 mg/kg diet based on corn-SBM for female Cobb broilers (Lee et al., 2003). When carboxylmethyl cellulose (CMC) was added in the corn-SBM diet as a means to increase intestinal viscocity, the addition of cinnamaldehyde or the commercial preparation partly counteracted the negative effect of CMC on broiler BWG during the first 21 days of age (Lee et al., 2004b). However, when basal diets based in rye instead of corn were used, the rye induced suppression of weight gain between 1 and 14 days of age was partially overcome by the addition of cinnamaldehyde (Lee et al., 2004c). Oregano essential oil supplementation at 300 mg/kg wheat-SBM basal diet fed to Cobb birds that were infected at 14 days of age with Eimeria tenella, resulted in a significantly better BWG and FCR compared to the infected non-supplemented control treatment. In addition, performance levels were comparable to the non-infected control treatment but significantly lower compared to the respective performance levels of a coccidiostat (i.e. lasalocid) supplemented infected group (Giannenas et al., 2003). The supplementation of two commercial 3-component mixtures (i.e. one consisting of oregano, cinnamon and pepper and the other consisting of sage, thyme and rosemary) phytogenic EO products in wheat-corn-SBM basal diet at 200 mg/ kg and 5000 mg/kg levels, did not improve overall BWG, FI and FCR compared to the unsupplemented control or an avilamycin treatment (Hernandez et al., 2004). On the contrary, anise EO supplementation in basal diets at 400 mg/kg diet, fed to Ross broilers, resulted in a significant improvement in BWG and FCR throughout the experiment compared to the control and lower inclusion levels of anise oil (i.e. 100 and 200 mg/kg). Interestingly, the improvement in growth parameters was even better compared to a treatment having avilamycin used as an AGP at 10 mg/kg diet (Ciftci et al., 2005). Supplementation of corn-SBM or wheat-barley-SBM diets fed to male Hubbard Hi-Ye broilers with 100 mg/kg of a plant extract containing carvacrol, cinnamaldehyde and capsicum oleoresin significantly improved FCR by 3.9% on the maize diet (Jamroz et al., 2005). The addition of rosemary powder at 500 mg/kg diet resulted in higher BWG compared to the control treatment (Spernakova et al., 2007). Feed inclusion of five herbs (i.e. thyme, oregano, marjoram, rosemary or yarrow) or their associated EO at 10 g/kg and 1 g/kg respectively, in a wheat-SBM diet for female Ross broilers had variable effects on chick performance. In particular, the oregano herb and oil treatments had reduced average BWG and FI values and were ranked among the poorest in terms of performance. On the contrary the thyme oil treatment performed best. In addition, from the herbs tested, the yarrow herb treatment performed best (Cross et al., 2007). The inclusion of anise seeds at 0.5–0.75 g/kg cornSBM diet administered to Hubbard broilers for 6 weeks, improved BWG, performance index and relative growth rate of broilers, while there were no significant effects on FI and FCR when compared to the control. The highest inclusion level of anise seeds (1.5 g /kg diet) reduced growth performance (Soltan et al., 2008).

K.C. Mountzouris et al. 103

Effects on gut function and nutrient digestibility Among the mechanisms purported to influence gut function are effects on gut transit time, digestive secretions and enhancement of digestive enzyme activities. In turn, the combination of all these effects will impact nutrient digestibility. A wide range of dietary spices (e.g. curcumin, capsaicin, ginger and piperine) were found to stimulate pancreatic digestive enzymes (Platel and Srinivasan, 2000) and shorten the feed transit time in adult female rats (Platel and Srinivasan, 2001). In broilers phytogenic compounds were shown to enhance the intestinal activities of trypsin, lipase and amylase (Lee et al., 2003; Jamroz et al., 2005). The addition of plant extracts to feed mixtures for 41 d old broiler chicken enhanced lipase activity by 38–46% (Jamroz et al., 2005). Phytogenic compounds enhanced mucus production and thickness in the stomach and jejunum suggesting a potential protective effect against colonisation by gut pathogens (Jamroz et al., 2006). In addition, potential effects of phytogenics on gut morphological characteristics have been reported (Jamroz et al., 2006). In an experiment with female Cobb broilers fed a corn-SBM basal diet supplemented with thyme, cinnamaldehyde or a commercial EO preparation at 100 mg/kg diet, there were no differences between the non-supplemented control and the phytogenic treatments regarding the apparent ileal digestibilities of crude protein (CP) and starch as well as for the total tract fat digestibility at the age of 21 and 40 days (Lee et al., 2003). Similarly, the apparent ileal digestibilities of crude protein and starch were not affected even when CMC was added in the diet as a means to increase intestinal viscocity. While the CMC addition reduced the total tract fat digestibility, possibly via reduced availability of bile salts, cinnamaldehyde or the commercial EO treatments partly reversed this negative effect, possibly due to stimulation of bile secretion (Lee et al., 2004b). On the contrary, in a rye based diet, none of the phytogenics above reversed the reduction in total tract apparent fat digestibility (Lee et al., 2004c). In a wheat-corn-SBM basal diet, fed to Ross male broilers, inclusion of two 3 component mix commercial EO products improved the ileal digestibility of dry matter (DM) and starch and the total tract apparent digestibility of DM, CP and fat (Hernandez et al., 2004) compared to the un-supplemented control. Interestingly, the improvement in the digestibility coefficients of the nutrient components above did not differ from the respective ones of the avilamycin treatment used as AGP. Feed inclusion of a plant extract containing carvacrol, cinnamaldehyde and capsicum oleoresin in cornSBM or wheat-barley-SBM diets fed to male Hubbard Hi-Ye broilers did not significantly increase the apparent ileal digestibility of nutrients (i.e. CP, crude fibre and amino acids) compared to the un-supplemented controls (Jamroz et al., 2005). In the study by Cross et al. (2007), none of the five herb or their respective EO treatments tested had an effect on the apparent metabolisable energy and the total tract apparent digestibility coefficients of dry and organic matter. In addition, none of the dietary treatments affected the intestinal endogenous losses determined by the concentration of sialic acid in the excreta. Mountzouris et al. (2008) reported that supplementation of corn-SBM diets with a blend of essential oils derived from oregano, anise and citrus at 125 mg/kg diet increased ileal apparent fat digestibility in male Cobb broilers.

104 Phytogenics in broiler nutrition Effects on gut microflora The antimicrobial activity of phytogenic compounds is perhaps their most well documented property. Various phytogenic compounds have been shown to be active against a wide range of foodborne pathogens such as Listeria monocytogenes, Salmonella typhimurium, S. enteritidis, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus (Lambert et al., 2001; Burt, 2004; Chorianopoulos et al., 2004; Penalver et al., 2005; Si et al., 2006). The antibacterial properties of EO could be attributed mainly to their phenolic components (Burt 2004; Penalver et al., 2005; Si et al., 2006). It has therefore been suggested that their mechanism of action would be similar to other phenolics, generally considered to create disturbance of the cytoplasmic membrane, disruption of the proton motive force, electron flow and active transport and coagulation of cell contents (Lambert et al., 2001; Burt, 2004). In addition, an anticoccidial effect of oregano EO against Eimeria tenella infection has been reported (Giannenas et al., 2003). Data from six field trials utilising Ross broilers fed commercial corn-based diets have shown that specific blends of EO could control the proliferation of Clostridium perfringens, considered as the main causative agent of necrotic enteritis in the broiler gut (Mitsch et al., 2004). It is generally accepted that most EO have a slightly higher antibacterial activity against Gram positive bacteria compared to Gram negative ones (Burt, 2004). To what extent such an antibacterial activity could have a negative impact on the beneficial gut microflora, apart from pathogens such as C. perfringens, remains an open question. A strong bactericidal effect of very high oregano EO test levels was noted against five lactobacilli strains isolated from chicken excreta (Horosova et al., 2006). However, other studies indicate that it is possible to select EO compounds with a strong antimicrobial action against gut pathogens whilst not harming beneficial bacteria such as bifidobacteria and lactobacilli (Si et al., 2006). Addition of a commercial EO blend in corn-SBM diet reduced E.coli and increased Lactobacillus spp. levels in the small intestine of broilers (Jamroz et al., 2005). Similarly, a reduction in E. coli concentration combined with a reduction of C. perfringens levels was seen. In this case, however, Lactobacillus levels were also reduced in the intestine of 41-d old broilers (Jamroz et al., 2005). In the study by Cross et al. (2007) none of the five herbs or the five respective EO tested had a significant effect on the caecal and excreta populations of coliforms, lactic acid bacteria, total anaerobes and C. perfringens. Similarly, the addition of a commercial phytogenic feed additive in piglets did not have an effect on intestinal and fecal microflora composition (Muhl and Liebert, 2007). Feed inclusion of a specific commercial EO blend derived from oregano, anise and citrus at 125 and 250 mg/ kg diet in corn-SBM basal diet containing no coccidiostats resulted in changes in broiler gut microflora composition revealed by the increased caecal levels of bifidobacteria and lactobacilli (Mountzouris et al., unpublished data).

Effects on immune function Anise seed supplementation resulted in improved blood parameters, while a non specific immunostimulatory effect was postulated by the increased phagocytic activity and

K.C. Mountzouris et al. 105

lymphocyte number (Soltan et al., 2008). However, there is still an obvious lack of studies regarding the immuno modulatory potential of phytogenic feed additives (Windisch et al., 2008; Applegate, Chapter 3).

Effects on carcass meat safety and quality As it was mentioned earlier, there is an extensive list of studies supporting a clear in vitro antimicrobial activity of many extracts against a range of gut and food-borne pathogens. It is also known that addition of phytogenic compounds (e.g. herbs, spices or EO) in foods of animal origin contributes to food microbiological safety and quality upon food storage in raw or cooked stage, via their antimicrobial and antioxidant properties (Ruberto et al., 2002; Chouliara et al., 2007; Georgantelis et al., 2007a and 2007b; Fasseas et al., 2008). Therefore, in principle, the dietary intake of phytogenic feed additives could contribute to food safety in a couple of ways. The first would be through the reduction of pathogens in the gut, thus promotion of a healthy gut environment, which in turn could contribute to reduction of carcass contamination at slaughter. According to the European Food Safety Authority (EFSA) this should be considered as one of the most effective ways of reducing the contamination of foodstuffs and the subsequent number of foodborne illnesses in humans. In addition to this, phytogenic compound applications for decontamination of poultry carcasses have been reported (Gulmez et al., 2006). The second way would be via the growth inhibition of spoilage or pathogenic bacteria as a result of potential accumulation of EO active components or metabolites in tissues. In this sense, incorporation of oregano EO in the diet at 100 mg/kg level exerted an inhibitory effect on microbial growth, determined as total viable counts and Pseudomonas spp counts, on turkey breast fillets during refrigerated storage for 12 days (Govaris et al., 2005). From a quality point of view, the dietary intake of phytogenic compounds has been reported to have a beneficial effect on stored meat quality, an effect related to phytogenic antioxidant properties, in terms of reducing or delaying lipid oxidation. Examples of phytogenic compounds tested in this respect include: rosemary and sage extracts (Lopez-Bote et al., 1998), oregano oil (Botsoglou et al., 2002; Govaris et al., 2005), oregano (Young et al., 2003), rosemary (Govaris et al., 2007) and rosemary powder (Spernakova et al., 2007). In terms of carcass yield there are no indications of significant beneficial effects of phytogenic feed additives. Plant extract addition enhanced the breast muscle proportion of the eviscerated carcass by only 1.2% in comparison to the control birds (Jamroz et al., 2005), while supplementation of anise seeds did not improve the carcass dressing percentage (Soltan et al., 2008).

Safety issues Most of the phytogenic compounds have a long association with human history as part of our foods and medicines and therefore could be regarded as generally safe. In terms of handing however, due to their high activity, strong odour and irritating potential that can cause allergic contact dermatitis (Burt, 2004), appropriate caution measures should

106 Phytogenics in broiler nutrition always be taken. Although toxicological studies have been contacted for a number of phytogenic active components (reviewed by Lee et al., 2004a), it is recommended that before phytogenic usage levels increase, potential toxicity issues to be further addressed (Burt, 2004). Despite the fact that accumulation of essential oils in the body is unlikely due to their fast metabolic conversion and excretion, the continuous intake in chickens may result to deposition of phytogenic constituents in various tissues (Lee et al., 2004a), which needs further investigation. Concerns have been raised whether phytogenic extensive feeding could cause a selective pressure on gut microflora similar to antibiotic feeding that need to be addressed. For example E. coli chicken isolates from broilers fed an oregano EO for 36 d at high inclusion levels of 480–1200 mg/kg diet were found to have significantly increased minimum inhibitory concentration (MIC) values for amikacin, apramycin, streptomycin and neomycin compared with the control (Horosova et al., 2006). Similar concerns for microbial selective pressure have been expressed for the use of organic acids (acidifiers) in animal nutrition and meat processing (Theron and Lues, 2007).

Conclusions and further considerations The effect of supplementation of feed with phytogenics on broiler growth parameters is the most well studied parameter. Generally, the studies reviewed herein support the notion that phytogenic compounds could serve as natural non-antibiotic growth promoters in broiler nutrition. However, evidence of beneficial effects in nutrient digestibility and/or gut function and microflora composition in broilers is still rather weak. Regarding the latter, there is some limited evidence that intake of phytogenics could depress pathogen growth in the gut. However, there is clearly the need to further research of phytogenic effects on broiler gut microflora. In addition, there is a clear lack of studies regarding the immuno modulatory potential of phytogenic components. Whereas the antimicrobial effect of phytogenic components added to foods is well documented, there is still lack of studies regarding effects of phytogenic dietary intake on carcass meat safety in terms of growth inhibition of spoilage and pathogenic microorganisms. On the contrary, the beneficial effect of dietary intake of phytogenics on carcass meat quality is very well documented. Since the continuous phytogenic intake in chickens may result to deposition of phytogenic constituents in various tissues (Lee et al., 2004a), the effects of such an accumulation on the sensory properties of chicken meat products should be assessed. As in the case of other bioactive compounds (e.g. enzymes, organic acids, prebiotics) and microorganisms (e.g. probiotics) used as feed additives, it is very difficult to directly assess different studies using phytogenics. The efficacy of phytogenic applications in poultry depends on many factors. Differences in phytogenic composition and phytogenic feed inclusion level, bird genetics, overall diet composition and overall farm management could be considered among the most important ones. In addition, storage and stability (e.g. oxidation, volatility) issues of phytogenics in feed still remain to be addressed.

K.C. Mountzouris et al. 107

It could be suggested that in order to better exploit the antimicrobial potential of phytogenics in feed, one would need to consider increasing feed inclusion levels to exceed the relevant MIC (Burt, 2004). However, in practical terms this is not currently possible since the MIC values of phytogenic compounds obtained in vitro correspond to levels that considerably exceed dietary doses of phytogenic compounds (Windisch et al., 2008). In addition, as parts of the active substances are highly odorous or may taste hot or pungent, high inclusion levels may result in feed refusal and, therefore, restrict their use in animal feeding programs (Windisch et al., 2008). It is known that, due to the large variation in composition, the potential biological effects of EO may differ. In order to face the uncertainty stemming from the high variability, an alternative approach to the use of undefined or partially defined phytogenic compounds could be the use of proprietary phytogenic products with standardised compositions and quality control. However, depending on each individual application an optimisation study regarding feed inclusion levels should be carried out since, apart from the phytogenic EO composition, broiler response to phytogenics could be dose-related (Zhang et al., 2005; Soltan et al., 2008). It is clear that a prerequisite for the design of highly efficacious phytogenic products is the advancement of our knowledge and understanding of the complex poultry gut ecosystem in order to be able to fully explore the precise mode(s) of action of phytogenic compounds.

References Bampidis VA, Christodoulou V, Florou Paneri P, Christaki E, Chatzopoulou PS, Tsiligianni T and Spais AB (2005) Effect of dietary dried oregano leaves on growth performance, carcase characteristics and serum cholesterol of female early maturing turkeys. British Poultry Science 5: 595–601. Botsoglou NA, Florou Paneri P, Christaki E, Fletouris DJ and Spais AB (2002) Effect of dietary oregano essential oil on lipid oxidation in raw and cooked chicken during refrigerated storage. Meat Science 62: 259–265. Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods - a review. International Journal of Food Microbiology 94:: 223–253. Chorianopoulos N, Kalpoutzakis E, Aligiannis N, Mitaku S, Nychas GJ and Haroutounian SA (2004) Essential oils of Satureja, Origanum, and Thymus species: chemical composition and antibacterial activities against food borne pathogens. Journal of Agricultural and Food Chemistry 52: 8261–8267. Ciftci M., Guler T, Dalkilic B and Ertas ON (2005) The effect of anise oil (Pimpinella anisum) on broiler performance. International Journal Poultry Science 4: 851–855. Cross DE, Mcdevith RM, Hillman K and Agamovic T (2007) The effect of herbs and their associated essential oils on performance, digestibilities and gut microflora in chickens 7 to 28d of age. British Poultry Science 4: 496–506. Chouliara E, Karatapanis A, Savvaidis IN and Kontominas MG (2007) Combine effect of oregano essential oil and modified atmosphere packaging on shelf life extension of

108 Phytogenics in broiler nutrition fresh chicken breast meat stored at 4°C. Food Microbiology 24: 607–617. Chun SS, Vattem DA, Yuang TL and Sety K (2005) Phenolic antioxidants from clonal oregano (Origanum Vulgare) with antimicrobial activity on Helicobacter Pylori. Process Biochemistry 40: 809–816. Daferera J D, Ziogas, NB and Polissiou MG (2003) The Effectiveness of plant essential oils on Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis. Crop Protection 22: 39–44. Fasseas MK, Mountzouris KC, Tarantilis PA, Polissiou M and Zervas G (2008) Antioxidant activity in meat treated with oregano and sage essential oils. Food Chemistry 106: 1188–1194. Georgantelis D, Blekas G, Katikou P, Ambrosiadis I and Fletouris DJ (2007a) Effect of rosemary extract, chitosanand a-tocopherol on lipid oxidation and colour stability during frozen storage of beef burgers. Meat Science 75: 256–264. Georgantelis D, Ambrosiadis I, Katikou P, Blekas G and Georgakis SA (2007b) Effect of rosemary extract, chitosanand a-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4°C. Meat Science 76: 172–181. Giannenas I, Florou Paneri P, Papazahariadou M, Christaki E, Botsoglou NA and Spais AB (2003) Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition 57: 99–106. Govaris A, Botsoglou E, Florou Paneri P, Moutas A and Papageorgiou G (2005) Dietary supplementation of oregano essential oil and α–tocopheryl acetate on microbial growth and lipid oxidation of turkey breast fillets during storage. International Journal of Poultry science 4: 969–975. Govaris A, Florou-Paneri P, Botsoglou E, Giannenas I, Ambrosiadis I and Botsoglou N (2007) The inhibitory potential of feed supplementation with rosemary and/or α-tocopheryl acetate on microbial growth and lipid oxidation of turkey breast during refrigerated storage. LWT - Food Science and Technology 40: 331–337. Gulmez M, Oral N and Vatansever L (2006) The effect of water extract of Sumac (Rhus coriaria L) and lactic acid on decontamination and shelf life of raw broiler wings. Poultry Science 85: 1466–1471. Hernandez F, Madrid J, Garcia V, Orengo J and Megias MD (2004) Influence of two plant extracts on broiler performance digestibilities and digestive organ size. Poultry Science 83: 169–174. Horosova K, Bujnakova and Kmet V (2006) Effect of oregano essential oil on chicken lactobacilli and E. coli. Folia Microbiology 51: 278–280. Jamroz D, Wiliczkiewicz A, Wertelecki T, Orda J and Scorupinska J (2005) Use of active substances of plant origin in chicken diets based on maize and domestic grains. British Poultry Science 46: 485–493. Jamroz D, Wertelecki T, Houszka M and Kamel C (2006) Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal

K.C. Mountzouris et al. 109

Physiology and Animal Nutrition 90: 255–268. Kamel C (2000) A novel look at a classic approach of plant extracts. Feed Mix 11: 19–21. Kokkini S, Karousou R, Dardiotis A, Krigas N and Lanaras T (1997) Autumn essential oils of greek oregano. Phytochemistry 44: 883–886. Lambert RJW, Skandamis PN, Coote PJ and Nychas GJE (2001) A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology 91: 453–462. Lee KW, Kappert HJ, Frehner M, Losa R and Beynen AC (2003) Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broiler chickens. British Poultry Science 44: 450–457. Lee KW, Everts H, Kappert HJ, Wouterse H, Frehner M and Beynen AC (2004b) Cinnamaldeheyde but not thymol, counteracts the carboxymethyl cellulose induced growth depression in female broiler chickens. International Journal Poultry Science 3: 608–612. Lee KW, Everts H, Kappert HJ, Van der Kuillen J, Gemmens AG, Frehner M and Beynen AC (2004c) Growth performance, intestinal viscosity, fat digestibility and plasma cholesterol in broiler chickens fed a rye-containing diet without or with essential oil components. International Journal of Poultry Science 3: 613–618. Lee KW, Everts H, and Beynen AC (2004a) Essential oils in broiler nutrition. International Journal of Poultry Science 3: 738–752. Lopez-Bote CJ, Gray JI, Gomaa EA and Flegal CJ (1998) Effect of dietary administration of oil extracts from rosemary and sage on lipid oxidation in broiler meat. British Poultry Science 39: 235–240. Lopez-Bote CJ (2004) Bioflavonoids’ effects reach beyond productivity Feed Mix 12: 12–15. Mert A, Kirici S and Ayanoglu F (2002) The effects of different plant densities on yield, yield components and quality of Atremisia annua L. ecotypes. Journal of Herbs, Spices & Medicinal Plants 9: 413–418. Mitsch P, Zitterl-Eglseen K, Kohler B, Gabler C, Losa R and Zimpernik I (2004) The effect of two different blends of essential oil components on the proliferation of Clostridium perfigrens in broiler chicks. Poultry science 83: 669–675. Mountzouris KC, Tsirtsikos P, Paraskevas V and Fegeros K (2008) Evaluation of the effect of a phytogenic essential oils product on broiler performance and nutrient digestibility. In: World’s Poultry Congress, August 10–15, 2008, Brisbane, Australia. P 444. Muhl A and Liebert F (2007) Growth and parameters of microflora in intestinal and faecal samples of piglets due to application of a phytogenic feed additive. Journal of Animal Physiology and Animal Nutrition 91: 411–418. Penalver P, Huerta B, Borge C, Astorga R, Romero R and Perea R (2005) Antimicrobial activity of five essential oil against animal origin strains of the Enterobacteriaceae family. Acta Pathologica et Immunologica Scandinavica 113: 1–6. Platel K and Srinivasan K (2000) Influence of dietary spices and their active principles on

110 Phytogenics in broiler nutrition pancreatic digestive enzymes in albino rats. Nahrung 44: 42–46. Platel K and Srinivasan K (2001) Studies on the influence of dietary spieces on food transit time in experimental rats. Nutrition Research 21: 1309–1314. Ruberto G, Barrata MT, Sari M and Kaabehe M (2002) Chemical composition and antioxidant activity of essential oils from Algerian Origanum Glandulosum Desf. Flavour and Fragrance Journal 17: 251–254. Russo M, Galletti G, Bocchini P and Carnacini A. (1998) Essential oil chemical composition of wild populations of italian oregano spice (Origanum vulgare ssp. hirtum (Link) Ietswaart): A preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. Journal of Agriculture and Food Chemistry 46: 3741– 3746. Spernakova D, Mate D, Rozanska H and Kovac G (2007) Effects of dietary rosemary extract and a-tocopherol on the performance of chickens, meat quality, and lipid oxidation in meat storage under chilling conditions. Bulletin of the Veterinary Institute in Pulawy 51: 585–589. Soliman KM and Badeaa RI (2002) Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food and Chemical Toxicology 40: 1669–1675. Soltan MA, Shewita RS and El-Katcha MI (2008) Effects of diary anise seeds supplementation on growth performance, immune response, carcass traits and some blood parameters of broiler chickens. International Journal of Poultry Science 7: 1078–1088. Si W, Gong J, Tsao R, Zhou, Yu H, Poppe C, Johnson R and Du Z (2006) Antimicrobial activity of essential oils and structally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology 100: 296–305. Tarantilis PA and Polissiou MG (1997) Isolation and Identification of the Aroma Components from Saffron (Crocus sativus). Journal of Agriculture and Food Chemistry 45: 459–462. Theron MM and Lues JFR (2007) Organic acids and food preservation: A review. Food Reviews International 23: 141–158. Van den Bogaard AE and Stobberingh EE (2000) Epidemiology of resistance to antibiotics. Links between animals and humans. International Journal of Antimicrobial Agents 14: 327–335. Wenk C (2003) Herbs and botanicals as feed additives in monogastric animals. AsianAustralasian Journal of Animal Sciences 16: 282–289. Windisch W, Schedle K, Plitzner C and Kroismayr A (2008) Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86: 140–148. Young JF, Stagsted J, Jensen SK, Karlsson AH and Heckel P (2003) Ascorbic acid a-tocopherol and oregano supplements reduce stress induced deterioration of chicken meat quality. Poultry Science 82: 1343–1351.

C. Benchaar et al. 111

7 ESSENTIAL OILS AS FEED ADDITIVES IN RUMINANT NUTRITION Chaouki Benchaar1, Alexander N. Hristov2 and Henry Greathead3 1 Agricuture and Agri-Food Canada, Dairy and Swine Research and Development Centre, 2000 College Street - P.O. Box 90, STN-Lennoxville, Sherbrooke, Quebec, Canada, J1M 1Z3, e-mail [email protected]; 2Pennsylvania State University, Department of Dairy and Animal Science, 352 Agricultural Sciences and Industries Building, University Park, PA16802, USA; 3University of Leeds, Faculty of Biological Sciences, Leeds, LS2 9JT, UK

Abstract Over the last few years, the use of plant bioactive compounds for animal health and productivity has been of increasing research interest. This recent surge of interest has been stimulated by the search for alternatives to growth-promoting antibiotics in livestock production. Public concern over the routine use of antibiotics in animal feed has increased in recent years because of their possible contribution to emergence of antibiotic resistant bacteria. Plants produce an array of diverse secondary metabolites such as essential oils that, when extracted and concentrated, may exert antimicrobial activities against a wide variety of microorganisms including bacteria, protozoa, fungi and viruses. Accordingly, considerable research effort has been devoted towards exploiting the antimicrobial properties of essential oils to manipulate rumen microbial fermentation in order to improve nutrient utilization in the animal and reduce the environmental impact of livestock production systems. Most of the studies conducted to date have been laboratory based (i.e. in vitro) and of short-term nature. Results from in vitro batch and continuous culture studies showed that at high doses, essential oils reduced ammonia nitrogen concentration and methane production, but in many cases this was associated with a decrease in total volatile fatty acid concentration and diet fermentability. Evidence for in vivo antimicrobial activity of essential oils is equivocal and in most studies conducted to date, no beneficial effects on nutrient utilisation and animal performance (milk and meat) have been observed. Literature data suggest that rumen microbial populations may adapt when exposed continually to essential oils. Such a response represents a major challenge for commercial application of this feed additive technology. More in vivo research is required to fully assess the potential use of essential oils as feed additives in ruminant nutrition. 111

112 Essential oils as feed additives in ruminants

Introduction In livestock production, antibiotics (e.g. ionophores) at sub-therapeutic levels are commonly used to improve the efficiency of converting feeds to gain (milk and meat) and/or to prevent disease and metabolic disorders. Ionophore antibiotics such as monensin have been the most effective manipulators of rumen microbial populations and fermentation in practical feeding conditions. Typically, effects include decreased rumen ammonia and increased propionate concentrations, reduced dry matter intake and improved feed efficiency (Nagaraja et al., 1997). A recent metaanalysis reported improvement in energy metabolism (Duffield et al., 2008a), decreased dry matter intake, increased milk yield, improved milk production efficiency, increased body condition score and increased conjugated linoleic acid content of milk fat (Duffield et al., 2008b) in dairy cows supplemented with monensin. Over all the trials analysed, monensin decreased the risk of ketosis, displaced abomasums and mastitis (Duffield et al., 2008c). Despite these positive effects on animal productivity and health, the use of feed antibiotics in animal nutrition has become increasingly controversial because of the emergence of multi-drug resistant bacteria that may pose a risk to human health. Consequently, in recent years, public pressure to restrict or even ban the widespread use of antibiotics in animal nutrition has increased. It is likely that such public pressure would eventually force producers to produce milk and meat with less or no antibiotic feed supplements in animal diets. For example, a recent report of the Pew Commission on Industrial Farm Animal Production in the United States (PCIFAP, 2008) recommended restricting the use of antimicrobials in food animal production (to reduce the risk of antimicrobial resistance to medically most-used antibiotics). Plant natural products are potential alternatives to antibiotic feed additives and it is hoped they may permit a reduction in the use of synthetic antimicrobial drugs. Therefore, the interest in the medicinal properties of natural products (essential oils, herbs, spices, botanicals) as animal feed supplements with the potential of improving animal productivity and health and to mitigate the environmental impact of animal feeding operations has dramatically increased in recent years. A search in the CAB (Commonwealth Agricultural Bureau) International database (CABI, 2008; animal sciences subject area) with essential oil as a keyword returned 345 references between 1970 and 1990 and 3174 references since 1991. Research has been particularly intensive in Europe after the European Union ban in January 2006 of the use of antibiotics in animal feed (OJEU, 2003). This chapter presents the current knowledge on the use of plant-derived essential oils as feed additives in ruminant nutrition. Mechanisms of action, effects on rumen microbial fermentation (protein metabolism, volatile fatty acid production, methane production) and ruminant performance (milk and meat) are discussed.

Historical use of essential oils For thousands of years humans have used plants and extracts thereof and the first records of such usage date back to ca. 2600 BC from Mesopotamia (Bager et al., 1997; Newman et al., 2000) (Figure 1). However, the earliest written account of a preparation of an essential oil dates back only as far as the Greek historian Herodotus (484–425 BC) (Urdang, 1948). The essential

C. Benchaar et al. 113

oil was that of turpentine, which is extracted from the turpentine tree (Pistacia terebinthus) and other coniferous trees. Interestingly, the oil of turpentine happens to be associated with almost every event in the history of essential oils. The relative youth of the essential oils industry is probably a consequence of its dependence on distillation technology, which has its origins in the East, most notably Egypt, Persia and India (Urdang, 1948). It wasn’t until the late 13th early 14th century that the extraction and use of essential oils was first properly described. Arnald de Villanova (ca 1235–1311), a Catalan physician, described oils of rosemary and sage in addition to turpentine in his book ‘Opera Omnia’. In the 16th century essential oils began being traded in the City of London (Burt, 2004) and this is regarded as being the start of the widespread use of essential oils in Europe. However, even then, only a few essential oils are described in relevant texts of the time. For example Brunschwig (1450–1534), a Strassburg physician, in his book on distillation, ‘Liber De Arte Distillandi’, only mentions four essential oils, namely the oils of turpentine, juniper wood, rosemary and spike (lavender) (Urdang, 1948). A turning point in the uptake of essential oils is thought to have been a publication in 1551 by the German physician Adam Lonicer (1528–1586), in which the medicinal value of essential oils was stressed and another (‘De Destllatione libre IX’) by Giovanni Battista della Porta published in 1563, in which methods of essential oil preparation and separation, including apparatus required, were described (Urdang, 1948). At the start of the 17th century the French physician Joseph Du Chesne wrote in his ‘Pharmacopoea Dogmaticorum Restituta’ that preparations of essential oils were well known to ‘everyone’ and that up to 20 different essential oils were stocked in pharmacies. In 1881, a scientist by the name of De la Croix is reported to have made the first experimental measurement of the bactericidal properties of essential oils (Burt, 2004). During the 19th century, the great advances in chemistry were successfully applied to essential oils as described by Urdang (1948). In 1887, Otto Wallach proposed the isoprene rule for terpenoids (Banthorpe, 1994), which preceded Jacques Julien Houton de la Billardière’s report in 1818 of the 5:8 ratio of carbon to hydrogen in the oil of turpentine, the molecular formula for isoprene (Urdang, 1948). By 1894, the structure of camphor and α-pinene had been elucidated (Banthorpe, 1994). As the 19th century progressed, the mainstream use of essential oils for medicinal purposes became secondary to their use as flavouring and fragrance agents (Urdang, 1948) and this continues to this day with almost 90% of the world’s essential oil production being used by the flavour and fragrance industries (Holmes, 2007). Over the last decade a growing proportion of the market share has been for medicinal uses (both human and animal) and performance enhancing feed additives for animals.

Current interest in the use of essential oils in ruminants While the use of essential oils in ethno-veterinary is primarily aimed at restoring health to animals and thus indirectly production efficiency, current interest in mainstream animal production industry is focused on trying to improve production efficiency in animals that are not necessarily displaying any symptoms of ill health. Over the last 60 years or so one of the ways in which the latter has been achieved has been via the inclusion of sub-therapeutic

114 Essential oils as feed additives in ruminants

Figure 1. A timeline of some of the important events in the history of essential oils (based on Urdang, 1948)

C. Benchaar et al. 115

levels (2.5–125 mg/kg feed, i.e. 5–10 × lower than therapeutic levels) of antibiotics in the feeds of animals, which can improve the efficiency of growth by up to 10% (Russell and Houlihan, 2003). The antibiotics most commonly used in ruminant production are monensin, lasalocid and laidlomycin propionate, which are all ionophore antibiotics. They are used mainly in beef cattle production, especially feedlot beef. Use in dairy cattle is limited by the risks of contaminating milk with residues. They function by dissipitating ion gradients across cell membranes of susceptible bacteria (principally Gram-positive bacteria), which are selected against, resulting in beneficial changes to rumen fermentation (Callaway et al., 2003; Russell and Houlihan, 2003; Tedeschi et al., 2003). The proportion of propionate relative to acetate is increased, which is associated with a concomitant reduction in methane production and the degradation of dietary protein in the rumen is reduced, both of which contribute to increased feed conversion efficiency. They also help reduce the incidence of acidosis and feedlot bloat. However, due to concerns about the development of antibiotic resistance in bacteria, antibiotic growth promoters have been banned from use in the European Union (EU). The key events and legislation that led to this ban in January 2006 (Directive 1831/2003/EC), starting with Directive 70/524/EEC, which was based on the recommendations of the Swann Report (Swann, 1969), in which a list of allowable feed additives with directions for use was published, are presented in Table 1. Although this ban is currently limited to the EU, where use of ionophores had always been limited, the consequences for the animal production industry are significant considering ionophores are estimated to save the industry, globally, $1 billion per annum (Callaway et al., 2003). Therefore, alternatives to antibiotic growth promoters are being sought. Plant essential oils, due to there well documented antimicrobial actions (Hulin et al., 1998; Dorman and Deans, 2000; Burt, 2004), are one of these alternatives.

Essential oils Definition Essential oils are oily liquids composed of low molecular weight, hydrophobic (lipophilic) secondary metabolites extracted from plants. They have low boiling points, which renders them volatile and hence they are also referred to as volatile oils. They are referred to as ‘essential’, not in the ‘nutritional’ sense of the word, but because they are the ‘essence’ of plants – they are responsible for the fragrance and the flavour of the plants. Whilst most essential oils are complex mixtures of secondary metabolites, there are usually one or two major components that are responsible for the oils character, in terms of its essence. For example, the dominant compounds in the essential oil of oregano (Oreganum vulgare ssp. hiritum) are the terpenoids carvacrol and thymol, which together can account for more than 90% of the total essential oil content (Vokou et al., 1993) (Table 2).

116 Essential oils as feed additives in ruminants Table 1. Significant events associated with the ban on the use antibiotic growth promoters in the European Union (EU)

Date 1969

Legislation

Comment Swann Report recommends that use of antibiotic growth promoters should be restricted

23/11/70

70/524/EEC

Primary legislation for feeding stuffs introduced in which a list of allowable feed additives with their maximum and minimum dosages, withdrawal period and animal species in which they could be used was published

1986

Sweden banned all antibiotics for growth promotion

1993

Initial report on the isolation of vancomycin (glycopeptides) resistant enterococci, which showed cross-resistance with avoparcin, a glycopeptide antibiotic used only as a growth promoter in animals, published (Bates et al., 1993)

1997

Occurrence of glycopeptide-resistant enterococci in food animals associated with use avoparcin (glycopeptides antibiotic) in Europe (Bager et al., 1997)

30/1/97

97/6/EC

Use of avoparcin as an antibiotic feed additive banned

18/3/98

98/19/EC

Ronidazole banned from use in turkeys

17/12/98

EC 2821/98

Tylosin, virginiamycin, bacitracin zinc and spiramycin banned from use as animal growth promoters

22/12/98

EC 2788/98

Carbadox and olaquinadox banned from use as growth promoters

1/1/06

EC 1831/2003

All antibiotic growth promoters, with the exception of coccidiostats and histomonostats, banned

1/1/13

Coccidiostats and histomonostats to be banned from use as feed additives

Essential oils are located in resin ducts, lactifers, trichomes (glandular hairs) and/or on the cuticle of plants (Wink, 2001). Trichomes not only store essential oils, but have also been shown to synthesise them, both terpenes and phenylpropenes (Gershenzon et al., 1992; Gang et al., 2001). Plant secondary metabolites are so named because their distribution is limited to specific plants, unlike the ubiquitous primary metabolites. They are thought to be an important means by which plants interact with their environment, affording protection from biotic and abiotic stressors and acting as attractants to organisms that pollinate and disperse seeds (Wink, 1999). The production of many secondary metabolites in plants is inducible, which explains the considerable variation in essential oil yield and composition from plants grown under different climatic conditions and on different soils (Table 2) and from plants exposed to differing levels of attack from herbivores (Chen, 2008). Essential oils can be

C. Benchaar et al. 117 Table 2. Chemical composition (% of total oil) of Oreganum vulgare ssp. hirtum* essential oil samples from different regions of Greece (adapted from Vokou et al., 1993)

Constituent

Athos Peninsula (650 m) 46.7 11.9 12.0 16.0 0.8 0.6 0.1 0.4 0.1 2.2 0.1 2.5

Kriti Island (550 m) 0.8 74.2 9.1 4.1 1.0 1.0 0.1 0.2 0.1 2.2 0.1 1.2 0.3 0.2

Mount Taygetos (400 m) 30.0 51.0 7.6 5.2 0.5 0.2

Thymol Carvacrol p-Cymene γ-Terpinene α-Thujene α-Pinene Camphene 1-Octen-3-ol 0.7 3-Octanol 0.3 Myrcene 0.9 α-Phellandrene 0.1 α-Terpinene 0.9 ß-Phellandrene Limonene ß-Phellandrene + 0.3 0.1 limonene 0.7 0.6 0.7 trans-Sabinene hydrate Terpinolene 0.2 0.4 0.2 0.2 cis-Sabinene hydrate Borneol 0.2 0.2 0.2 Naphthalene 0.4 Terpinen-4-ol 1.3 0.6 α-Terpineol 0.1 Methylthymol 3.5 ß-Caryophyllene 1.0 0.8 0.2 Farnesene 0.1 2.2 0.4 Caryophyllene oxide * Plants were sampled at the flowering stage, air-dried and then distilled.

Evoia Island (260 m) 90.2 2.5 3.8 0.6

0.7 0.1

0.4 0.1 0.1 0.2

1.3

extracted from all parts of a plant, from the roots to the flowers. However, there can be considerable variations in yield and composition of yield from different parts of plants and consequently extraction is often targeted at specific parts of plants depending upon the intended use (Burt, 2004). Methods of extraction include distillation (boiling water and steam), liquid solvent extraction, supercritical CO2 extraction and expression. Method of extraction affects the composition of the final product and thus affects the properties of the oil (Anitescu et al., 1997). Distillation, for example, involves high temperatures which can destroy temperature sensitive compounds within essential oils and solvent extraction can leave residues in the extracted product. Packiyasothy and Kyle (2002) have shown that solvent (hexane) extracted essential oils exhibit greater antimicrobial activity than essential oils extracted by steam distillation from the same plant material.

118 Essential oils as feed additives in ruminants

Chemistry The building blocks of secondary metabolites are derived from primary metabolism, principally the processes of photosynthesis, glycolysis and the citric acid cycle. The secondary metabolites that make up essential oils are normally either terpenes or phenylpropenes. When these compounds contain oxygen in their structure they are referred to as terpenoids and phenylpropanoids, although the terms can be regarded as synonyms, as they are used interchangeably in the literature. Terpenes and phenylpropenes are groups of secondary metabolites that are derived from common pathways and synthesised using common structural units. While compounds representative of both groups may be present in a plants essential oil, normally compounds from one group dominate. For example, the major constituents of clove (Eugenia caryophyllus) essential oil are the phenylpropenes eugenol, eugenol acetate and ß-caryphyllene (75–90%, 10–15% and 3% of typical composition, respectively; Dewick, 2002), while the major constituents of oregano (Oreganum vulgare) essential oil are terpenes (Table 2).

(a) (b)

Figure 2. The basic building units of the terpenes and phenylpropenes, the C5 isoprene unit (a) and the C6C3 phenylpropyl unit (b), respectively

The basic building blocks of phenylpropenes are C6C3 phenylpropyl units (a 6-carbon aromatic ring with a 3-carbon chain attached to it; Figure 2), which are derived from the carbon skeletons of the aromatic amino acids phenylalanine and tyrosine. These amino acids, including tryptophan, are synthesised by the shikimate pathway (Figure 3), so named because shikimic acid is a key intermediate in the pathway. The pathway is found only in microorganisms and plants (Sangwan et al., 2001). The first step in the synthesis of shikimic acid is the condensation of phosphoenolpyruvate (intermediate of glycolysis) and erythrose 4-phosphate (intermediate of the pentose phosphate pathway). Shikimic acid then condenses with a further molecule of phosphoenolpyruvate to form chorismate from which the aromatic amino acids phenylalanine, tyrosine and tryptophane are synthesised. The first committed step to phenylpropene synthesis is the deamination of amino acids

C. Benchaar et al. 119

phenylalanine and tyrosine yielding cinnamic acid (the basic C6C3 penylpropyl building block) and 4-coumaric acid, respectively. While all plants can apparently deaminate phenylalanine, a reaction catalysed by phenylalanine ammonia lyase, the same is not true for tyrosine. Those that cannot transform tyrosine synthesise 4-coumaric acid from cinnamic acid. Hydroxylation and methylation of 4-coumaric acid yields caffeic, ferulic and synaptic acids, all collectively referred to as cinnamic acids. Reduction of these cinnamic acids yields cinnamyl alcohols, which are used for the synthesis of the phenylpropenes, e.g. cinnamaldehyde, eugenol, anethole, myristicin and safrole (Figure 4).

Figure 3. An overview of the pathways responsible for synthesising terpenes and phenlypropenes, the principle metabolites found in plant essential oils

120 Essential oils as feed additives in ruminants

Figure 4. Chemical structures of some of the commonly encountered secondary metabolites found in essential oils

The basic building blocks of the terpenes, the C5 isoprene units (Figure 2) isopentenyl diphosphate and dimethylallyl diphosphate (hemiterpenes), are derived from the mevalonate and deoxyxylulose pathways. The mevalonate pathway involves the synthesis of isopentenyl diphosphate via the intermediate mevalonic acid. Mevalonic acid is synthesised from three molecules of acetyl-CoA; two molecules of acetyl-CoA condense to form acetoacetyl CoA, which then reacts with a further molecule of acetyl-CoA to yield 3-hydroxy-3-methyl-

C. Benchaar et al. 121

glutartyl CoA (HMG CoA), which is then reduced forming mevalonic acid. Mevalonic acid is then phosphorylated and decarboxylated in a series of reactions yielding isopentenyl diphosphate. The deoxyxylulose pathway, which is thought not to be present in animals, involves the synthesis of isopentenyl diphosphate via the intermediate deoxyxylulose phosphate, which is formed from the combination of the glycolytic intermediates glyceraldehyde 3-phosphate and pyruvate. Deoxyxylulose phosphate is then converted to isopentenyl diphosphate via a series of reactions that are not yet fully understood. Plants use both the mevalonate and deoxyxylulose pathways to synthesise isopentenyl diphosphate (Dewick, 2002). Isomerisation of the hemiterpene isopentenyl diphosphate yields the other isoprene unit and hemiterpene dimethylallyl diphosphate. Combination of two yields geranyl diphosphate, a monterpene (C10), from which the monterpenes linalyl diphosphate and neryl diphosphate are formed. From these three compounds a range of linear and cyclic (mono- and bi-) monterpene are formed, which may be hydrocarbons (e.g. ß-myrcene and limonene), alcohols (e.g. gereniol, linalool, thymol and carvacrol), aldehydes (e.g. citronellal, neral, carvone) and esters (e.g. geranyl acetate) (Figure 4). Most monterpenes are optically active. Some essential oils contain both enantiomeric forms, e.g. (+)- and (–)-limonene are both components of the essential oil of peppermint (Mentha × piperita), while other essential oils contain only one enantiomeric form, e.g. caraway (Carum carvi) essential oil contains only (+)-carvone, whilst spearmint (Mentha soicata) essential oil contains only (–)-carvone. Different enantiomeric forms can have quite different smells, for example (+)-carvone gives caraway its characteristic smell, whereas (–)-carvone smells of spearmint. Addition of another isopentenyl diphosphate to geraniol diphosphate (C10) yields farnesyl diphosphate, a sesquiterpine (C15), which is precursor to many other sesquiterpines, both linear and cyclic (mono-, bi- and tri-), e.g. zingerberine, α-bisabolol, α-santonin, artemesinin and α-cadinene. Sesquiterpenes are in general less volatile that the monterpenes. It must be appreciated that not all secondary metabolites found in essential oils are phenylpropenes and terpenes. For example the metabolites found in garlic oil, which include allicin, diallyl sulphides, ajoenes and vinyldithiins, are derived from the amino acid cystein via γ-glutamylS-allyl cysteine (Amagase et al., 2001).

Mechanisms of action (antimicrobial activity) Essential oils and their constituent secondary metabolites often exhibit antimicrobial activity. This activity appears to stem entirely from their hydrophobicity, which enables them to accumulate in the lipid bilayer of the microbial plasma membrane from where they orchestrate their effects, which vary according to type of secondary metabolite. Some alter membrane permeability, some interact with membrane proteins and others may interact

122 Essential oils as feed additives in ruminants directly with cytoplasmic components from within the plasma membrane or by diffusing into the cytoplasm itself (Figure 5). As essential oils are mixtures of many different secondary metabolites it is likely that a number of mechanisms of action are employed.

Figure 5. Schematic overview of some of the sites and mechanisms of antimicrobial activity of essential oils in the bacterial cell

It is generally accepted that of the secondary metabolites found in essential oils those with the greatest antimicrobial activity are phenolic compounds, i.e. compounds with a hydroxyl group (-OH) attached to a phenyl ring (Cosentino et al., 1999; Dorman and Deans, 2000; Lambert et al., 2001). Examples of phenolic secondary metabolites found in essential oils are the monoterpenes carvacrol and thymol and the phenylpropene eugenol. The hydroxyl

C. Benchaar et al. 123

group, in addition to being involved in the transport of ions across the plasma membrane (Ultee et al., 2002), is also thought to be involved in the inactivation of microbial enzymes (Burt, 2004). The primary antimicrobial mechanism of action of oregano essential oil is reported to be damage to the plasma membrane leading to loss of cell contents and finally lysis (Di Pasqua et al., 2007; Paparella et al., 2008). The major components of oregano essential oil are the phenolic monterpenes thymol and carvacrol and these appear to be the principle bioactive compounds in the oil. Depending upon treatment concentration and/or type of microorganism, both have been shown to cause microbial cell death or limit growth (Helander et al., 1998; Ultee et al., 1999; Ultee et al., 2000a; Ultee et al., 2000b). They have both been shown to increase the permeability of the plasma membrane (Xu et al., 2008) and have been reported to dissipate H+ and K+ ion gradients leading to depletion of intracellular ATP concentration, due either to inhibition of ATP synthesis or increased rates of ATP hydrolysis (Lambert et al., 2001). A proton transfer mechanism has been proposed by Ultee (2002) in which the hydroxyl group acts as a trans-membrane carrier of H+ and K+ (Figure 5). The fatty acid composition of the plasma membranes of bacteria exposed to thymol and carvacrol have a higher ratio of saturated:unsaturated fatty acids (Di Pasqua et al., 2007). Whether this is a bacterial response to treatment or a treatment effect through interactions of thymol and carvacrol with proteins (enzymes) is unclear. The phenylpropene eugenol (a major component of clove essential oil) is a phenolic compound and it, like the phenolic monoterpenes carvacrol and thymol, appears to exert its antimicrobial effects by disrupting the plasma membrane, resulting ultimately in cell lysis, presumably by similar mechanism as described for carvacrol and thymol (Thoroski et al., 1989; Di Pasqua et al., 2007). Eugenol has also been reported to inhibit production of microbial enzymes and to inhibit enzyme activity by binding to them (Burt, 2004). Non-phenolic secondary metabolites found in essential oils have variable antimicrobial actions. The monoterpenes, p-cymene and γ-terpinene appear to have limited antimicrobial activity compared to the phenolic monoterpenes (Dorman and Deans, 2000). Cosentino et al. (1999) were unable to report any antimicrobial activity for these compounds. Ultee (2002) has suggested the antimicrobial activity of p-cymene is brought about by their accumulation in the plasma membrane, causing the membrane to expand allowing leakage of ions. Cristani (2007) has suggested that non-phenolic monoterpenes are able to diffuse through the plasma membrane into the cytoplasm of microorganisms, which supports evidence indicating that they potentiate the antimicrobial effects of the phenolic monoterpenes by facilitating there transport across the plasma membrane (Ultee et al., 2002). Cinnamaldehyde (a major component of cinnamon essential oil), a non-phenolic phenylpropene, does exhibit antimicrobial activity (Helander et al., 1998). This is reported to be achieved by binding and inactivating microbial enzymes rather than through disintegration of the of the plasma membrane (Burt, 2004). The antimicrobial activity of essential oils tends to be selective against the Gram-positive bacteria. For example, there is evidence to suggest that Gram-positive bacteria may be more

124 Essential oils as feed additives in ruminants sensitive to oregano essential oil and its constituent phenols than Gram-negative bacteria (Cosentino et al., 1999; Lambert et al., 2001) and Smith-Palmer et al. (1998) has reported similar observations for other essential oils. It has been hypothesised that the differences in sensitivity between these bacterial groups is due to the differences in the cell envelope, in that access of essential oils to the membrane is more restricted in Gram-negative bacteria (Helander et al., 1998). It has been suggested that essential oils active against Gram-negative bacteria contain active secondary metabolites that are small enough to pass through porin proteins in the outer membrane and so are able to gain access to the plasma membrane (Nikaido, 1994; Dorman and Deans, 2000). It has been claimed that, unlike with conventional antibiotics, it is not possible for microorganisms to develop resistance to antimicrobial plant extracts, e.g. essential oils. Reasons include: essential oils are complex mixtures of chemical compounds, many of which posses antimicrobial activity with different modes of action making it impossible for microorganisms to establish resistance mechanisms (Briskin, 2000); the mode of action of plant extracts against microorganisms is structural rather than at the level of DNA (Mellor, 2000). These claims are wrong! Microbial resistance is not limited to a single mode of action and neither is it dependent on the mode of action being at the level of DNA. Theoretically all that is required for the creation of resistant bacteria is for bacteria to randomly acquire a genotype that confers a survival advantage to the bacteria in the presence of the compound(s) with antimicrobial properties of interest, be it an antibiotic or an essential oil. Creation of resistant bacterial populations is facilitated by exchange of genes, in particular those responsible for resistance, which are carried on plasmids, between bacteria of the same and related species by a process of conjugation or bacterial mating. As yet there has been little research to investigate bacterial resistance to herbs and spices with antibacterial properties. However, Brul and Coote (1999) have reviewed this topic and Nelson (2000) has reported how Staphylococcus aureus is able to develop resistance to tea tree (Melaleuca alternifolia) essential oil.

Effects of essential oils on rumen microbial fermentation Understandably, most of the research with essential oils up-to-date has been conducted in batch or continuous culture in vitro systems. As these compounds are relatively novel supplements in animal nutrition (Wallace, 2004), their effects on rumen fermentation and overall nutritional effects are unknown. It is impractical and technically impossible to screen in vivo the vast number of essential oils available (more than 3000; Van de Braak and Leijten, 1999). Therefore, researchers have heavily relied upon in vitro models to investigate effects of essential oils and their main constituents on rumen fermentation and consequently predict in vivo effects. However, in vitro systems have their limitations. Ultimately, all rumen in vitro systems are intended to simulate, as close as possible, the in vivo conditions in the rumen, such as viable and dynamic microbial populations, constant inflow and outflow of nutrients, relatively

C. Benchaar et al. 125

constant pH, constant temperature and continuous mixing. However, these conditions can never be completely simulated in vitro. Perhaps the most apparent limitation of any in vitro system is the inability to replicate the diversity and viability of the microbial population in the rumen. All rumen-based in vitro systems utilize rumen inoculum, which at least in the initial stages of the incubation, is representative of the microbial population in vivo. However, due to various factors inherent to the in vitro systems, the original microbial community degenerates and protozoa disappear (Slyter and Putnam, 1967; Mansfield et al., 1995), which is the primary reason for contrastingly different fermentations parameters in vitro compared with in vivo. The in vitro systems, for example, are unable to achieve fibre degradability rates comparable to in vivo conditions (Mansfield et al., 1995). Rumen bacteria normally low in numbers in vivo (e.g. Streptococcus bovis) may proliferate and lactate may accumulate. Unfortunately, lactate concentrations and lactate-producing bacterial counts are rarely reported for continuous culture experiments. Slyter and Putnam (1967), for example, did not observe Streptococcus bovis in vivo, but the bacterium made up from 2 to 9% of the total strains examined on certain days of the continuous culture fermentation. Mansfield et al. (1995) reported a large increase in the proportion of amylolytic species of the total viable count (from 3.3 to 28.2%, in vivo and continuous culture, respectively). Simultaneously, the proportion of cellulolytic bacteria decreased from 5.4 (in vivo) to 1.7% (continuous culture) of the total viable count. In both studies, protozoa were dramatically lower in vitro than in vivo. Often, the question is which in vitro system is more appropriate for studying rumen effects of various bioactive compounds. Each of the two types of rumen-based in vitro fermentation systems, batch and continuous culture, have advantages and disadvantages regarding simulation of the in vivo rumen. As indicated by the father of rumen microbiology (Dehority, 2005), Hungate (1966), referring to an earlier work by Markoff (1913), shortterm incubations provide more reliable estimates of fermentation parameters in the rumen than long-term in vitro incubations. The exact text quoted by Hungate from Markoff (1913) states: “…….the most exact results are obtained, when the rumen contents are removed as quickly as possible and the fermentation allowed to proceed for only a few hours”. Hungate continues: “In vitro experiments as usually conducted do not provide reliable estimates of the rates at which the phenomena under study occur in the rumen. Very short-term in vitro methods can provide such information” and “The theory behind this technique is that a sample of rumen contents removed from the rumen continues to function as in the rumen until accumulation of fermentation products, exhaustion of substrate, availability of new foods, or other factors cause it to change. This method is theoretically always applicable and in many cases practicable for evaluating microbial activity in the rumen. In a short-term in vitro incubation of a few seconds to an hour, changes in the composition of the rumen microbiota in the sample lie within the range characteristics of the rumen, particularly if anaerobiosis, proper temperature, and suitable acidity are maintained”. In in vitro incubations beyond “a few hours”, however, fermentation end-products begin to accumulate and the microbial population begins to change. Thus, the inability to adequately remove fermentation end products from the in vitro batch culture system is a significant disadvantage (compared with

126 Essential oils as feed additives in ruminants the continuous culture fermentation), if fermentation is prolonged beyond “a few hours”. In the case of essential oils (as well as any other bioactive agent), one of the most important questions the researcher must address is the persistence of the effect and the adaptability of the rumen ecosystem to inactivate or enhance the effect of the compound studied. Apparently, these questions cannot be answered in batch culture incubation systems. It is naive to claim that a continuous culture system can adequately address these questions either. The longterm effects of a bioactive agent on rumen fermentation can be successfully studied only in vivo. In vitro systems can only orient the researcher about possible in vivo effects, but the ultimate test remains the in vivo experiment. Even in vivo, not all experimental designs are adequate to study rumen effects. Latin square (or cross-over) design experiments, for example, are usually not well-suited to answer the adaptability question. The adaptation period between experimental periods has to be sufficiently long (at least 4 wks) to allow proper manifestation of the effects of the compound tested. With some types of animal, e.g. lactating dairy cows, this is not always feasible as stage of lactation becomes another possible confounding factor. Despite of its disadvantages, an in vitro system remains the only practical way of screening large number of compounds, as is the case with essential oils. Once the number of compounds of interest is narrowed down to a few, experimentation must be taken to the next level, an in vivo test.

Effects on protein metabolism Ruminant animals are relatively inefficient utilisers of dietary nitrogen (N). The efficiency of transfer of feed N into milk protein has been determined to be on average 24.7 ± 0.14% with minimum and maximum of 13.7 and 39.8%, respectively (Hristov et al., 2005), as the remaining N is being lost to the environment with urine and faeces. Thus, N not used for milk protein synthesis or growth contributes to ground and surface water and air pollution. Environmental issues of significant public concern such as contamination of groundwater aquifers, eutrophication of lakes and rivers due to hypoxia, acid rain and formation of fine particles (PM2.5) can all be partially attributed to farm animals and related to inefficient utilization of dietary N. As ruminally degradable N in excess of microbial requirements is largely lost to the animal and along with the metabolisable protein unutilised for production needs is mostly excreted with the urine, improving the efficiency of ruminal N use is a primary goal in ruminant nutrition with far-reaching production and environmental implications. In recent years, in vitro studies have been conducted to determine the effects of essential oils and their components on rumen microbial fermentation. A large number of these studies used short-term batch culture techniques and examined a wide range of essential oils and essential oil compounds, dose rates and diets, and not surprisingly, results have been variable. Research work by the Rowett group suggested selective inhibition of rumen bacteria by a specific mixture of essential oil compounds (MEO) that included thymol, eugenol, vanillin and

C. Benchaar et al. 127

limonene (McIntosh et al., 2003). Using 48 h in vitro batch culture incubations, McIntosh et al. (2003) observed reduced (− 9%) deaminative activities of rumen fluid collected from dairy cows fed a silage-based diet supplemented with 1 g/day of the specific blend of MEO. The inhibitory effect of this particular mixture on deaminative activity was later confirmed in vitro (24 h batch cultures) by Newbold et al. (2004) who reported a decrease (−25%) in deaminative activities of rumen fluid collected from sheep fed 110 mg/day of MEO (Table 3). However, in the later study, in vivo rumen concentration of ammonia N was not affected by the addition of the specific commercial mixture. In further investigations, McIntosch et al. (2003) observed that MEO inhibited the growth of some hyper-ammonia producing (HAP) bacteria such as Clostridium sticklandii and Peptostreptococcus anaerobius while the growth of other HAP bacteria (e.g. Clostridium aminophilum) were not affected. Castillejos et al. (2005, 2007) did not find MEO effective in modifying rumen N metabolism (concentrations of ammonia N, large peptide N, small peptide plus amino acid N, bacterial and dietary N flows, degradation of N and efficiency of microbial protein synthesis) when added at the concentrations of 1.5, 5, 50 and 500 mg/l in a continuous culture fermenter maintained at constant pH (6.4 ± 0.05). The lack of effectiveness of the blend of essential oil compounds in those studies may be attributed to the dosage rates used, which may not have been high enough to affect microbial populations. In fact, McIntosh et al. (2003) reported that a minimal concentration of 40 mg/l of MEO is required in order to inhibit the growth of predominant rumen bacterial species, including HAP bacteria (e.g. Clostridium sticklandii and Peptostreptococcus anaerobius). This concentration is higher than that likely to be achieved in vivo (Hart et al., 2008). In fact, results from in vivo studies (Benchaar et al., 2006a, 2007a) showed that feeding MEO (0.75 and 2 g/day) to dairy cows had no effects on rumen ammonia N concentration, retention and digestibility of N. Such feeding levels of 0.75 and 2 g/cow/day would have corresponded to a ruminal concentration of 3.1 and 8.3 mg/l, respectively, assuming a rumen volume of 100 L and an outflow rate of 10%/h for an average adult dairy cow. These rumen concentrations are indeed much lower than the reported concentration of 40 mg/l required for the essential oils mixture to alter substantially N metabolism in the rumen. Such feeding levels of 0.75 and 2 g/day would correspond to a maximum rumen concentration of 7.5 and 20 mg/l, respectively, assuming an average rumen volume of 100 L for an adult dairy cow. These rumen concentrations are indeed much lower than the reported concentration of 40 mg/l required for MEO to alter substantially N metabolism in the rumen (McIntosh et al., 2003). The effects of essential oils on rumen protein metabolism have also been assessed using the in situ bag technique and the results were variable among studies depending on the protein source tested, the composition of the diet and the dose of the product fed to animals. Molero et al. (2004) observed small reductions in the effective rumen degradabilities of protein in lupin seeds (− 3%), green peas (− 6%) and sunflower meal (− 5%) when growing heifers were supplemented for 10 days with 700 mg/day of MEO in a high-concentrate diet. When MEO was added to a low-concentrate diet, only the rumen degradability of green peas was slightly reduced (− 2%). Based on the results of Molero et al. (2004), Hart et al. (2008) speculated that the effect of these specific essential oil components on rumen degradation of protein seems to be selective with the effects being more pronounced with the rapidly degradable protein

128 Essential oils as feed additives in ruminants sources than with the more resistant substrates. Nevertheless, the observed reductions were too small to have any likely nutritional impact on rumen protein metabolism in the animal. Table 3. Proteolitic, peptidololytic and deaminative activities of rumen fluid collected from sheep fed a silage-based diet supplemented with 110 mg/day of a mixture of essential oil compounds (adapted from Newbold et al., 2004)

Control

Essential oil compounds mixture1 1.43

SED

1.40 0.045 Proteolytic activity (mg 14C casein degraded/mg protein/h) Peptidolytic activity Hydrolysis of dialanine (mmol/mg of protein/min) 1.19 1.06 0.173 Hydrolysis of penta-alanine (mmol/mg of protein/min) 2.62 2.71 0.171 204 155* 9.27 Deaminase acitivity (nmol NH3 produced/mg of protein/h 1 Essential oil compounds mixture (MEO) containing thymol, eugenol, vanillin, guaiacol and limonene *P < 0.05

There are some suggestions that essential oil supplements may be effective only after a prolonged period of exposure/adaptation of the rumen microorganisms to the active compounds. Castillejos et al. (2007) speculated that an adaptation time of 28 days is necessary to observe an effect of MEO on rumen N metabolism. However, in the study by Molero et al. (2004), when the adaptation period was extended to 28 days (versus 10 days), the addition of 700 mg/day of MEO to a high-forage diet did not modify the effective rumen degradability of soybean meal N, although MEO decreased the soluble and increased the potentially degradable N fractions. In another in situ study, Newbold et al. (2004) examined the effects of MEO on rumen degradation of soybean meal N incubated at different time intervals (0, 2, 4, 6, 8, 16, 24 and 48) in the rumen of adult sheep fed for 42 days a highforage diet supplemented with 110 mg/day of MEO. Results showed that rumen degradation of soybean meal N was only reduced (− 18%) at 2 h incubation. However, it is unlikely that this change would have had an impact on the overall effective rumen degradability of soybean meal N. More recently, Benchaar et al. (2006a, 2007a) reported no changes in protein degradability of soybean meal incubated in the rumen of lactating dairy cows fed MEO at 0.75 or 2 g/day. Collectively, results from continuous culture studies (Castillejos et al., 2005, 2007), in situ (Molero et al., 2004; Newbold et al., 2004; Benchaar et al., 2006a, 2007a) and in vivo (Benchaar et al., 2006a, 2007a) studies failed to confirm the positive effects of MEO additive on rumen N metabolism reported in short-term batch culture incubation studies (McIntosh et al., 2003; Newbold et al., 2004). This discrepancy between studies using different experimental approaches is a clear illustration that shortterm in vitro studies have limitations and that the ultimate value of essential oils for altering rumen microbial fermentation must be assessed in vivo.

C. Benchaar et al. 129

The range of essential oils available is extensive and many of these substances have yet to be examined for their antimicrobial effects against rumen microbes. In addition to studies investigating the effects of commercial mixtures of essential oils, other studies evaluated in depth the potential of single, naturally occurring essential oils and their main components to modulate rumen microbial fermentation. Most of these studies are laboratory based (batch culture or continuous culture systems) and originate from the group of the University of Barcelona (Spain). The effects on rumen N metabolism were variable depending on the essential oil or the essential oil compound being tested and the dose used. Cardozo et al. (2004) reported that the addition (0.22 mg/l) of cinnamon bark essential oil (Cinnamonum cassia; 59% cinnamaldehyde) in a continuous culture fermenter maintained at constant pH did not affect ammonia N concentration, but increased the concentration of peptide N and numerically decreased that of amino acid N, suggesting that peptidolysis was inhibited. Surprisingly, in subsequent studies from the same laboratory, no changes were observed in N metabolism (i.e. concentrations of ammonia N, large peptide N and small peptide plus amino acid N) when cinnamaldehyde was supplied at higher concentrations (i.e. 2.2, 31.2 and 312 mg/l of culture fluid) in continuous culture systems (Busquet et al., 2005a; 2005b). Using in vitro batch culture incubations (24 h), Busquet et al. (2006) observed that high doses of cinnamon oil (3000 mg/l) and cinnamaldehyde (300 and 3000 mg/l) strongly inhibited ammonia N concentration, but the effects were non-existent at low doses (i.e. 3 and 30 mg/l). However, the decreased rumen ammonia N concentration was associated with a reduction in total VFA concentration, suggesting a reduction in overall fermentation of the diet. Few studies have investigated in vivo the effects of cinnamon oil and cinnamaldehyde on rumen microbial fermentation. Chaves et al. (2008a) observed no changes in rumen ammonia N concentration in lambs fed barley- or corn- based diets supplemented with cinnamaldehyde (200 mg/kg of dry matter intake). More recently, Benchaar et al. (2008) reported that supplementing lactating dairy cows with 1 g/day of cinnamaldehyde (i.e. 43 mg/kg of dry matter intake) had no effects on rumen concentration of ammonia N and in situ degradation of soybean meal N. Compounds with phenolic structures have a broad spectrum of activity against a variety of both Gram-positive and Gram-negative bacteria (Kim et al., 1995; Helander et al., 1998; Dorman and Deans, 2000; Lambert et al., 2001). A number of in vitro studies have examined the effects of phenolic compounds (eugenol, carvacrol and thymol) or essential oils with high concentrations of phenolic compounds on rumen N metabolism. Reported effects have been variable among studies, apparently related to the dosage level and the in vitro technique used (batch vs. continuous culture). For instance, Busquet et al. (2005c) reported that addition of clove bud essential oil (Syzygium aromaticum; containing 85% of eugenol) at 2.2 mg/l to a continuous culture fermenter markedly decreased (− 80%) large peptide N concentration, but had no effect on ammonia N concentration, suggesting that clove bud essential oil inhibited the peptidolytic activity of rumen bacteria. However, the addition of eugenol (main component of clove bud essential oil) at the same concentration had no effect on N metabolism, suggesting that the anti-peptidolytic activity of clove bud

130 Essential oils as feed additives in ruminants essential oil is not due to its main component, eugenol, but results from unidentified compounds within the essential oil fraction. Using a 24 h in vitro batch culture, Busquet et al. (2006) reported that, when supplied at the concentration of 3000 mg/l, both oregano essential oil and its major constituent carvacrol reduced the concentration of ammonia N, indicating that carvacrol is responsible for the majority of the antimicrobial activity in oregano essential oil. Controversial results for cinnamon leaf essential oil (containing 76% of eugenol) were reported by Fraser et al. (2007). The addition of cinnamon leaf essential oil (500 mg/l) in the Rusitec (Rumen Simulation Technique) fermenter decreased ammonia N concentration and molar proportions of branched-chain volatile fatty acids (end-products fermentation of branched amino acid catabolism in the rumen), whereas no effects were observed in the dualflow system. More recently Benchaar et al. (2008) and Chaves et al. (2008b) reported that eugenol (800 mg/l) and cinnamon leaf essential oil (250 mg/l) had no effects on deaminative activity (expressed as µg ammonia N/mg bacterial N/min) of rumen bacteria and ammonia N concentration in vitro. Thymol has been extensively investigated for its antimicrobial properties against different types of microorganisms, including rumen microbes (Calsamiglia et al., 2007). An early report by Borchers (1965) showed that the addition of thymol (1000 g/l) to rumen fluid containing casein resulted in an accumulation of amino acid N and a decrease in ammonia N concentration, suggesting inhibition of amino acid deamination by rumen bacteria. Castillejos et al. (2006; 2008) conducted a series of batch culture and continuous culture studies to assess the potential of thyme oil (Thymus vulgaris) and its main constituent thymol, to favourably alter rumen microbial fermentation. In general, at low doses (5 and 50 mg/l) thymol had no effects on rumen N metabolism. At high doses of thymol (500 and 5000 mg/l) results on N metabolism were inconsistent depending on the in vitro system used. At these doses, thymol decreased ammonia N and branched-chain volatile fatty acids concentrations in 24 h batch cultures, which is consistent with the inhibition of the deamination process. However, when added at the concentration of 500 mg/l in a continuous culture system, thymol increased the concentration of large peptide N and small peptide plus amino acid N, but had no effect on ammonia N concentration. The accumulation of large peptides N and small peptide plus amino acid N is an indication that both proteolysis and peptidolysis processes were stimulated by thymol. At the concentrations of 5, 50 and 500 mg/l, thyme oil reduced ammonia N concentration but had no effect on that of branched-chain volatile fatty acids in 24 h in vitro batch culture fermentation (Castillejos et al., 2008). Hristov et al. (2008) examined the effects of 40 essential oils (at 10 and 100 mg/l final medium concentration) on rumen fermentation in short-term (4 h) in vitro batch culture incubations. Of the 40 essential oils evaluated, very few had statistically significant effects on ammonia N concentration. However, the observed effects were subtle and the authors concluded that it was unlikely that these moderate in vitro effects would correspond to any substantive impact on rumen N metabolism in vivo. Additive, antagonistic and synergistic effects have been observed between components of essential oils (Burt, 2004). This suggests that combinations of essential oils of different composition or specific combinations of essential oil secondary metabolites may result in additive and/or synergetic effects that may enhance efficiency of rumen microbial fermentation

C. Benchaar et al. 131

and nutrient utilization in ruminants. For example, Cardozo et al. (2006) evaluated the effects of feeding a mixture of cinnamaldehyde and eugenol in beef cattle fed a diet consisting of 90% of concentrate and 10% of barley straw. The combination of these two essential oil compounds at two feeding rates (180 mg/day of cinnamaldehyde + 90 mg/day of eugenol; 600 mg/day of cinnamaldehyde + 300 mg/day of eugenol) affected N metabolism in the rumen by increasing the concentration of small peptide plus amino acid N and decreasing ammonia N concentration, suggesting that deamination was inhibited.

Effects on volatile fatty acid production It may be energetically favourable to the animal if essential oils increase volatile fatty acid (VFA) production, alter VFA profile such that proportionally more propionate and less acetate are produced and decrease methane production during rumen fermentation. The effects of essential oils and their constituents on volatile fatty acid production have been inconsistent among studies ranging from no change to increased or decreased total rumen VFA concentration. Castillejos et al. (2005) observed that the addition 1.5 mg/l of MEO increased total VFA concentration without affecting proportions of individual VFA in a continuous culture system maintained at constant pH. However, the increase in total VFA concentration was not consistent with the lack of effects of MEO additive on organic matter digestibility. In a later continuous culture study by the same group (Castillejos et al., 2007), supplementation with MEO at 5 mg/l increased total VFA concentration and shifted VFA pattern towards more acetate and less propionate, although again there was no concomitant increase in organic matter digestibility. When MEO was added at higher concentrations (50 and 500 mg/l) in the same study, there were no effects on total VFA concentration, VFA pattern and digestibility of nutrients. The authors offered no explanation for this lack of effects. Castillejos et al. (2007) suggested that rumen microbes need to be exposed to the MEO for at least 6 days to observe changes in VFA. However, in their study, total VFA concentration was not affected both in vivo and in vitro in rumen fluid collected from sheep adapted for 4 weeks to MEO. Other in vivo studies (Newbold et al., 2004; Beauchemin and McGinn, 2006; Benchaar et al., 2006a) reported no changes in total VFA concentration and VFA proportions when MEO was fed to sheep (110 mg/day), beef cattle (1 g/day) and lactating dairy cows (2 g/day). Benchaar et al. (2007a) observed that rumen total VFA concentration tended to increase (+ 5%) in dairy cows fed an alfalfa silage-based diet supplemented with MEO (750 mg/day), but tended to decrease (−10%) when the diet contained corn silage. Although the observed changes were small, such results may suggest that effects of MEO on total VFA concentration may be diet depend. The effects of pure essential oils and their main components on total VFA concentration have been shown to be dose dependent. Busquet et al. (2006) screened several essential oils (anise, cade, capsicum, cinnamon, clove, bud, dill, garlic, ginger, oregano and tea tree) and essential oil compounds (anethol, benzyl salicylate, carvacrol, carvone, cinnamaldehyde and

eugenol) for rumen effects when supplied at 3, 30, 300 and 3000 mg/l in 24 h batch culture fermentations. At low (3 mg/l) and moderate (30 mg/l) doses, none of the essential oils or essential oil compounds affected total VFA concentration. At high doses (300 and 3000 mg/l) most of treatments decreased total VFA concentration. Castillejos et al. (2006) also observed that high doses (500 to 5000 mg/l of culture fluid) of some essential oil compounds (eugenol, guaiacol, limonene, thymol and vanillin) strongly decreased total VFA concentration in 24 h batch cultures of rumen fluid. A reduction in total VFA production may be a reflection of reduced diet fermentability and would generally be viewed as nutritionally unfavourable because VFA are the main source of metabolizable energy to ruminants. A number of in vitro studies have shown that some essential oils and essential oil compounds produce desirable changes in rumen fermentation by shifting VFA profile towards more propionate and less acetate. For example, in a continuous culture study, Busquet et al. (2005b) reported that at high doses (31.2 and 312 mg/l), cinnamaldehyde decreased the proportion of acetate and increased the proportions of propionate and butyrate. Other in vitro studies reported that garlic oil also reduced acetate proportion and increased propionate and butyrate proportions (Busquet et al., 2005b; 2006). High butyrate concentration as a result of supplementation with cinnamaldehyde and garlic oil may indicate that these secondary metabolites act differently from monensin, but similarly to other methane inhibitors (Calsamiglia et al., 2007). While previous studies have shown that the use of some essential oils may result in beneficial effects on VFA profile, other studies, however, revealed that the use of some essential oils and essential oil constituents results in undesirable changes in the proportions of individual VFA. For instance, in a batch culture study Castillejos et al. (2006) observed that at 500 mg/l, thymol decreased total VFA concentration, increased acetate proportion and reduced that of propionate. Benchaar et al. (2007b) reported that carvacrol (400 mg/l) and eugenol (800 mg/l) reduced the molar proportion of propionate without affecting total VFA concentration in batch culture incubations. It is possible that the effect of essential oils on VFA profile may be diet and pH dependent as shown in an in vitro batch culture study by Cardozo et al. (2005). For example, at pH 7.0 cinnamaldehyde and capsicum increased the acetate to propionate ratio, while at pH 5.5 the acetate to propionate ratio was lower with cinnamaldehyde and capsicum. Overall, supplementation with essential oils and or their main constituents caused either a decrease or no change in total VFA concentration in most studies. In some studies essential oils alter favourably VFA pattern, whereas in others essential oils produce undesirable changes in the proportions of individual VFA. The challenge is to identify the dose rates for various essential oils or essential oil compounds that favourably alter aspects of rumen metabolism without reducing total VFA concentrations. Microbial populations exhibit a remarkable capacity to adapt to and/or degrade a wide variety of plant secondary metabolites such as saponins and tannins (Newbold et al., 1997; Makkar et al., 1995; Makkar, 2003). Similarly with essential oils, there appears to be adaptation of rumen microbial populations, particularly at low dosage rates in vitro. Indeed, rumen microbes were able to adapt to essential oils when these secondary metabolites were

C. Benchaar et al. 133

administered at low doses (Cardozo et al., 2004: 0.22 mg/l; Busquet et al., 2005a: 2.2 mg/l), but at higher doses (Busquet et al., 2005b: 300 mg/l; Fraser et al., 2007: 500 mg/l) the effect of essential oils appears to be sustained over time (e.g. nine days of continuous culture fermentation). However, such doses (≥ 300 mg/l) are higher than likely to occur in vivo. Indeed, assuming an average rumen volume of 100 L, a dilution rate of 10%/h and therefore a daily rumen fluid outflow of 240 l, an adult dairy cow should be fed 72 g/day of a given essential oil (i.e. 300 mg/l × 240 l/day) to achieve a final concentration of 300 mg/l in rumen fluid. Such an amount is extremely high and if fed to the animal is likely to adversely affect the efficiency of rumen microbial fermentation and animal performance. However, such concentrations (≥ 300 mg/l) are higher than likely to occur in vivo and would correspond to impractical feeding rates that if applied would adversely affect the efficiency of rumen microbial fermentation and animal performance. Results from these studies provide evidence that under practical feeding conditions (i.e. normal feeding rates), microbial populations are able to adapt to essential oil over time, which presents a challenge for commercial application of this feed additive technology.

Effects on methane production Being an integral part of substrate (primarily carbohydrate) fermentation, VFA production and hydrogen disposal in the rumen, methane formation represents a net loss of energy to the host animal. Energy lost as methane from cattle ranges from 2 to 12% of gross energy intake (Johnson and Johnson, 1995). Methane is also a potent greenhouse gas with a significant environmental impact. In recent years, however, the discussion on methane production by livestock has entirely shifted towards its contribution to climate change and global warming. Indeed, methane has a global warming potential 23 times that of CO2. According to a FAO report (Steinfeld et al., 2006), livestock contributes 37% of the global anthropogenic methane emissions with most of it from enteric fermentation by ruminants. It is worthwhile pointing out, however, that in developed countries, emissions from livestock (methane and nitrous oxide) may still be a relatively small proportion of the total greenhouse gas emissions. In the United States., for example, livestock emissions make up a mere 2.2% of the total greenhouse gas emissions (EPA, 2005). Nevertheless, the interest in reducing methane production in the rumen is well-justified from nutritional and environmental standpoints. Mitigating methane emissions from ruminants will have shortterm economic (i.e. improved feed efficiency) and long-term environmental (i.e. decreasing agriculture’s contribution to greenhouse gas emissions) benefits. A number of in vitro studies have evaluated the potential of essential oils to inhibit rumen methanogenesis. The reported effects varied with the type and the dose of the essential oil used. Evans and Martin (2000) observed no effects on methane concentration when thymol was used at 50, 100 and 200 m/l of culture fluid in 24 h incubations of mixed rumen bacteria. However, at high concentration (400 mg/l), thymol strongly decreased methane concentration, but acetate and propionate concentrations were also reduced. Busquet et al. (2005b) studied the effects of high concentration (300 mg/l) of garlic essential oil and four of its main components

134 Essential oils as feed additives in ruminants (diallyl sulfide, diallyl disulfide, allyl mercaptan and allicin) in batch culture fermentation (17 h). Garlic oil and diallyl disulfide drastically reduced methane production (−74 and −69% respectively), but diet digestibility and total VFA concentration were also depressed. In the same study, the inhibitory effect of monensin on methane was less pronounced (−42%) than garlic essential oil and diallyl sulphate. Based on these observations, Busquet et al. (2005b) suggested that contrarily to monensin, which specifically inhibits rumen Gram-positive bacteria, the antimethanogenic effect of garlic and its main components was the result of a direct inhibition of Archaea microorganisms in the rumen. In an in vitro short-term incubation study (6 h), Chaves et al. (2008b) observed that cinnamon leaf oil (250 mg/l), garlic oil (100 and 250 mg/l), juniper berry oil (20 mg/l) and p-cymene (20 mg/l) reduced the methanogenic activity of rumen bacteria (expressed as µmol of methane/g bacterial N/min) and methane concentration in the fermentation gases, without altering total VFA. Tatsuoka et al. (2008) investigated the effects of essential oil (cineol, eucalyptus, menthol, peppermint, thyme and wasabi) cyclodextrin (CD, α or β) complexes on in vitro short-term (6 h) rumen fermentation. Eucalyptus-αCD (10 and 20 mg equivalent oil/60 mL of culture fluid) reduced methane production, increased total VFA concentration and the molar proportion of propionate. Wasabi oil (as α- or βCD, equivalent to 10 mg oil/60 mL of culture fluid) drastically reduced methane production and increased concentration of propionate. The other essential oil-CD showed no significant effect on reducing methane production. Only few studies have examined in vivo the effect of essential oils and their main components on enteric methane emission by ruminants. Mohammed et al. (2004) observed that at high levels of feed incorporation (20 g/kg of dietary dry matter), encapsulated (α- cyclodextrin) horseradish oil decreased (−19%) methane production in steers without affecting diet digestibility. The reduced methane production was accompanied by a shift in VFA profile towards proportionally more propionate and less acetate and a decrease in the total numbers of methanogens. In another in vivo study, Beauchemin and McGinn (2006) observed no change in methane production, although feed digestibility decreased in beef cattle supplemented with MEO (1 g/day) in a high-forage diet. McIntosh et al. (2003) observed that the inhibition of the growth of the methanogen Methanobrevibacter smithii occurred only when the concentration of MEO exceeded 1000 mg/l. This level was 33-times higher (33 mg/l of rumen fluid) than that fed in the in vivo study reported by Beauchemin and McGinn (2006), a feeding rate that is not practical, due to potentially adverse effects on efficiency of rumen fermentation and diet digestibility. Based on findings from these studies, it appears that some essential oils and essential oil compounds have the potential to reduce enteric methane emission in ruminants. However, the challenge is to identify essential oils and components that selectively inhibit rumen methanogenesis without depressing feed digestion.

Effects on rumen ciliate protozoa The concept of improving N utilization by ruminants has evolved into two main objectives: (1) to maximize microbial protein synthesis in the rumen and (2) to maximize the supply of

C. Benchaar et al. 135

essential amino acids to the small intestine of the host animal. As protozoa make up 40 to 50 % of total microbial biomass in the rumen, their ability to assimilate and convert both dietary and microbial proteins plays a significant role in the N economy of the ruminant animal (Hristov and Jouany, 2005). Reducing protozoal numbers often lowers rumen methanogenesis because ciliate protozoa have a symbiotic relationship with methanogenic bacteria. About 25% of rumen methanogens live in association with protozoa (Newbold et al., 1995). Thus, control of rumen protozoa population may offer a way to improve N and energy utilisation in ruminants. However, the approach is not without controversy as protozoa contribute significantly to fibre degradation and pH stability in the rumen. Therefore, partial reduction in protozoal counts or inhibition of protozoal activities, rather than complete defaunation, might be more beneficial to the overall animal performance (Greathead, 2003; Hristov and Jouany, 2005). Few studies have examined the effects of essential oils on rumen ciliate protozoal populations. Ando et al. (2003) reported a strong decrease in the total numbers of protozoa (−50%), as well as the numbers of certain protozoal species including Entodinum (−58%), Isotrica (−30%) and Diplodium (−70%), in rumen fluid from steers supplemented with 200 g/day (i.e. 54 g/kg of dry matter intake) of sun-dried peppermint (Mentha × piperita L.). Mohammed et al. (2004) observed no modification in the number of rumen cilate protozoa when cyclodextrin encapsulated horseradish was included as a supplement at high doses both in vitro (0.17 to 1.7 g/l of culture fluid) and in vivo (20 g/kg of dietary dry matter). In a longterm (16 days incubation) continuous culture fermentation study, Fraser et al. (2007) observed that at 500 mg/l of culture fluid, cinnamon leaf oil (76% eugenol) reduced protozoa numbers in the RUSITEC and in a dual-flow fermerter. Cardozo et al. (2006) reported no change in entodiniomorphs and an increase in holotrichs numbers in beef heifers fed a high-concentrate based diet supplemented with a mixture of cinnamaldehyde (24 mg/kg of dry matter intake) and eugenol (12 mg/kg of dry matter intake). However, no effects were observed when the mixture contained higher concentrations of cinnalmadehyde (77 mg/kg of dry matter intake) and eugenol (38 mg/kg of dry matter intake). In the same study, feeding anise oil at 2 g/day (i.e. 250 mg/kg of dry matter intake) decreased the counts of entodiniomorphs and holotrichs whereas feeding capsicum oil at 1 g/day (i.e. 120 mg /kg of dry matter intake) had no effects. More recently, Benchaar et al. (2008) reported that supplementing dairy cow diets with 1 g/ day of cinnamaldehyde (i.e. 43 mg/kg of dry matter intake) had no effect on total numbers of protozoa as well as the numbers of Dasytricha, Diplodinium, Entodinium and Polyplastron. However, numbers of Isotricha increased. Newbold et al. (2004) and Benchaar et al. (2007a) reported that rumen protozoa counts were not affected when sheep and dairy cows were fed 110 and 750 mg/day of MEO, respectively. Rasmussen et al. (2005) reported that at the concentration of 100 mg/l, rosemary (Rosmarinus officinalis) essential oil had no effect on protozoa viability whereas at 10000 and 40000 mg/l, the essential oil greatly decreased (−90%) protozoal viability. However, again, these levels are very high and impractical in terms of feeding due to potentially negative effects on the efficiency of fermentation in the rumen. These studies are compelling evidence that essential oils have no marked effects on rumen ciliate protozoa when supplied at low or moderate concentrations and it seems that high concentrations are required to exert an effect.

136 Essential oils as feed additives in ruminants The effects of essential oils and their main components on rumen microbial fermentation are inconsistent and dose-dependent. The effects on N metabolism in the rumen are small and variable and in most cases occur only after a strong inhibition of overall fermentation as evidenced by a reduction in total VFA concentration. The effects on total and individual VFA production are inconclusive. Only few studies examined the effect of essential oils and their compounds on rumen methanogens and methane production. In most cases, it appears that essential oils have a potential (through direct inhibition of methanogenic bacteria or inhibition of rumen protozoa) to inhibit methane production in the rumen, although this effect is accompanied by a reduction in diet digestibility.

Effects of essential oils on performance While a large number of in vitro studies have been published on the effects of essentials oils and their main constituents on rumen microbial fermentation, only few studies have been carried out to determine their effects on ruminant performance. Several commercial products are currently available on the market that claim to improve feed efficiency and performance (milk and gain) when included in ruminant diets. Among these products, the MEO supplement is perhaps the most investigated. Benchaar et al. (2006a; 2007a) observed no changes in dry matter intake, milk production and milk constituents when the MEO additive was fed to dairy cows at the rates of 0.75 (i.e. 43 mg/kg of dry matter intake) or 2 g (i.e. 87 mg/kg of dry matter intake) daily. It is possible that the effect of MEO on milk composition is diet dependent. Yield of fat corrected milk (4% FCM) was not affected when MEO was added to alfalfa silage- or grass silage- based diets (Benchaar et al., 2006a; Benchaar et al., 2007a), but it was depressed when the diet contained corn silage (Benchaar et al., 2006a). Kung et al. (2008) reported higher dry matter intakes for lactating cows fed 1 g/day (i.e. 42 mg/kg of dietary dry matter) of the MEO. However, milk production was not significantly (P = 0.16) affected, although it was numerically increased (1.9 ± 0.9 kg/day) in cows fed the supplemented diet. In the later study by Kung et al. (2008), cows supplemented with MEO produced more fat-corrected milk than did the cows fed the control diet. Yang et al. (2007) observed that feeding garlic oil at 5 g/day (i.e. 245 mg/kg of dry matter intake) and juniper berry oil at 2 g/day (i.e. 98 mg/kg of dry matter intake) to lactating dairy cows had no effect on intake, milk production and milk composition. More recently, Benchaar et al. (2008) reported no changes in dry matter intake, milk production and milk components of dairy cows fed cinnamaldehyde at the dose of 1g/day (i.e. 43 mg/kg of dry matter intake). Few studies have examined the effects of essential oils and their main constituents on growth, carcass composition and meat quality. Benchaar et al. (2006b) evaluated growth performance of beef cattle supplemented with 2 (i.e. 240 mg/kg of dry matter intake) or 4 g/day (i.e. 460 mg/kg of dry matter intake) of the MEO in a silage-based diet. Results showed no changes in dry matter intake and average daily gain, but feed conversion (i.e. gain to dry matter intake ratio) was affected quadratically with a dose of 2 g/day maximizing

C. Benchaar et al. 137

feed efficiency. Bampidis et al. (2005) observed no change in dry matter intake, gain and efficiency of feed utilisation when growing lambs were fed diets supplemented with oregano leaves (Origanum vulgare L.) providing the equivalent of 144 or 288 mg of oregano oil (containing 85% of carvacrol) per kilogram of diet dry matter. Chaves et al. (2008a) reported that the addition (200 mg/kg of dietary dry matter) of cinnamadehyde or carvacrol to a barley- or corn-based diet had no effects on dry matter intake, gain, feed efficiency, carcass characteristics and meat quality of growing lambs. In another study by the same authors (Chaves et al., 2008b), supplementation (200 mg/kg of dietary dry matter) of a barley concentrate- based diet with cinnamaldehyde, garlic oil or juniper berry oil had no effects on feed intake. However, feeding cinnamaldehyde or juniper berry oil increased average daily gain and numerically improved feed conversion compared to feeding a diet containing no additives. The addition of cinnamaldehyde, garlic or juniper berry essential oils did not affect carcass characteristics and meat quality. Recently, plant secondary metabolites such as essential oils have been suggested as potential means to manipulate bacterial populations involved in rumen biohydrogenation in order to improve the fatty acid composition of ruminant-derived food products such as milk and meat. For instance, Durmic et al. (2008) observed that ethanolic extracts and essential oils from some Australian plants selectively inhibited the growth of pure cultures of some bacteria (e.g. Clostridium proteoclasticum) involved in rumen biohydrogenation and that some can inhibit the saturation of linoleic acid (C18:2), conjugated linoleic acid and vaccenic acid in batch culture incubations. This demonstrated the potential of plant extracts to increase output of conjugated linoleic and vaccenic acids from the rumen and to enhance the concentrations of these potentially beneficial unsaturated fatty acids in ruminant-derived food products. Only few studies have reported the effects of essential oils and their main components on the fatty acid composition of milk. Supplementing dairy cows with 750 mg/day of MEO did not affect milk fatty acid profile (Benchaar et al., 2007a). However, higher inclusion rates (i.e. 2 g/day; Benchaar et al., 2006a) enhanced milk fat content of conjugated linoleic acid (cis9 trans11 C18:1 isomer). Data on the effects of feeding essential oils on meat fatty composition are almost non-existent. In one study, Chaves et al. (2008c) reported no changes in fatty acid profile (including the concentrations of conjugated linoleic acids) of back fat of growing lambs fed a barley-based diet supplemented (200 mg/kg of dry matter intake) with cinnamaldehyde, garlic oil or juniper berry essential oils. The effects of essential oils and their main components on ruminant performance are small, which is not surprising considering the equivocal effects of these plant extracts on dry matter intake and rumen fermentation characteristics. In some studies, the greater performance observed when animals were supplemented with essential oils has been related to higher intakes rather to a modification in rumen microbial fermentation and nutrient utilization. In vitro data suggest that there may be potential to select essential oils that selectively inhibit rumen bacterial populations involved in the process of biohydrogenation of unsaturated

138 Essential oils as feed additives in ruminants fatty acids, which may enhance the concentrations of potentially health-promoting fatty acids (e.g. conjugated linoleic acid) in ruminant derived products. However, further research is required to assess the potential of essential oils to improve fatty acid composition of milk and meat.

Conclusions The antimicrobial activity of essential oils against a variety of microorganisms has been demonstrated in several studies. Essential oils and their constituents have been shown to inhibit the growth of several pathogenic bacteria such as Escherichia coli O157:H7, Salmonella spp and Staphylococcus aureus. This well documented antimicrobial activity of essential oils has recently prompted a plethora of researchers to examine their potential to modify rumen microbial populations to enhance efficiency of rumen fermentation and improve nutrient utilisation in ruminants. The resurgence of interest in using essential oils in ruminant nutrition and production has increased particularly in Europe after the ban on the use of growth-promoting antibiotics, including ionophores, in livestock production. In ruminant nutrition, the potential of essential oils to favourably alter microbial fermentation has been mostly assessed using batch or continuous culture in vitro systems. However, in vitro systems have limitations (i.e. difficulty of simulating the diversity and dynamics of microbial populations) and therefore, the ultimate value of essential oils for altering rumen microbial fermentation must be assessed in vivo. Results from in vitro studies show that the effects of essential oils and their main components on rumen microbial fermentation are variable and often contradictory, which can be related to differences in doses and the technique used (batch vs. continuous system). It appears that high doses of essential oils are required to alter rumen microbial fermentation and in most cases, the beneficial effects on N metabolism (i.e. reduction in protein degradation and ammonia N production) were counterbalanced by a decrease in total VFA concentration (i.e. feed degradation). When used at low and moderate doses, the effects were negligible, probably due to the adaptation of rumen microbes to essential oils, as has been shown in several continuous culture studies. The number of published papers on the effects of essential oils and their main components in vivo is surprisingly low. Results from the few studies published to date reveal no effects on ruminant performance (milk and growth), which is consistent with the lack of effects on rumen microbial fermentation characteristics (pH, VFA and ammonia N) and diet digestibility. Microbial populations exhibit a remarkable ability to adapt rapidly to a wide variety of antimicrobial agents such as ionophores, saponins and tannins and there is some evidence that the rumen microbial population also adapts to continual exposure to essential oils. This adaptive response represents a serious challenge for commercial application of this feed additive technology.

C. Benchaar et al. 139

Although little research has been conducted to investigate bacterial resistance to essential oils, some studies have shown that some pathogenic bacteria are able to develop resistance to essential oils. More research is warranted to determine the capacity of rumen bacteria to develop resistance to essential oils.

References Amagase H, Petesch BL, Matsuura H, Kasuga S and Itakura Y (2001) Intake of garlic and its bioactive components Journal of Nutrition 131: 955S-962S. Ando S, Nishida T, Ishida M, Hosoda K and Bayaru E (2003) Effect of peppermint feeding on the digestibility, ruminal fermentation and protozoa Livestock Production Science 82: 245–248. Anitescu G, Doneanu C and Radulescu V (1997) Isolation of coriander oil: comparison between steam distillation and supercritical CO2 extraction Flavour and Fragrance Journal 12: 173–176. Bager F, Madsen M, Christensen J and Aarestrup FM (1997) Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms Preventive Veterinary Medicine 31: 95112. Bampidis VA, Christodoulou V, Florou-Paneri P, Christaki E, Spais AB and Chatzopoulou PS (2005) Effect of dietary dried oregano leaves supplementation on performance and carcass characteristics of growing lambs Animal Feed Science and Technology 121: 285–295. Banthorpe DV (1994) Terpenoids. In Natural products: their chemistry and biological significance pp 289–359 Eds J Mann, RS Davidson, JB Hobbs, DV Banthorpe and JB Harborne. Longman Scientific and Technical, Harlow. Beauchemin KA and McGinn SM (2006) Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil Journal of Animal Science 84: 1489–1496. Benchaar C, Petit HV, Berthiaume R, Whyte TD and Chouinard PY (2006a) Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production and milk composition in dairy cows Journal of Dairy Science 89: 4352–4364. Benchaar C, Duynisveld JL and Charmley E (2006b) Effects of monensin and increasing dose levels of a mixture of essential oil compounds on intake, digestion and growth performance of beef cattle Canadian Journal of Animal Science 86: 91–96. Benchaar C, Petit HV, Berthiaume R, Ouellet DR, Chiquette J and Chouinard PY (2007a) Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition in dairy cows fed alfalfa silage or corn silage Journal of Dairy Science 90: 886–897. Benchaar C, Chaves AV, Fraser GR, Wang Y, Beauchemin KA and McAllister TA (2007b)

140 Essential oils as feed additives in ruminants Effects of essential oils and their components on in vitro rumen microbial fermentation Canadian Journal of Animal Science 87: 413–419. Benchaar C, McAllister TA and Chouinard PY (2008) Digestion, ruminal fermentation, ciliate protozoal populations, and milk production from dairy cows fed cinnamaldehyde, quebracho condensed tannin, or Yucca schidigera saponin extracts Journal of Dairy Science 91: 4765–4777. Borchers R (1965) Proteolytic activity of rumen fluid in vitro Journal of Animal Science 24: 1033–1038. Briskin DP (2000) Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health Plant Physiology 124: 507–514. Brul S and Coote P (1999) Preservative agents in foods - mode of action and microbial resistance mechanisms International Journal of Food Microbiology 50: 1–17. Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods - a review International Journal of Food Microbiology 94: 223–253. Busquet M, Calsamiglia S, Ferret A and Kamel C (2005a) Screening for the effects of natural plant extracts and secondary plant metabolites on rumen microbial fermentation in continuous culture Animal Feed Science and Technology 123: 597–613. Busquet M, Calsamiglia S, Ferret A, Cardozo PW and Kamel C (2005b) Effects of cinnamaldehyde and garlic oil on rumen microbial fermentation in a dual flow continuous culture Journal of Dairy Science 88: 2508–2516. Busquet M, Calsamiglia S, Ferret A and Kamel C (2005c) Screening for the effects of natural plant extracts and secondary plant metabolites on rumen microbial fermentation in continuous culture Animal Feed Science and Technology 123: 597–613. Busquet M, Calsamiglia S, Ferret A and Kamel C (2006) Plant extracts affect in vitro rumen microbial fermentation Journal of Dairy Science 89: 761–771. CABI, Commonwealth Agricultural Bureau International (2008). http://217.154.120.06/ CABDIRECT/select-database.nsp (accessed 16 November 2008). Callaway TR, Edrington TS, Rychlik JL, Genovese KJ, Poole TL, Jung YS, Bischoff KM, Anderson RC and Nisbet DJ (2003) Ionophores: their use as ruminant growth promotants and impact on food safety Current Issues in Intestinal Microbiology 4: 43–51. Calsamiglia S, Busquet M, Cardozo PW, Castillejos L and Ferret A (2007) Essential oils as modifiers of rumen microbial fermentation Journal of Dairy Science 90: 2580–2595. Cardozo PW, Calsamiglia S, Ferret A and Kamel C (2004) Effects of natural plant extracts on ruminal protein degradation and fermentation profiles in continuous culture Journal of Animal Science 82: 3230–3236. Cardozo PW, Calsamiglia S, Ferret A and Kamel C (2005) Screening for the effects of natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle Journal of Animal Science 83: 2572–2579. Cardozo PW, Calsamiglia S, Ferret A and Kamel C (2006) Effects of alfalfa extract, anise, capsicum, and a mixture of cinnamaldehyde and eugenol on ruminal fermentation

C. Benchaar et al. 141

and protein degradation in beef heifers fed a high-concentrate diet Journal of Animal Science 84: 2801–2808. Castillejos L, Calsamiglia S, Ferret A and Losa R (2005) Effects of a specific blend of essential oil compounds and the type of diet on rumen microbial fermentation and nutrient flow from a continuous culture system Animal Feed Science and Technology 119: 29–41. Castillejos L, Calsamiglia S, and Ferret A (2006) Effect of essential oils active compounds on rumen microbial fermentation and nutrient flow in in vitro systems Journal of Dairy Science 89: 2649–2658. Castillejos L, Calsamiglia S, Ferret A and Losa R (2007) Effects of dose and adaptation time of a specific blend of essential oils compounds on rumen fermentation Animal Feed Science and Technology 132: 186–201. Castillejos L, Calsamiglia S, Martìn-Tereso J and Ter Wijlen H (2008) In vitro evaluation of effects of ten essential oils at three doses on ruminal fermentation of high concentrate feedlot-type diets Animal Feed Science and Technology 145: 259–270. Chaves AV, Stanford K, Gibson L, McAllister TA and Benchaar C (2008a) Effects of carvacrol and cinnamaldehyde on intake, rumen fermentation, growth performance, and carcass characteristics of growing lambs Animal Feed Science and Technology 145: 396–408. Chaves AV, He ML, Yang WZ, Hristov AN, McAllister TA and Benchaar C (2008b) Effects of essential oils on proteolytic, deaminative and methanogenic activities of mixed ruminal bacteria Canadian Journal of Animal Science 88: 117–122. Chaves AV, Stanford K, Dugan MER, Gibson LL, McAllister TA, Van Herk F and Benchaar C (2008c) Effects of cinnamaldehyde, garlic and juniper berry essential oils on rumen fermentation, blood metabolites, growth performance, and carcass characteristics of growing lambs Livestock Science 117: 215–224. Chen MS (2008) Inducible direct plant defense against insect herbivores: A review Insect Science 15: 101–114. Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V, Arzedi E and Palmas F (1999) In vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils Letters in Applied Microbiology 29: 130–135. Cristani M, D’Arrigo M, Mandalari G, Castelli F, Sarpietro MG, Micieli D, Venuti V, Bisignano G, Saija A and Trombetta D (2007) Interaction of four monoterpenes contained in essential oils with model membranes: Implications for their antibacterial activity Journal of Agricultural and Food Chemistry 55: 6300–6308. Dehority BA (2005) In memoriam: Robert Edward Hungate (1906–2004) Journal of Eukaryotic Microbiology 52: 396–397. Dewick PM (2002) Medicinal Natural Products, Second Edition. John Wiley and Sons Ltd., Chichester. Di Pasqua R, Betts G, Hoskins N, Edwards M, Ercolini D and Mauriello G (2007) Membrane toxicity of antimicrobial compounds from essential oils Journal of Agricultural and Food Chemistry 55: 4863–4870.

142 Essential oils as feed additives in ruminants Dorman HJD and Deans SG (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils Journal of Applied Microbiology 88: 308–316. Duffield TF, Rabiee AR and Lean IJ (2008a) A meta-analysis of the impact of monensin in lactating dairy cattle. Part 1. Metabolic effects Journal of Dairy Science 91: 1334–1346. Duffield TF, Rabiee AR and Lean IJ (2008b) A meta-analysis of the impact of monensin in lactating dairy cattle. Part 2. Production effects Journal of Dairy Science 91: 1347–1360. Duffield TF, Rabiee AR and Lean IJ (2008c) A meta-analysis of the impact of monensin in lactating dairy cattle. Part 3. Health and reproduction Journal of Dairy Science 91: 2328–2341. Durmic Z, McSweeney CS, Kemp GW, Hutton P, Wallace RJ and Vercoe PE (2008) Autralian plants with potential to inhibit bacteria and processes involved in ruminal biohydrogenation of fatty acids Animal Feed Science and Technology 145: 271–284. EPA (2005) Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990-2005. http:// epa.gov/climatechange/emissions/usinventoryreport.html (accessed 16 November 2008). Evans JD and Martin SA (2000) Effects of thymol on ruminal microorganisms Current Microbiology 41: 336–340. Fraser GR, Chaves AV, Wang Y, McAllister TA, Beauchemin KA and Benchaar C (2007) Assessment of the effects of cinnamon leaf oil on rumen microbial fermentation using two continuous culture systems Journal of Dairy Science 90: 2315–2328. Gang DR, Wang JH, Dudareva N, Nam KH, Simon JE, Lewinsohn E and Pichersky E (2001) An investigation of the storage and biosynthesis of phenylpropenes in sweet basil Plant Physiology 125: 539–555. Gershenzon J, McCaskill D, Rajaonarivony JIM, Mihaliak C, Karp F and Croteau R (1992) Isolation of secretory-cells from plant glandular trichomes and their use in biosynthetic-studies of monoterpenes and other gland products Analytical Biochemistry 200: 130–138. Greathead H (2003) Plants and plant extracts for improving animal productivity Proceedings of the Nutrition Society 62: 279–290. Hart KJ, Yanez-Ruiz DR, Duval SM, McEwan NR and Newbold CJ (2008) Plant extracts to manipulate rumen fermentation Animal Feed Science and Technology 147: 8–35. Helander M, Alakomi H, Latva-Kala K, Mattila-Sandholm T, Pol I, Smid EJ, Gorris LGM and Wright AV (1998) Characterization of the action of selected essential oil components on Gram-negative bacteria Journal of Agricultural and Food Chemistry 46: 3590–3595. Holmes CA (2007) IENICA European Summary Report 2000-2005. http://www.ienica.net/ reports/ienicafinalsummaryreport2000-2005.pdf (accessed 10 December 2008). Hristov AN and Jouany J-P (2005) Factors affecting the efficiency of nitrogen utilization in the rumen. In Nitrogen and Phosphorus Nutrition of Cattle: Reducing the

C. Benchaar et al. 143

Environmental Impact of Cattle Operations pp 117–166 Eds E Pfeffer and Hristov AN. CAB International, Wallingford, U.K. Hristov AN, Price WJ, and Shafii B (2005) A meta-analysis on the relationship between intake of nutrients and body weight with milk volume and milk protein yield in dairy cows Journal of Dairy Science 88: 2860–2869. Hristov AN, Ropp JK, Zaman S and Melgar A (2008) Effects of essential oils on in vitro ruminal fermentation and ammonia release Animal Feed Science and Technology 144: 55–64. Hulin V, Mathot AG, Mafart P and Dufosse L (1998) Les propriétés anti-microbiennes des huiles essentielles et composés d’arômes [Antimicrobial properties of essential oils and flavour compounds] Sciences des Aliments 18: 563–582. Hungate RE (1966) The rumen and its microbes. Academic Press, New York. Johnson KA and Johnson DE (1995) Methane emissions from cattle. Journal of Animal Science 73: 2483 –2492. Kim J, Marshall MR and Wei CI (1995) Antibacterial activity of some essential oil compounds against five food-borne pathogens Journal of Agricultural and Food Chemistry 43: 2839–2845. Kung LJr, Williams P, Schmidt RJ and Hu W (2008) A blend of essential plant oils used as an additive to alter silage fermentation or used as a feed additive for lactating dairy cows Journal of Dairy Science 91: 4793–4800 Lambert RJW, Skandamis PN, Coote PJ and Nychas GJE (2001) A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol Journal of Applied Microbiology 91: 453–462. Makkar HPS (2003) Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds Small Ruminant Research 49: 241–256. Makkar HPS, Becker K, Abel HJ and Szegletti C (1995) Degradation of condensed tannins by rumen microbes exposed to quebracho tannins (QT) in rumen simulation technique (RUSITEC) and effects of QT on fermentation processes in the RUSITEC Journal of the Science of Food and Agriculture 69: 495–500. Mansfield HR, Endres MI, and Stern MD (1995) Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture Animal Feed Science and Technology 55: 47–66. Markoff I (1913) Biochemische Zeitschrift 57 1–70 (as cited by Hungate, RE (1966) The rumen and its microbes. Academic Press, New York. McIntosh FM., Williams P, Losa R, Wallace RJ, Beever DA and Newbold CJ (2003) Effects of essential oils on ruminal microorganisms and their protein metabolism Applied and Environmental Microbiology 69: 5011–5014. Mellor S (2000) Herbs and spices promote health and growth Pig Progress 16: 27–30. Mohammed N, Ajisaka N, Lila ZA, Mikuni K, Hara K, Kanda S and Itabashi H (2004) Effect of Japanese horseradish oil on methane production and ruminal fermentation in vitro and in steers Journal of Animal Science 82: 1839–1846.

144 Essential oils as feed additives in ruminants Molero R, Ibara M, Calsamiglia S, Ferret A and Losa R (2004) Effects of a specific blend of essential oil compounds on dry matter and crude protein degradability in heifers fed diets with different forage to concentrate ratios Animal Feed Science and Technology 114: 91–104. Nagaraja TG, Newbold CJ, Van Nevel CJ and Demeyer DI (1997) Manipulation of ruminal fermentation. In The Rumen Microbial Ecosystem pp 523-623 Eds PN Hobson and Steward CS. Blackie Academic and Professional, London. Nelson RRS (2000) Selection of resistance to the essential oil of Melaleuca alternifolia in Staphylococcus aureus Journal of Antimicrobial Chemotherapy 45: 549–550. Newbold CJ, Lassalas B and Jouany JP (1995) The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro Letters in Applied Microbiology 21: 230–234. Newbold CJ, El Hassan SM, Wang J, Ortega ME and Wallace RJ (1997) Influence of foliage from African multipurpose trees on activity of rumen protozoa and bacteria British Journal of Nutrition 78: 237–249. Newbold CJ, McIntosh FM, Williams P, Losa R and Wallace RJ (2004) Effects of a specific blend of essential oil compounds on rumen fermentation Animal Feed Science and Technology 114: 105–112. Newman DJ, Cragg GM and Snader KM (2000) The influence of natural products upon drug discovery Natural Product Reports 17: 215–234. Nikaido H (1994) Prevention of drug access to bacterial targets: Permeability barriers and active efflux Science 264: 382–388. OJEU (2003) Regulation (EC) No 1831/2003 of the European Parliament and the Council of 22 September 2003 on additives for use in animal nutrition Official Journal of European Union Page L268/36 in OJEU of 10/18/2003. Packiyasothy EV and Kyle S (2002) Antimicrobial properties of some herb essential oils Food Australia 54: 384–387. Paparella A, Taccogna L, Aguzzi I, Chaves-Lopez C, Serio A, Marsilio F and Suzzi G (2008) Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes Food Control 19: 1174–1182. Pew Commission on Industrial Farm Animal Production (2008) Putting Meat on the Table: Industrial Farm Animal Production in America. http://www.pewtrusts.org/ news_room_detail.aspx?id=38438 (accessed 16 November 2008). Rasmussen M, Franklin S, McNeff C and Carlson S (2005) Control of pathogens using defaunation. In Proceedings of the Third International Rushmore Conference: Strategies in the Prevention of Enteric Disease and Dissemination of Food-Bourne Pathogens p 35. Rapid City, South Dakota, USA. Russell JB and Houlihan AJ (2003) Ionophore resistance of ruminal bacteria and its potential impact on human health FEMS Microbiology Reviews 27: 65–74. Sangwan NS, Farooqi AHA, Shabih F and Sangwan RS (2001) Regulation of essential oil production in plants Plant Growth Regulation 34: 3–21. Slyter LL and Putnam PA (1967) In vivo vs. in vitro continuous culture of ruminal microbial

C. Benchaar et al. 145

populations Journal of Animal Science 26: 1421–1427. Smith-Palmer A, Stewart J and Fyfe L (1998) Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens Letters in Applied Microbiology 26: 118–122. Steinfeld H, Gerber P, Wassenaar T, Castel V, Rosales M and de Haan C (2006) Livestock’s Long Shadow. Environmental Issues and Options. ftp://ftp.fao.org/docrep/fao/010/ A0701E/A0701E00.pdf (accessed 16 November 2008). Swann MM (1969) Report of Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine. HMSO, London. Tatsuoka N, Hara K, Mikuni K, Hara K, Hashimoto H, Itabashi H (2008) Effects of the essential oil cyclodextrin complexes on ruminal methane production in vitro Animal Science Journal 79: 68–75. Tedeschi LO, Fox DG and Tylutki TP (2003) Potential environmental benefits of ionophores in ruminant diets Journal of Environmental Quality 32: 1591–1602. Thoroski J, Blank G and Biliaderis C (1989) Eugenol induced-inhibition of extracellular enzyme production by Bacillus cereus Journal of Food Protection 52: 399–403. Ultee A, Kets EPW and Smid EJ (1999) Mechanisms of action of carvacrol on the foodborne pathogen Bacillus cereus Applied and Environmental Microbiology 65: 4606–4610. Ultee A, Kets EPW, Alberda M, Hoekstra FA and Smid EJ (2000a) Adaptation of the food-borne pathogen Bacillus cereus to carvacrol Archives of Microbiology 174: 233–238. Ultee A, Slump RA, Steging G and Smid EJ (2000b) Antimicrobial activity of carvacrol toward Bacillus cereus on rice Journal of Food Protection 63: 620–624. Ultee A, Bennik MHJ and Moezelaar R (2002) The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus Applied and Environmental Microbiology 68: 1561–1568. Urdang G (1948) The origin and development of the essential oil industry. In The Essential Oils pp 3–13 Ed E Guenther. Van Nostrand Reinhold Company, New York. Van de Braak SAAJ and Leijten GCJJ (1999) Essential oils and oleoresins: A survey in the Netherlands and other major markets in the European Union. CBI, Centre for the Promotion of Imports from Developing Countries, Rotterdam. Vokou D, Kokkini S and Bessiere JM (1993) Geographic variation of Greek oregano (Origanum vulgare ssp hirtum) essential oils Biochemical Systematics and Ecology 21: 287–295. Wallace RJ (2004) Antimicrobial properties of plant secondary metabolites Proceedings of the Nutrition Society 63: 621-629. Wink M (1999) Functions of plant secondary metabolites and their exploitation in biotechnology. In Annual Plant Reviews pp 304 Ed M Wink. Sheffield Academic Press, Sheffield. Wink M (2001) Secondary metabolites: deterring herbivores. In Encyclopedia of Life Sciences John Wiley and Sons, Inc.

146 Essential oils as feed additives in ruminants Xu J, Zhou F, Ji BP, Pei RS and Xu N (2008) The antibacterial mechanism of carvacrol and thymol against Escherichia coli Letters in Applied Microbiology 47: 174–179. Yang WZ, Benchaar C, Ametaj BN, Chaves AV, He ML and McAllister TA (2007) Effects of garlic and juniper berry essential oils on ruminal fermentation and on the site and extent of digestion in lactating cows Journal of Dairy Science 90: 5671–5681.

P. Encarnação 147

8 THE POTENTIAL OF PHYTOGENIC COMPOUNDS IN AQUACULTURE Pedro Encarnação BIOMIN Singapore Pte Ltd, 3791 Jalan Bukit Merah #08-08, E-Centre@Redhill, Singapore 159471, e-mail: [email protected]

Background The global aquaculture production has grown rapidly during the past decades, contributing significant quantities to the world’s fish supply for human consumption. As aquaculture expands and develops, an increase in problems and challenges arises. Some of the most relevant problems currently confronting the aqua industry are related to the widespread occurrence of disease, e.g. parasitic infestation, bacterial and viral infections, which can lead to heavy losses to the industry. In this particular aspect, we need to consider that the host-microbe interactions are often qualitatively as well as quantitatively different for aquatic and terrestrial species. The aquatic environment is rich in microorganism (up to 105 to 106 per ml), with hosts and microorganisms sharing the same ecosystem. Thus, much more than terrestrial animals, aquatic farmed animals are surrounded by an environment that supports their pathogens independently of the host animals, as such, (opportunistic) pathogens can reach high densities around the animal. Surrounding bacteria are continuously ingested either with the feed or when the host is drinking, causing a natural interaction between the microbiota of the ambient environment and the gut environment. If the bacterial challenge exceeds a certain level, the health of the animal is in danger, as the animal alone cannot defend itself sufficiently (Verschuere et al., 2000). As such, health management is one of the most important factors for the control of specific pathogens responsible for high mortalities and also other organisms responsible for reduced growth, increased FCR or any other effect that reduces the commercial value of the fish/shrimp being produced. Different strategies have been used to face the bacterial and viral threats, chemotherapy being the most used approach, using large amounts of antibiotics and chemical products. Nonetheless, the development of drug resistance in bacteria, the accumulation of chemicals in the environment and in shrimp/fish muscle have led to strict regulations that limit the use of antibiotics and other chemicals in aquaculture operations. In addition, from mammalian research it is well known that the gastrointestinal tract is responsive and sensitive to a wide range of stressors. Some of the more common features are degeneration of the intestinal 147

148 Phytogenics in aquaculture mucosa and perturbation of its barrier function and uptake mechanisms (Ringø and Olsen, 1999). Closely connected with the state of health of the gut is a well balanced intestinal micro-flora, which helps the digestive and absorptive process and protects the host against invading pathogens. During the last decade, there has been an improvement in understanding the importance of intestinal microbiota in fish. Floral health is a new concept, which underlines the importance of the microbiota to the intestinal health and performance. As a result, there is increasing evidence that the complex microbial ecology of the intestinal tract provides both nutritional benefit and protection against pathogens, and it is vital in modulating interactions with the environment and the development of beneficial immune responses. Several studies also have shown that different feed ingredients and changes in diet composition can affect gut structure and microbiota balance influencing digestive and absorptive functions (Ringø and Olsen, 1999). Replacing marine ingredients with plantbased ingredients exposes fish to a series of “foreign” components, for example, starch and anti-nutrients that may upset natural processes occurring in the intestine. Plant components, such as lectins, saponins, phyto-oestrogens, phytic acid, tannins and others, which do not exist in the natural feed of wild fish, may disturb digestive processes and affect health (Lilleeng, 2008). Alteration of the intestinal microbiota composition and consequent reduction of protective gut microflora may contribute to pathogenesis in the gut (Ringø and Olsen, 1999). Plant ingredients also introduce proteins that may stress the immune system of the intestine (Lilleeng, 2008). Management of the gut flora is, therefore, an important issue to achieve a good feed efficiency, animal growth and animal health. Management means selection of beneficial strains, control of their numbers, minimizing number of negative or potential pathogenic strains. Nowadays, we have learned about more sustainable ways to manage fish health and performance using nutriceuticals or functional foods to modulate the health of farmed animals. The gut is the main entry point for bacterial and viral infections. The microbiota associated with the fish intestinal system can be affected by products and substances that, directly or indirectly, give an advantage to a particular group of microorganisms, reduce or even inhibit the growth of other bacterial groups. The possibility to modulate the gut microbiota by a choice of natural solutions is real, considering the documented ability of particular ingredients to alter the intestinal structure, modulate the innate and adaptive immune response, increase stress resistance and directly affect the growth of potential pathogens, reduce their ability to colonize the gastrointestinal tract or affect them in a way to reduce their growth depressing effects (Rojas, 2007).

Phytogenics Also in aquaculture phytogenics represent a relatively young class of feed additives and the knowledge regarding their mode of action and application strategies is rather

P. Encarnação 149

fragmented. Phytogenics are plant-derived products which are added to the feed in order to improve palatability of feeds or animal performance. These plant active ingredients (e.g. phenolic compounds and flavanoids) can exert multiple effects on the organisms, including improvement of feed efficiency and digestion, reduction of nitrogen excretion and improvement of gut flora and health status (Kroismayr, 2007). Many mechanisms have been proposed for the beneficial actions of phytogenics in different species. These range from direct reduction of gut bacteria and stimulation of growth and acid production by beneficial species such as Lactobacillus, to the enhancement of specific elements of both humeral and cell-mediated arms of the immune system (Cardozo et al., 2008). Phytogenic feed additives are an extremely heterogeneous group of feed additives originating from leaves, roots, tubers or fruits of herbs, spices or other plants. They are either available in a solid, dried or ground form or as extracts or essential oils. Within phytogenic feed additives, the content of active substances in products may vary widely, depending upon the plant part used (e.g., seeds, leaf, root, and bark), harvesting season, and geographical origin (Steiner, 2006). Phytogenics can have antioxidative and/or antimicrobial activity.

Essential oils Essential oils are odoriferous, secondary plant products which contain most of the plant’s active compounds (e.g. alcohol, aldehydes, ketones, phenolic compounds, etc). Processing modifies the active substances and associated compounds within the final product (e.g. by cold expression, steam distillation, or extraction with non aqueous solvents). The plant family of Labiatae has received most interest with thyme, oregano and sage as the most popular representatives (Steiner, 2006). Research on the antimicrobial activity of essential oils demonstrated their beneficial effects on Salmonella typhimurium, E. coli, and Listeria monocytogenes, among others (Rojas, 2007). Higher inhibitory capacity has been observed in the oils with higher percentage of phenolic components (carvacol and thymol) in comparison with oils containing monoterpenic alcohol linalol (Rojas, 2007). These phenolic compounds are known to be powerful antimicrobial agents due to their toxic effects on the bacterial cell wall. The antimicrobial mode of action is considered to arise mainly from the potential of hydrophobic essential oils to intrude into the bacterial cell membrane, to disintegrate membrane structures and cause ion leakage (Kroismayr, 2007). Many essential oils components are generally recognized as safe (GRAS) and have been used for many years in the food, cosmetic and pharmaceutical industries. Among the herbs and spices used in animal nutrition, oregano is probably used most frequently, it is rich in carvacrol, thymol and other active principles, which are known to have strong antibacterial and antioxidant properties and described as acting synergistically (Burt, 2004). Essential oils extracted from rosemary (Rosmarinus officinalis) also seem particularly interesting due to their high concentration of components such as carnasol and carnosic acid, which have strong antioxidant properties (Abutbul et al., 2004).

150 Phytogenics in aquaculture

Effects of essential oils in aquaculture species Effects of phytogenics in fish In animal nutrition, phytogenics are an interesting category of feed additives due to their different beneficial effects on palatability of feeds and performance. Most studies on application of essential oils in animal nutrition have been conducted in swine and poultry, however, there is increasing evidence that the application of phytogenics can also be beneficial for some aquaculture species, such as fish and shrimp. A series of trials conducted at the Aquaculture Center of Applied Animal Nutrition (ACAN), Bangkok, Thailand, confirmed that essential oils derived from oregano, anise and citrus peel (Biomin® P.E.P.) have a positive effect on Pangasius catfish (P. hypothalamus) (Figure 1) and red tilapia (Orechromis niloticus × O. Mussambicus.) growth performance (Table 1). When included in a Pangasius commercial-type feed at levels of 125 g/ton, fish showed improved growth rates (6%) and improved FCR (1.32 vs. 1.38). Similar increases in

Weight gain (g/fish)

40

b %

a

35

+6.0

30

25 Control

PEP

Figure 1. Effect of an essential oils based product (PEP) on growth performance of Pangasius hypothalamus after a 4 weeks trial

Table 1. Growth performance (weight gain, FCR) of red tilapia after fed different concentrations of an essential oils based product during an 8 week trial

Diet 1 2 3

PEP 125 g/t 0 90 120

Initial BW (g/fish) 134.8 137.3 135.8

4 210 137.2 DGC: Daily Growth Coefficient

1

Final BW (g/fish) 181.8 187.6 186.6

Gain (g/fish) 48.8 50.4 50.8

Feed (g/fish) 109.6 108.9 108.3

FCR 2.26 2.17 2.14

DGC1 (%) 1.28 1.35 1.37

188.0

50.9

108.9

2.19

1.36

P. Encarnação 151

performance was achieved in tilapia where improvements in weight gain and FCR were obtained when the same mixture of essential oils (Biomin®PEP125) was supplemented at levels of 90, 120 and 240 ppm in the diet (Table 1). The best results however, were obtained at levels of inclusion of 120 ppm, which resulted in an increase of 8.0% in weight gain and a reduction of 5.3% in FCR (Table 1). Several other studies tested the application of essential oils of different sources in aqua species (Cardozo et al., 2008; Rojas, 2007; Abutbul et al., 2004; Athanassopoulo et al., 2004). These studies focused mainly on the therapeutic effects of essential oils against pathogenic bacteria and parasites. Rojas (2007) reported that the application of thyme essential oil through feed using a preventive dosage (dosage not stated in report) improved the survival of Atlantic salmon (Salmo salar) when challenged with Saprolegnia parasitica after 30 days of feeding compared to the control (8 vs. 48% mortality). A curative dosage of thyme essential oil applied after the appearance of the first signs of disease was not as effective as the preventive treatment but still better than the control (8 vs. 23% mortality). In another study, Abutbul et al. (2004) investigated the effects of rosemary (Rosmarinus officinalis) extracts as a treatment for Streptococcus iniae in tilapia. The in vitro bactericidal tests revealed that all tested R. officinalis extracts showed antibacterial activity against S. iniae with the ethyl acetate extract giving the strongest inhibitory effect. A significant reduction in mortality of infected tilapia was then obtained when fish were fed a diet containing ethyl acetate extract of R. officinalis (1:24 w/w) or leaf powder. Moreover, no significant differences in mortality of fish were found between the two R. officinalis treatments and the oxytetracycline treatment. In European sea bream (Sparus aurata), Athanassopoulo et al. (2004) tested the application of oregano essential oil along with other anti-parasitic drugs for treatment of fish infected with parasitic myxosporean (Polysporoplasma sparis). Oregano essential oils have been found to have inhibitory effects on microorganisms (Athanassopoulou et al., 2000) and spore-forming organisms (Mejiholm and Dalgaard, 2002), however, this was the first time that they were tested against myxosporean infections in fish. One land-based experiment and one experimental cage trial were performed for this purpose (Athanassopoulo et al., 2004). In the land-based experiment, 25 and 50 g fish infected with the same parasite were treated with oregano essential oils, Toltrazuril with propylene glycol, Amprolium, and a combination of Salinomycin 12% + Amprolium (SA). In the field trials, 15 and 155 g S. aurata infected with the same parasite were treated with SA, oregano essential oils and Fumagillin. In all trials the drugs were incorporated into feed (by dilution in cod liver oil and top-coated onto commercial pellets with a mechanical mixer) and administered according to the selected schemes, while their efficacy was evaluated in terms of mortality, pathology and prevalence rate of P. sparis (Athanassopoulo et al., 2004). The most efficient treatment for 25 g fish were SA and oregano oil at a dosage of 2.4 ml/kg BW (statistically different from control treatment, P < 0.05, but not between these treatments). These products reduced prevalence from 50 to 0–4%, whereas untreated control fish showed a final prevalence of 11%. For the 155 gfish, SA was more efficient, but the application of oregano essential oil significantly reduced the prevalence of P. sparis in the fish (Athanassopoulo et al., 2004).

152 Phytogenics in aquaculture Effects of phytogenics in shrimp In shrimp, Cardozo et al. (2008) tested the application of an encapsulated combination of two active ingredients of Origanum vulgare (thymol and carvacrol) under normal and stress challenge conditions. The thymol/carvacol mixture was tested in white shrimp (Litopenaeus vannamei) diets at a concentration of 30 ppm. After a 28-day feeding trial there were no significant differences in growth, but the group fed the phytogenic substances showed an improved FCR (1.21 vs. 1.29). On day 29 the shrimp were then exposed 24 h to virulent Vibrio harveyii (106 CFU/ml). On day 56 the shrimp fed the thymol/carvacol diet sowed significant higher survival rates as compared with the control treatment (96.9 vs. 84.4%). Shrimp fed the phytogenic diet also had a significant better FCR and average daily gain when compared to the control treatment. The performance and survival benefits of the thymol/carvacol mixture correlated directly with improvements in the immune parameters with a significant increase in shrimp phagocytosis index and prophenoloxidase activity, indicative of a more resistant immune system against V. harveyii. The effects of six phytogenic extracts in juvenile shrimp (Penaeus indicus) were investigated by Immanuel et al. (2004). In this study, extracts of Ricinus communis, Phyllanthus niruri, Leucus aspera, Manihot esculenta, Ulva lactuca and Sargassum wightii were enriched in Artemia franciscana (brine shrimp) which were then fed to juveniles inoculated with the shrimp pathogen Vibrio paramhaemolyticus at 107 CFU/ml. Although the highest survival rate and best growth performance was observed in non-inoculated shrimp without herbal treatment, all plant extracts significantly increased survival and growth, and reduced pathogen load in comparison to the infected control. This may reflect the antimicrobial efficacies of these herbs against V. paramhaemolyticus and their potential immune-stimulating effects in shrimp.

In vitro immune-stimulating effects of phytogenics A stimulation of the immune system by essential oils was also observed in the non-specific immune system of common carp (Cyprinus carpio) during an in vitro trial project testing several natural extracts in carp’s head kidney and blood cell (Jeney, unpublished). In this trial, nitric oxide production in head kidney cells was significantly enhanced by a commercial phytogenic blend of essential oils derived from oregano, anis and citrus peel (Biomin® P.E.P.) at concentrations of 0.1, 1.0 and 5.0 μg/ml, and was higher than that observed for yeast extracts at doses of 1.0 and 5.0 μg/ml (Figure 2). Moreover, the respiratory burst was significantly increased in the treatment with the phytogenic blend at the three lowest concentrations (0.1, 1.0 and 5.0 μg/ml), while no significant changes were measured in other treatments. These in vitro results attest the capacity of essentials oils to stimulate the non-specific immune system in fish.

P. Encarnação 153

Phytogenic blend 1 Control 50 µg/ml LPS 0.1 µg/ml phytogenic blend 1 1.0 µg/ml phytogenic blend 1 5.0 µg/ml phytogenic blend 1 10.0 µg/ml phytogenic blend 1 30.0 µg/ml phytogenic blend 1 60.0 µg/ml phytogenic blend 1

70 60

µΜ ΝΟ

50 40

* ** *

30 20 10 0

Yeast extract 1

Control 50 µg/ml LPS 0.1 µg/ml yeast cell wall 1 1.0 µg/ml yeast cell wall 1 5.0 µg/ml yeast cell wall 1

40

µΜ ΝΟ

30

*

20

*

10

0

40

µΜ ΝΟ

30

20

Yeast extract 2

* * *

Control 50 µg/ml LPS 0.1 µg/ml yeast cell wall 2 1.0 µg/ml yeast cell wall 2 5.0 µg/ml yeast cell wall 2

10

0

Figure 2. Changes in nitric oxide production in macrophages isolated from head kidney following in vitro incubation with different doses of test substances. Significant differences (P < 0.05) from negative control are shown by asterisks.

Conclusions Sustainable aquaculture development requires the use of safe and effective solutions to tackle the industry challenges. There is increasing evidence that natural products such as essential

154 Phytogenics in aquaculture oils could have application in aquaculture, including prophylactic and therapeutically agents to control some major bacterial and fungal diseases and also for growth promotion.

References Abutbul S, Golan-Goldhirsh A, Barazani O and Zilberg D (2004) Use of Rosmarinus officinalis as a treatment against Streptococcus iniae in tilapia (Oreochromis sp.). Aquaculture 238: 97–106. Athanassopoulou F, Kotou E, Watsos E and Giagnisi M (2000) Study of the bacteriostatic ability of an Angelica sp. derivedcompound used for the enrichment of live feed of marine fish. Journal of the Hellenic Veterinary Association 51: 293–296. Athanassoupoulou FA, Karagouni E, Dotsika E, Ragias V, Tavla J and Christofilloyanis P (2004) Efficacy and toxicity or oral administered anticoccidial drugs for innovative treatments of Polysporoplasma sparis (Sitja-Bobadilla and Alvarez-Pelliteroi 1985) infection in Sparus aurata L. Journal of Applied Ichthyology 20: 345–354. Burt S (2004) Essential oils: their antibacterial properties and potential applications in food - a review. International Journal of Food Microbiology 94: 223–253. Cardozo P, Kamel C, Greathead HMR and Jintasataporn O (2008) Encapsulated plant extracts as dietary enhancers of growth, feeding efficiency and immunity in white shrimp (Litopenaeus vannamei) under normal and stress conditions. Aqua 2008. X Congresso Ecuatoriano de Acuicultura & Aquaexpo. October 6–9. Guayaquil. Ecuador. Abstract. Immanuel G, Vincybai VC, Sivaram V, Palavesam A and Marian MP (2004) Effect of butanolic extracts from terrestrial herbs and seaweeds on the survival, growth and pathogen (Vibrio parahaemolyticus) load on shrimp Penaeus indicus juveniles. Aquaculture 236: 53–65. Jeney G (unpublished) Effect of different immune-stimulant substances on non-specific immune system activity of common carp (Cyprinus carpio): in vitro study. Project report. Research Institute for Fisheries, Aquaculture and Irrigation (HAKI). Szarvas, Hungary. Krosmayr A (2007). Experimental studies of the gastrointestinal effects of essential oils in comparison to avilamycin in weaned piglets. PhD dissertation. Universität für Bodenkultur Wien. Mejiholm O and Dalgaard P (2002) Antimicrobial effect of essential oils on the seafood spoilage micro-organism Photobacterium phosphoreum in liquid media and fish products. Letters in Applied Microbiology 34: 27–31. Ringø E and Olsen RE (1999) The effect of diet on aerobic bacterial flora associated with intestine of Artic charr (Salvelinus alpinus L.). Journal of Applied Microbiology 86: 22–28. Rojas A (2007) Potential essential oil applications within the salmon industry in Chile. International Aqua Feed. September-October. Pp. 32–36.

P. Encarnação 155

Steiner T (2006) Managing Gut Health. Natural Growth Promoters as a Key to Animal Performance. Nottingham University Press. 98 pp. Verschuere L, Rombaut G, Sorgeloos P and Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Reviews 64: 655–671.

156 Phytogenics in aquaculture

T. Steiner 157

9 APPLICATION AND BENEFITS OF PHYTOGENICS IN EGG PRODUCTION Tobias Steiner BIOMIN Holding GmbH, Industriestrasse 21, 3130 Herzogenburg, Austria, e-mail: [email protected]

Abstract The use of plants, plant extracts or their active ingredients in diets for laying hens has been investigated in a limited number of in vivo trials. Depending on the type and dosage of the substance used, a down-regulation of feed consumption, associated with an increased feed efficiency, was observed in several trials. Moreover, there is evidence that dietary addition of plants or plant extracts affects the oxidative stability of egg yolks, which might be an interesting subject to the egg processing industry. Effects on egg quality traits, such as yolk composition, shell thickness or Haugh Unit rating, were reported in a few studies only. Future research should focus on the identification of the most suitable phytogenic substances to develop compound feed additives which may positively affect gut health and performance of laying hens, thus contributing to the overall productivity in egg production.

Introduction Within the last decades, production performance of modern commercial laying hens has improved considerably, including an increase in egg production and a reduction in feed conversion. Various factors, such as genetics, housing, vaccination, lighting, nutrition, moulting, ambient temperature and processing, may affect the productivity in egg production (Alodan and Mashaly, 1999; Amerah et al., 2007; Franco-Jimenez et al., 2007; Singh et al., 2009). Among these factors, optimal feeding strategies are mandatory to meet the huge metabolic demand of modern laying hens. Undoubtedly, the requirement for energy, nutrients, trace minerals and vitamins of these high-performing birds must be met by implementation of adequate feed formulations, adapted to the bird’s requirement as closely as possible. Furthermore, it must be considered that the digestive tract is a vital organ in which feed is reconstituted biochemically into performance, i.e. egg mass. Unquestionably, only a healthy digestive tract is capable of dealing with this effort in a competitive biodiverse environment and under sometimes challenging external conditions. Therefore, a 157

158 Phytogenics in egg production fundamental objective in feeding the modern laying hen is to keep this important organ healthy, thereby maintaining its functionality on a high and efficient level. Adequate energy intake and utilization is a prerequisite to maintain production on a high level throughout the production cycle. It is well known that the bird regulates its feed intake based on the energy concentration in the feed (Harms et al., 2000; Valkonen et al., 2008). However, reduced energy intake and/or utilization may cause drops in egg production and egg size. The period between housing and peak production is probably the most critical time, which is often associated with reduced feed consumption. In addition, heat stress has a negative impact on feed intake and conversion (Franco-Jimenez et al., 2007; Grizzle et al., 1992). As a matter of fact, egg producers are looking for strategies to optimize feed efficiency of their flocks, especially in times of volatile feed prices. Suitable feed additives represent one potential option to help achieving this target (Steiner, 2006). As reported in the previous chapters, there is considerable potential to support gut health and positively influence performance using phytogenics in swine, ruminants, broilers, fish and shrimp. Whilst for these animal species the number of studies pertaining to the application of phytogenics is increasing rapidly, relatively little information is available as to the application and potential benefits of phytogenics in laying hens.

Effects of phytogenics on performance In broilers performance parameters were improved by supplementation of diets with plants, plant extracts, their single active principles or blended additives (Chapters 4 and 6). Furthermore, the in vivo mode of action of phytogenic compounds has been investigated in several studies (Chapter 2). With regard to laying hens, there is a comparatively small number of studies that investigated the effects of phytogenic substances on production performance as well as their gastrointestinal effects. Experimental results reported so far are summarised in Table 1. Similarly to other species, a direct comparison among these studies is difficult due to the use of phytogenic preparations which differed in terms of their composition, physical form, content of active principles and dosages. Moreover, experimental conditions, as well as genetics and age of the birds may markedly have affected the results observed in the various trials. The preparations used in these experiments included intact herbs, ground plant material, parts of plants and essential oils, with inclusion levels ranging between 0.002 and 1% of finished feed. In several studies feed intake was markedly and significantly reduced (Bölükbaı et al., 2007, 2008; Abd El-Motaal et al., 2008) upon dietary supplementation with essential oils derived from thyme, sage or rosemary, as well as thyme powder. A significant stimulation of feed intake was not obtained in any of the studies. The lower feed consumption was accompanied by a reduction in feed conversion ratio (FCR), which was significant in five out of ten studies, indicating an increased efficiency of production. Indeed, egg production, as well as egg weights, were significantly increased in some of the experiments when hens were fed phytogenics (Bölükbaı et al., 2007; Bölükbaı and Erhan, 2007; Bölükbaı et al., 2008; Radwan et al., 2008). In contrast, no significant effects of phytogenics on performance parameters were reported by other authors (Botsoglou et al.,

T. Steiner 159

2005a; Florou-Paneri et al., 2005, 2006). A lack of effect on performance might be explained by excellent hygienic conditions in these experiments (Botsoglou et al., 2005a). However, additional experiments are urgently required to further elucidate the potential performance-enhancing effects of phytogenics in laying hens. The efficacy of phytogenics seems to be dose-related. In studies by Abd El-Motaal et al. (2008), Bölükbaı and Erhan (2007) and Radwan et al. (2008), a clear relation between the inclusion level of the substances under test and their effect on feed intake, FCR and egg production was noted. In the study by Abd El-Motaal et al. (2008), sixty brown Hy-Line layers were fed commercial diets supplemented with 0, 0.1, 0.2 or 0.3 % eucalyptus leaf powder from 46 to 54 weeks of age. Feed intake and FCR did not differ between birds fed eucalyptus up to a dosage of 0.2% However, with the inclusion of 0.3% there was a 5.1 and 9.1% reduction in feed intake and FCR, respectively. Moreover, the number of eggs produced and egg mass were also significantly higher in these birds. As indicated by Çabuk et al. (2006), inclusion of phytogenics might be useful to maintain laying performance in hot climatic conditions. The efficacy of a blend of essential oils, derived from oregano, laurel leaf, sage, myrtle, fennel seeds and citrus peel, was examined at temperatures reaching 37.8 °C in a trial with 54-week old Nick-Brown hens. Inclusion of the blend of essential oils in the diets significantly increased egg production and reduced FCR as compared to birds fed the control diet and a diet containing the antibiotic growth promoter Avilamycin at 10 mg/kg. Additionally, the number of cracked-broken eggs was reduced by 16%. Increased performance may be due to the various modes of action, including a modified gut microflora (Mitsch et al., 2004), stimulated secretion or activity of digestive enzymes (Jang et al., 2007), altered immune functions (Kroismayr et al., 2008) and histological changes (Jamroz et al., 2006). Supplementation of layer diets with 0.02% thyme, sage or rosemary oil reduced the faecal E. coli as well as coliform counts in a study with 24 week-old Lohmann-LSL laying hens (Bölükbaı et al., 2008). This is partly in agreement with Bölükbaı et al. (2007), who observed reduced faecal levels of E. coli, but not of coliforms, in response to feeding graded levels of thyme oil to laying hens. The counts of Clostridium perfringens in different sections of the digestive tract of broilers were lowered through dietary supplementation with a blend of thymol, eugenol, curcumin and piperin (Mitsch et al., 2004). Hence, a reduction of potentially pathogenic bacteria and a shift in the composition of the gut microflora towards more beneficial bacteria may reduce the competition for nutrients and dietary energy between the host and its microflora.

Effects of phytogenics on egg traits The effect of essential oils originating from thyme, sage or rosemary on egg quality traits was assessed by Bölükbaı et al. (2008) with 64 24-week-old Lohman-LSL hybrid hens. The oils were added to basal corn-soy-wheat-based diets at a dosage of 200 mg/kg. In addition to a reduction in feed consumption and FCR, essential oils reduced the proportion of yolk and increased the proportion of the egg shell, compared to the control treatment (Table 2).

160 Phytogenics in egg production Table 1. Effect (% increase/reduction vs. negative control) of dietary phytogenics on performance (Summary of trials) Reference

Compound

Abd El-Motaal et al. (2008) Eucalyptus leaf powder Eucalyptus leaf powder   Eucalyptus leaf powder Thyme oil 0.01% Bölükbaı et al. (2007) Thyme oil 0.02%   Thyme oil 0.03% Bölükbaı and Erhan (2007) Thyme powder Thyme powder   Thyme powder Thyme oil Bölükbaı et al. (2008) Sage oil   Rosemary oil Rosemary flowered tops Botsoglu et al. (2005a) and leaves Oregano flowered tops, leaves, stems and stalks   Saffron dried red stigmas Blend of essential oils2 Çabuk et al. (2006) Florou-Paneri et al. (2005) Oregano essential oil   Oregano essential oil Florou-Paneri et al. (2006) Rosemary (ground)   Rosemary (ground) Thyme Radwan et al. (2008) Thyme Oregano Oregano Rosemary Rosemary Curcuma longa Curcuma longa Nichol and Steiner (2008) Blend of essential oils3 Feed Conversion Ratio (kg feed/kg eggs)

Concentration in diet 0.1% 0.2% 0.3% 0.01%

Feed intake +0.3 -1.3 -5.1* -1.2

FCR1

0.02% 0.03% 0.1% 0.5% 1.0% 0.02% 0.02% 0.02% 0.5%

-6.1* -0.5 ±0.0 -2.1 -3.4* -5.4* -10.2* -12.0* +3.3

-5.4 -4.4 -2.4* -5.4* +3.1* -7.3* -5.6* -4.5* +1.7

+1.5* +4.6* +6.2* +5.2* -2.1 +2.4 +1.1 ±0.0 -2.4

+10.7* +10.1* +1.4 -3.6 +3.5 +10.8* +4.0* +14.3* +0.2

0.5%

-1.5

-1.1

+1.2

-1.7

0.002% 0.0024% 0.01% 0.01% 0.5% 1.0% 0.50% 1.0% 0.5% 1.0% 0.5% 1.0% 0.5% 1.0%

+3.4 +0.6 +2.1 -2.8 +3.0 +1.6 +0.2 +3.4 +1.5 +0.4 +2.5 +1.3 +1.9 +1.0

+2.8 -4.8* +1.7 -1.7 +4.7 ±0.0 -1.7 -3.9* +0.6 -5.5* ±0.0 -3.9* -4.6* -4.7*

-1.2 +5.4 +2.3 +1.3 -2.4 +2.4 +1.9 +6.9* +0.8 +6.1* +2.5 +4.6 +6.1* +5.2

+0.6 +5.3 -1.1 -1.2 +0.2 -1.1 +0.1 +0.5 ±0.0 +0.4 +0.1 +0.7 +0.4 +0.7

0.0125%

-1.8

-1.7

+1.0

 

-0.8 -2.5 -9.1* -7.8

Egg Egg production weight ±0.0 +0.1   ±0.0 +6.2* +6.6*

1

*Significant difference vs. negative control (P < 0.05) 2

Oregano (Origanum sp.), laurel leaf (Laurus nobilis L.), sage leaf (Salvia triloba L.), myrtle leaf (Myrtus communis), fennel seed (Foeniculum vulgare), citrus peel (Citrus sp.) 3 Essential oils derived from oregano (Origanum sp.), anise (Pimpinella anisum) and citrus peel (Citrus sp.)

In addition, hens fed thyme or rosemary oil showed significantly lower Haugh units in comparison with control hens, which is, however, in contrast to findings by Botsoglou et al. (2005a), Abd El-Motaal et al. (2008), Florou-Paneri et al. (2005) and Florou-Paneri et al. (2006), who did not observe an effect of phytogenic supplementation on Haugh Unit rating and yolk

T. Steiner 161 Table 2. Effect of phytogenics on egg traits (Bölükbaı et al., 2008)

Treatment Control Thyme oil Sage oil Rosemary oil P-value

Yolk (%)

Albumen (%)

Shell (%)

Haugh Unit

25.7a 23.3b 22.9c 22.5c < 0.01

61.8 61.4 62.9 62.6 > 0.05

12.1c 15.3a 14.3b 14.8b < 0.01

80.3b 76.9d 85.6a 78.7c < 0.05

a-d: Column means with no common superscript differ significantly

characteristics. However, according to Abd El-Motaal et al. (2008) egg breaking strength was slightly and significantly increased in the group fed 3 g/kg eucalyptus leaf powder. This is partly supported by results of a trial with commercial Lohmann Brown hens, in which egg shell thickness was significantly increased when the birds were fed a blend of essential oils derived from oregano, anis and citrus in comparison with control hens (0.48 vs. 0.42 mm) (Nichol and Steiner, 2008).

Effects of phytogenics on lipid and cholesterol levels It was speculated that phytogenics may influence hepatic enzymes, hence having an impact on egg yolk composition and possibly affecting cholesterol levels (Bölükbaı et al., 2008; Qureshi et al., 1983a, b). Indeed, the use of garlic and garlic-derived extracts for prevention of cardiovascular diseases is well known in human nutrition (Bordia, 1981; Silagy and Neil, 1994). However, results from experiments with layers were mixed. As reported by Qureshi et al. (1983a), garlic-supplemented diets fed to White Leghorn pullets suppressed several hepatic enzymes associated with cholesterol synthesis (e.g. 3-hydroxy-3-methylglutarylCoA reductase, cholesterol 7α-hydroxylase and others), resulting in decreased serum cholesterol levels. Similar observations were reported in trials with male broilers (Qureshi et al., 1983b). In another study, triglyceride and cholesterol concentrations were reduced in the serum but not in the egg yolk when hens were fed thyme, sage or rosemary essential oils (Bölükbaı et al., 2008). In addition, the authors reported changes in the composition of egg yolk proteins, as measured by the levels of apovitellenin, α-livetin, phosvitin and others. According to Case et al. (1995), White Leghorn cockerels fed carvacrol, thymol and β-ionone reduced serum cholesterol levels. In contrast, Sarica et al. (2005) did not obtain a significant effect of dietary supplementation with thyme or garlic powder on plasma cholesterol levels of broilers.

Antioxidant effects of phytogenics The oxidative stability of eggs is a quality parameter relevant for the nutritional value and sensory quality of eggs. While lipid oxidation is not a major concern in shell eggs, processed eggs are subject to oxidative deterioration, especially since the demand for eggs

162 Phytogenics in egg production with enhanced levels of poly-unsaturated fatty acids has increased (Botsoglou et al,, 1997). Vitamin E (α-tocopherol) is well known as an antioxidant agent in this regard (Qi and Shim, 1998; Cherian et al., 1996). In addition, antioxidative properties of phytogenic compounds are documented in in vitro experiments (Economou et al., 1991; Schwartz et al., 1996). The potential of including specific plants and/or plant extracts into diets for laying hens in order to affect the oxidative stability of eggs was subject to several studies (Botsoglu et al., 2005a, b; Florou-Paneri et al., 2005, 2006; Botsoglou et al. 1997). Florou-Paneri et al. (2005) investigated the effects of oregano essential oil (50 or 100 mg/ kg) or vitamin E (200 mg/kg tocopheryl-acetate) supplementation in comparison with a negative control treatment in Lohmann layers. Although performance parameters and egg traits did not differ between treatments, the extent of lipid oxidation measured in shell eggs by the formation of malondialdehyde (MDA) was affected by supplementation with oregano essential oil. Moreover, there was no impact of storage time (0–60 days) on MDA concentrations. Oregano reduced the level of MDA in a dose-dependent manner. However, the lowest concentration of MDA was found in the Vitamin E treatment. Similar effects were reported by Florou-Paneri et al. (2006), who observed that supplementation of diets with 5 or 10 mg/kg ground rosemary significantly lowered the MDA concentrations in refrigerated yolks in a dose-dependent manner. Botsoglou et al. (1997) reported that, in a study with similar design, addition of 3% ground thyme inhibited oxidation in liquid yolk. Interestingly, addition of pure thymol to the samples did not increase oxidative stability to the same extent as thyme extract and dietary thyme, indicating that there might be active ingredients other than thymol responsible for the antioxidative activity of thyme. As reported by Bölükbaı et al. (2006), addition of thyme oil reduced the concentrations of saturated and polyunsaturated fatty acids of the leg and breast tissues in broilers, whereas the concentrations of monounsaturated fatty acids increased. They pointed out that additional thyme components were involved in the overall antioxidant effect, too. Botsoglou et al. (2005a) reported that lipid oxidation was inhibited by phytogenics in a study with 32-week old Lohmann hens. Among the phytogenic substances used in this study, saffron (Crocus sativus L.) exhibited higher anti-oxidative efficacy than oregano and rosemary. The antioxidant effect of saffron was confirmed in a similar study using 38-week old Lohmann hens (Botsoglou et al., 2005b). The authors indicated that a transfer of antioxidant constituents into the bird might inhibit the chain reaction involved in oxidation of dietary lipids. Liver parameters were also investigated by Abd El-Motaal et al. (2008). In an experiment with 46-week old, brown Hy-Line layers, dietary supplementation with 3 g eucalyptus leaf powder per ton of finished feed increased plasma globulin and calcium levels and reduced GOT (Glutamat-Oxalacetat-Transaminase) levels as compared to the negative control treatment, which was seen as an indication of potentially reduced liver function. Moreover, a reduction in the ratio of heterophils to lymphocytes, as well as an increased response to phytohemagglutinin-P injection in hens fed 2 or 3 g/kg eucalyptus powder was noticed, which was regarded as an indication of reduced response to stressors and overall improved immunity status.

T. Steiner 163

Application of phytogenics in feeding programs for laying hens An increasing number of commercial phytogenic feed additives are available in the market. The majority of these additives are based on mixtures of plant extracts. Dosages may vary greatly depending on the raw materials used. Generally, the use of highly-concentrated extracts (e.g. essential oils) allows for low inclusion levels, whereas less concentrated materials (e.g. whole, dried plants) are added to the diets at higher levels. In order to guarantee a continuous quality of these products, strict standardization of active ingredients is mandatory. This is not always easy since the levels of active principles in plants or plant extracts may vary considerably. As summarized by Hüsnü Can Baser (2002), for example, the oil content of Origanum vulgare ssp. hirtum harvested in Turkey ranged between 2.3 and 5.4%, whereas the carvacrol content may range between 52 and 61%. The use of synthetic compounds, such as carvacrol, thymol, limonen or cinnamaldehyde, may be considered as an alternative to using natural extracts. Choosing the most suitable combination of ingredients requires extensive research, hence implementing broad in vitro testing as well as sophisticated feeding experiments under standardized conditions. Phytogenic feed additives may be applied either in the feed or in the drinking water, depending on the technical possibilities. Addition of powdered or granulated phytogenic feed additives in mash or crumbled layer diets allows for accurate inclusion levels and usually guarantees a steady supply of the active principles in the feed. On the other hand, application of liquid phytogenic formulas in the drinking water has the advantage of high flexibility in terms of application time and dosage. Provided that suitable dosing equipment is available on the farm, the liquid additive may be applied either continually or specifically at times of enhanced stress, e.g. feed change, housing or vaccination.

Conclusions and future outlook According to the few reports available, supplementation of layer diets with phytogenics may affect performance parameters such as feed intake, FCR, egg production and egg weight. Depending on the type and dosage of the substance used, a reduction in feed consumption, associated with an increased feed efficiency, may be expected. Moreover, there is evidence that dietary plants/plant extracts affect the oxidative stability of egg yolks, which might be an interesting subject to the egg processing industry. However, the mode of action underlying this effect needs further research as of today. Effects on egg quality traits, such as yolk composition, shell thickness or Haugh Unit rating, were reported in few studies only, whereas the majority of reports did not identify substantial effects. Future research should focus on the identification of the most suitable phytogenic substances to develop blended feed additives which may positively affect gut health and performance of modern laying hens.

164 Phytogenics in egg production

References Abd El-Motaal AM, Ahmed AMH, Bahakaim ASA and Fathi MM (2008) Productive performance and immunocompetence of commercial laying hens given diets supplemented with eucalyptus. International Journal of Poultry Science 7: 445–449. Alodan MA and Mashaly MM (1999) Effect of induced molting in laying hens on production and immune parameters. Poultry Science 78: 171–177. Amerah AM, Ravindran V, Lentle RG and Thomas DG (2007) Influence of feed particle size and feed form on the performance, energy utilization, digestive tract development, and digesta parameters of broiler starters. Poultry Science 86: 2615–2623. Bölükbaı SC, Erhan MK and Özkan A (2006) Effect of dietary thyme oil and vitamin E on growth, lipid oxidation, meat fatty acid composition and serum lipoproteins of broilers. South African Journal of Animal Science 36: 189–196. Bölükbai SC, Erhan MK and Kanyar Ö (2007) Effect of dietary thyme oil on laying hens performance, cholesterol ratio of egg yolk and Escherichia coli concentration in feces. 3rd Joint Meeting of the Network of Universities and Research Institutions of Animal Science of the South Eastern European Countries, Thessaloniki 10-12 February. Bölükbai SC and Erhan MK (2007) Effect of dietary thyme (Thymus vulgaris) on laying hens performance and Escherichia coli (E. coli) concentration in feces. International Journal of Natural and Engineering Sciences 1: 55–58. Bölükbaı SC, Erhan MK and Kaynar Ö (2008) The effect of feeding thyme, sage and rosemary oil on laying hen performance, cholesterol and some proteins ratio of egg yolk and Escherichia Coli count in feces. Archiv für Geflügelkunde 72: 231–237. Bordia A (1981) Effect of garlic on blood lipids in patients with coronary heart disease. American Journal of Clinical Nutrition 34: 2100–2103. Botsoglou NA, Yannakopoulos AL, Fletouris DJ, Tserveni-Goussi AC and Fortomaris P (1997) Effect of dietary thyme on the oxidative stability of egg yolk. Journal of Agricultural and Food Chemistry 45: 3711–3716. Botsoglou NA, Florou-Paneri P, Botsoglou EN, Dotas V, Giannenas I, Koidis A. And Mitrakos P (2005a) The effect of feeding rosemary, oregano, saffron and α-tocopheryl acetate on hen performance and oxidative stability of eggs. South African Journal of Animal Science 35: 143–151. Botsoglou NA, Florou-Paneri P, Nikolakakis I, Giannenas I, Dotas V, Botsoglou EN and Aggelopoulos S (2005b) Effect of dietary saffron (Crocus sativus L.) on the oxidative stability of egg yolk. British Poultry Science 46: 701–707. Çabuk M, Bozkurt M, Alçiçek A, Çatlı AU and Baer KHC (2006) Effect of a dietary essential oil mixture on performance of laying hens in the summer season. South African Journal of Animal Science 36: 215–221. Case GL, He L, Mo H and Elson CE (1995) Induction of geranyl pyrophosphate pyrophosphatase activity by cholesterol-suppressive isoprenoids. Lipids 30: 357–359. Cherian G, Wolfe FW and Sim JS (1996) Feeding dietary oils with tocopherols: effects on

T. Steiner 165

internal qualities of eggs during storage. Journal of Food Science 61: 15–18. Economou KD, Oreopoulou V and Thomopoulos CD (1991) Antioxidant properties of some plant extracts of the Labiatae family. Journal American Oil Chemists Society 68: 109–113. Florou-Paneri P, Nikolakakis I, Giannenas I, Koidis A, Botsoglou E, Dotas V and Mitsopoulos I (2005) Hen performance and egg quality as affected by dietary oregano essential oil and α-tocopheryl acetate supplementation. International Journal of Poultry Science 4: 449–454. Florou-Paneri P, Dotas D., Mitsopoulos I, Dotas V, Botsoglou E, Nikolakakis I and Botsoglou N (2006) Effect of feeding rosemary and α-tocopheryl acetate on hen performance and egg quality. The Journal of Poultry Science 43: 143–149. Franco-Jimenez DJ, Scheideler SE, Kittok RJ, Brown-Brandl TM, Robeson LR, Taira H, and Beck MM (2007) Differential effects of heat stress in three strains of laying hens. Journal of Applied Poultry Research 16: 628–634. Grizzle J, Iheanacho M, Saxton A and Broaden J (1992) Nutritional and environmental factors involved in egg shell quality of laying hens. British Poultry Science 33: 781–794. Harms RH, Russel GB and Sloan DR (2000). Performance of four strains of commercial layers with major changes in dietary energy. Journal of Applied Poultry Research 9: 535–541. Hüsnü Can Baser K (2002) The Turkish Origanum species. In: Oregano. The genera Origanum and Lippia. Pp.109–126. Kintzios SE (Ed). Taylor and Francis, London, United Kingdom. Jamroz D, Wertelecki T, Houszka M and Kamel C (2006) Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90: 255–268. Jang IS, Ko YH, Kang SY and Lee CY (2007) Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Animal Feed Science and Technology 134: 304–315. Kroismayr A, Sehm J, Pfaffl MW, Schedle K, Plitzner C and Windisch W (2008) Effects of Avilamycin and essential oils on mRNA expression of apoptotic and inflammatory markers and gut morphology of piglets. Czech Journal of Animal Science 53: 377–387. Mitsch P, Zitterl-Eglseer K, Köhler B, Gabler C, Losa R and Zimpernik I (2004) The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poultry Science 83: 669–675. Nichol R and Steiner T (2008) Efficacy of phytogenics in commercial Lohmann Brown layers. In: Feed Ingredients & Additives Asia Pacific Conference, March 5, 2008, Bangkok, Thailand. Qi GH and Sim JS (1998) Natural tocopherol enrichment and its effect in n-3 fatty acid modified chicken eggs. Journal of Agricultural and Food Chemistry 46: 920–1926.

166 Phytogenics in egg production Qureshi AA, Din ZZ, Abuirmeileh N, Burger WC, Ahmad Y Elson CE (1983a) Suppression of avian hepatic lipid metabolism by solvent extracts of garlic: impact on serum lipids. Journal of Nutrition 113: 1746–1755. Qureshi AA, Abuirmeileh N, Din ZZ, Elson CE and Burger WC (1983b) Inhibition of cholesterol and fatty acid biosynthesis in liver enzymes and chicken hepatocytes by polar fractions of garlic. Lipids 18: 343–348. Radwan Nadia L, Hassan RA, Qota EM and Fayek HM (2008) Effect of natural antioxidant on oxidative stability of eggs and productive and reproductive performance of laying hens. International Journal of Poultry Science 7: 134–150. Sarica S, Ciftci A, Demir E, Kilinc K and Yildirim Y (2005) Use of an antibiotic growth promoter and two herbal natural feed additives with and without exogenous enzymes in wheat based broiler diets. South African Journal of Animal Science 35: 61–72. Schwartz K, Ernst H and Ternes W. (1996) Evaluation of antioxidant constituents from thyme. Journal of the Science of Food and Agriculture 70: 217–223. Silagy C and Neil A (1994) Garlic as a lipid lowering agent – a meta-analysis. Journal of the Royal College of Physicians of London 28: 39–45. Singh R, Cheng KM, Silversides FG (2009) Production performance and egg quality of four strains of laying hens kept in conventional cages and floor pens. Poultry Science 88: 256–264. Steiner T (2006) Managing Gut Health – Natural Growth Promoters as a key to Animal Performance. Nottingham University Press, Nottingham, United Kingdom. Valkonen E, Venäläinen E, Rossow L and J. Valaja J (2008) Effects of Dietary Energy Content on the Performance of Laying Hens in Furnished and Conventional Cages. Poultry Science 87: 844–852.

T. Steiner 167

Conclusion

The feed industry is currently looking for efficacious, safe and cost-efficient additives with a clearly defined mode of action and proven benefits. Plant-derived compounds have a considerable potential to fulfil this demand. There is still a lack of knowledge in several aspects, especially regarding the consistency of trial results and mechanisms of action of the various phytogenic compounds and blends thereof. Thus, more in vivo trials are necessary to investigate how phytogenics may affect gut parameters and performance in pigs, poultry, ruminants and aquatic species. Based on the experiments reported herein, it appears that the mode of action of phytogenics is highly versatile and far from being based only on a simple flavouring or antimicrobial effect. It can be expected that the development of appropriate phytogenic additives will be further driven by extensive research carried out by institutes and companies willing to invest in research and development projects. As such, finding and combining the most suitable compounds for different applications is a main target of ongoing research. Standardised quality and ingredient composition of phytogenic materials will have a major impact on the in vivo efficacies of commercial products. The use of chemically defined, synthetic active principles instead of naturally distilled essential oils may be of increasing interest for future applications. In addition, the choice of a suitable carrier substances will be a subject to consideration by animal nutritionists and formulation experts. Moreover, besides technological aspects, the economical expenses associated with the inclusion of additives in animal diets must be taken into account. Finally, phytogenic additives need to fulfil strict registration guidelines in respect to safety and efficacy for the animal, the consumer and the environment.

167

168 Phytogenics in egg production

Index 169

Index

1,8-cineole 13, 70, 99 a-phellandrene 13, 117 a-pinene 13, 99, 113, 117 a-terpinene 13, 117 a-terpineol 13, 117 a-tocopheryl acetate 29, 102 a-toxin 66 ß-caryophyllene 13, 117 ß-glucan 50 ß-phellandrene 117 ß-pinene 13, 99 g-terpinene 13, 67, 99, 117, 123

A Absorption 8, 20, 28, 30, 43, 73, 77, 84 Acetyl-CoA 120 Achillea millefolium 14 Acidifiers 98, 106 Active principles 1, 11, 15, 149, 158, 163, 167, 170 Acute phase response 40, 41 Additive 11, 21–27, 43, 48, 49, 51, 74, 87, 88, 90–95, 104, 111, 116, 128, 130, 131, 133, 136, 138, 163 Ajowan 4 Albumen 161 Alcohols 2–5, 119, 121 Aldehydes 2, 3, 121, 149 Allergic 9, 29, 105 Almond 5 Amines 20, 26 Ammonia 25 Amylase 28, 103 Anacardiaceae 1, 6 Anethole 4, 13, 98, 119 Anethum graveolens 4, 7 Anise 4, 6, 22, 25, 98, 100-104, 113, 135, 150, 160 Annonaceae 1 Anthocyanins 67 Antibiotic 10, 14, 19, 20, 22, 25–28, 61–86, 106, 111, 112, 115, 116, 124 Antibiotic growth promoters 15, 19, 45, 63, 74, 77, 97, 99, 106, 115, 116 Antibody 28, 46, 48, 51 Anticoccidial 61, 63, 65, 69, 70, 72, 74, 104, 154

169

170 Index Antidepressant 14 Antifungal 12, 12–14, 67 Antigen 40, 41, 44, 46, 48, 50 Antimicrobial 8, 24, 26, 30, 39, 43, 45, 47, 61, 62, 66–69, 73, 74, 97, 98, 104–107, 111, 112, 115, 117, 121–124, 129, 130, 138, 149, 152, 167, 170 Antioxidant 12, 13, 29, 39, 74, 98, 105, 149, 162, 166 Antiparasitic 61, 62, 67, 74, 98 Antiviral 3, 14, 67, 98 Apiaceae 1, 6, 8, 12 Apoptosis 41, 43 Apparent digestibility 25 Aquaculture 147–156 Araceae 1, 6 Aristolochiaceae 1, 6 Aroma 1, 2, 11, 62 Aromatherapy 8, 10 Artemisia absinthium 4 Artemisia dracunculus 7 Ascophyllum nodosum 49, 58 Aspergillus 12, 101 Asteraceae 1, 6 Astragulus membranaceus 39, 46 Avilamycin 22, 25, 159, 165 Avoparcin 116

B Bacillus cereus 104, 145 Bacitracin 69, 116 Bacteria 14, 42, 44, 45, 48, 51, 61–63, 68, 69, 76, 78, 104, 105, 111, 112, 115, 123–127, 129, 130, 133–138, 147, 149, 151, 159 Bacteroides fragilis 69 Bark 4, 98, 129, 149 Batch culture 125–130, 132, 134, 137 Bay 6, 12 B cells 44 Benzenacetonitriles 65 Bergamot 6, 9 Berries 6 Bifidobacterium longum 69 Biogenic amines 20, 26 Biological activities 8, 67, 73, 75, 76, 98 Black Mustard 12 Black Pepper 12 Borneol 3, 117 Botanicals 19, 112 Brassica 5

Index 171 Broiler 26, 39, 61, 63–65, 69–74, 97, 98, 100, 102–104, 106, 107, 166 Burseraceae 1

C Cadaverin 25 Cade 131 Caecum 25, 26 Cajuput 4 Calycanthaceae 1, 6 Camphene 3, 13 Camphor 4, 70, 113 Cannabinaceae 1, 6 Capsiscum oleoresin 42 Caraway 4, 21 Carbadox 116 Carcass 97, 100, 105, 106, 136, 137, 141 Carp 152, 154 Carum carvi 4, 121 Carvacrol 13, 15, 25, 30, 42, 44, 47, 48, 50, 51, 67–69, 71, 98, 99, 101–103, 115, 121–123, 129–132, 137, 149, 152, 161, 163 Caspase 3 25 Cassia 4, 6, 12, 21, 23 Catfish 150 Cathehins 67 Cavities 5, 6 Cayenne 12 Cell membrane 26, 149 Chamomile 3, 6 Chemotherapeutics 64 Chenopodium 5 Chenopodium ambrosioides 5 Chicken 29, 48, 52, 61, 64, 65, 69–72, 78, 103, 104, 106, 166 Chinese rhubarb 22, 37 Cholesterol 161, 166 Chromatography 76 Chyme 25, 26, 28 Cinchona succirubra 46, 71 Cinnamaldehyde 42, 44, 47, 48, 50, 51, 68, 69, 98, 101–103, 119, 129, 131, 132, 135–137, 163 Cinnamomum camphora 4 Cinnamomum cassia 4, 82 Cinnamomum verum 4 Cinnamon 50, 69, 98, 100–102, 123, 129–131, 134, 135, 142 Citronellic acid 13 Citronellol 3, 13 Clostridium aminophilum 127 Clostridium perfringens 45, 65, 104, 159, 165

172 Index Clostridium sticklandii 127 Clove 70, 118, 123, 129, 131 Coccidiosis 39, 45, 61, 63, 64, 69, 72, 73, 85 Coccidiostat 46, 102 Colamin 25 Colon 25, 26, 44, 45, 47, 50, 51, 52 Colony counts 26, 30 Combretum woodii 72 Continuous culture 111, 124–133, 135, 138, 144 Coriander 98, 139 Cow 127, 133, 135 Crypt 26, 43, 45, 58 Cultivation 7 Cumin 6, 12 Cyclin D1 25, 26 Cymbopogon 3, 4, 7 Cyprinus carpio 152, 154 Cytokine 28, 40, 58 Cytoplasm 122, 123 Cytotoxicity 9

D Dairy cow 127, 133, 135 Dasytricha 135 Deaminase 128 Decarboxylation 26 Dermatitis 29, 105 Diarrhea 15, 39, 43, 46, 47, 65 Digesta 42, 48, 50, 51, 52, 65, 66, 164 Digestibility 25, 42, 52, 67, 97, 100, 103, 106, 127, 131, 134, 136, 138, 139 Digestive secretions 103 Dill 5, 131 Diplodinium 135 Disease 144 Distillation 8, 66, 67, 113, 117, 149 Dry matter 103, 112, 129, 134–137, 144

E Echinacea purpurea 51, 73 E. coli 39, 47-51, 68-69, 104, 106, 138, 149, 159 Egg production 15, 157–159, 163 Eggs 29, 159–162, 166 Eimeria 24, 45, 46, 64 Eimeria acervulina 82 Eimeria brunetti 64

Index 173 Eimeria maxima 46, 64, 71 Eimeria mitis 64 Eimeria necatrix 64 Eimeria praecox 64 Eimeria tenella 102, 104 Elemicin 13 Enterococci 48, 116 Enterocyte 41, 43, 53 Entodinium 135 Enzymes 14, 15, 26–28, 30, 45, 67, 76, 98, 103, 106, 123, 159, 161, 166 Epidermis 5 Escherichia coli 68, 104, 138, 164.  See also E. coli Essential oils 1–3, 5, 7–10, 12, 14, 15, 20, 22, 24–30, 43–45, 48–51, 61–63, 66–70, 72, 74–77, 88, 98, 101, 103, 106, 111–124, 126–138, 149–153, 158, 159, 161, 163, 167, 170 Esters 2–4, 121 Ethers 2 Eucalyptus 6–8, 134, 159, 160, 162 Eucalyptus citriodora 4 Eucalyptus globulus 4 Eugenol 4, 123, 145 European Food Safety Authority 10, 105 European Union 10, 63, 74, 98, 112, 115, 116, 145 Expression 8 Extraction 8 Extracts 20, 21, 23, 28, 39, 43–46, 61–64, 66–75, 77, 78, 98, 100, 101, 103, 105, 112, 124, 137, 149, 151, 152, 157, 158, 161–163, 166

F Farnesole 2 Farrowing 49, 87–90, 92, 94, 96 Feed additive 11, 22, 25–27, 43, 48, 49, 51, 74, 87–95, 104, 111, 116, 133, 138, 143 Feed conversion ratio (FCR) 20, 22, 64, 72, 101, 102, 147, 150–152, 158, 159, 163 Feed intake (FI) 15, 20, 22, 30, 40, 41, 87–92, 95, 101, 102, 137, 158, 159, 163 Fennel 20, 70, 159, 160 Fish 66, 147, 148, 150–152, 158 Flavones 67 Flavonols 67 Flavonones 67 Flavour 2, 11, 20, 29, 62, 113, 115, 143 Flowers 8, 117 Foeniculum vulgare 4, 160 Fragrance 8, 113, 115 Fusarium moniliforme 101

174 Index

G Garlic 11, 14, 43, 98, 121, 131–134, 136, 137, 161, 166 Gastrointestinal tract 26, 147, 148 Gaultheria procumbens 5 Generally recognized as safe (GRAS) 11, 73, 149 Geraniaceae 1, 6 Geraniol 13, 121 Geranium 3, 9 Geranyl acetate 13, 121 Geranyl formate 13 Geranyl pyrophosphate 2, 164 Ginger 6, 12 Gramineae 1 Gram-negative 63 Gram-positive 84 Gut health 26, 157, 158, 163

H Harvest 6, 7, 10, 99 Haugh Unit 157, 160, 161, 163 Hedysarum coronarium 45 Heifers 127, 135, 144 Herbs 1, 21, 24, 143 Histamin 25 Histomonostats 98, 116 Horsemint 4 Horseradish 50 Hyperaceae 1 Hypericum peforatum 14

I Idioblasts 6 Ileum 25-27, 66 Illicium verum 4 Immune system 40, 42, 44, 67, 97, 148, 149, 152, 154 Immunity 39, 40, 44, 73, 78, 162 Immuno-modulation 40 Incubation 125, 126, 128, 134, 135, 153 In situ 127–129 In vitro 24, 30, 48–50, 66, 68–70, 73, 105, 107, 111, 124–136, 138, 151–153, 162, 163 In vivo 22, 24, 26, 30, 39, 44, 47, 48, 61, 66, 69, 70, 72, 73, 78, 111, 124–131, 133–135, 138, 157, 158, 167, 170 Ionophore 112, 144 Isoeugenol 13

Index 175 Isolation 7–8, 49, 76, 116 Isomenthone 13 Isoprene 2, 98, 113, 118, 120, 121 Isopropylamin 25

J Jejunum 25, 26, 28, 51, 103, 165 Juniper 49, 113, 134, 136, 137, 146

K Ketones 2, 149

L Lactation 49, 51, 87–90, 91–95, 126 Lactobacillus acidophilus 69 Lamiaceae 1, 6, 12 Lauraceae 1, 6 Lavender 3, 6, 9 Layer 42, 44, 45, 159, 163 Laying hens 157–159, 162, 163, 166 Leaves 5, 15, 46, 70, 73, 74, 137, 149, 160 Leguminosae 1, 6 Lemon 4, 6, 9, 70 Lemon grass 4, 6 Leonotis leonutus 6 Levisticum officinale 14 Licorice 12 Lime 6, 9 Limonene 4, 13, 25, 117, 121, 127, 128, 132 Linalool 3, 13, 98, 121 Lipase 103 Liposomes 77, 84 Litopenaeus vannamei 152, 154 Litter 42, 46, 64, 65, 88–90, 93–96 Livestock 11, 19, 29, 30, 39, 40, 52, 98, 111, 112, 133, 138 Location 3, 7 Lymphocyte 40, 44, 50, 51, 105

M Macrophage 40, 44, 49, 153 Magnoliaceae 1, 6 Malondialdehyde (MDA) 162 Maltase 43 Marjoram 12, 47, 48, 101, 102

176 Index Meat quality 11, 15, 97, 105, 106, 136, 137 Medicine 8, 62, 66, 77, 85 Melaleuca 4, 6, 124, 144 Melia azedarach 70, 78 Mentha cardiaca 4 Mentha × piperita 7, 121, 135 Mentha piperita 4 Mentha spicata 4 Mesenteric lymph nodes 43, 44, 50, 51 Metabolites 20, 26, 30, 45, 52, 62, 70, 75, 105, 111, 115, 116, 118–124, 130, 132, 137, 145 Methane 111, 112, 115, 131–134, 136, 145 Methanobrevibacter smithii 134 Methylamin 25 Methyl eugenol 13 Microbiota 10, 19, 20, 24, 26, 41, 47, 69, 125, 147, 148 Microflora 41, 45, 47, 48, 52, 65, 67, 69, 70, 78, 100, 104, 106, 148, 159, 165 Milk production 87–89, 91–93, 95, 112, 136, 140 Minimum inhibitory concentration (MIC) 13, 106, 107, 143 Mint 5 Mode of action 20, 26, 28, 30, 62, 68, 73, 74, 124, 148, 149, 158, 163, 167, 170 Monarda punctata 4 Monoterpenes 2, 98, 122, 123, 142 Morphology 26, 27, 30, 50, 165 Mortality 15, 41, 45, 46, 48–50, 63–65, 71, 89, 94, 151 mRNA 25, 26, 28, 52, 165 Mucus 28, 30, 42–45, 103 Mustard 98 Myoporaceae 1, 6 Myrcene 117 Myristica fragrans 5, 16 Myrtaceae 1, 6

N Necrotic enteritis 45, 65 Nematodes 45, 67, 79 Neral 13, 121 NFκB 25, 28, 51 Nitric oxide 152, 153 Nitrogen (N) 3, 111, 126, 149 Non-ruminants 45, 82 Nutmeg 5, 6

O Ocimum basilicum 7 Odour 1, 11, 15, 105

Index 177 Olaquinadox 116 Oligosaccharides 73, 79 Orange 6, 9 Orchidaceae 1, 6 Oregano 6, 12, 21–24, 29, 99, 102, 151, 160, 162, 165 Organic acids 15, 19, 20, 26, 77, 98, 106 Origanum vulgare 7, 67, 99, 137, 152, 163 Osmophores 6 Otto of rose 3 Oxidation 11, 14, 29, 77, 105, 106, 161, 162, 164

P Palatability 20, 30, 39, 88, 99–101, 149, 150 Palmarosa 3 Pangasius 150 Pangasius hypothalamus 150 p-anis-aldehyde 13 Parsley 5 Passage rate 28 Pasteurella multocida 40, 57 Pathogen 26, 28, 30, 40, 61, 97, 106, 152, 154 p-cymene 13, 67, 99, 123, 134 Peel 9, 43, 44, 51, 88, 150, 152, 159, 160 Penaeus indicus 152, 154 Peppermint 4, 6, 9, 21, 23 Peptidolysis 129, 130 Peptostreptococcus anaerobius 127 Perfumery 1 Petroselinum sativum 5 Peucedanum soia 5 Peyers’ Patches 27, 44, 51 pH 7, 26, 43, 45, 125, 127, 129, 131, 132, 135, 138, 140 Phenols 2, 3, 5, 67, 68, 70, 124 Phenylalanine 118, 119 Phenylpropenes 98, 116, 118, 119, 121, 142 Phosphoenolpyruvate 118 Photosensitizers 9 Phytobiotics 19 Phytogenics 20, 26, 28–30, 97, 100, 101, 103, 106, 107, 148–150, 152, 158–163, 167, 170 Piglets 15, 19, 20, 21, 22, 24, 25, 27, 28, 30, 39, 44, 47, 49–52, 87, 93, 94, 104, 165 Pigs 43, 44, 49–51, 87, 89, 90, 93–95, 167, 170 Pimento 21 Pimpinella anisum 4, 160 Pinaceae 1, 6 Pine 3 Piperaceae 1, 6

178 Index Plant extracts 43–45, 62, 72, 98, 103, 124, 137, 152, 157, 158, 162, 163, 165 Plasma membrane 121–124 Plectranthus madagascariensis 6 Polyplastron 135 Polysporoplasma sparis 151, 154 Poppy-seed 12 Poultry 10, 14, 15, 19, 20, 23, 29, 30, 39, 40, 45–48, 52, 61–65, 67, 73, 74, 77, 78, 98, 105–107, 150, 167, 170 Prebiotics 72, 98, 106 Probiotics 79 Protein 11, 15, 25, 41–43, 51, 65, 66, 88, 93, 103, 112, 115, 126–128, 134, 138, 144 Protozoa 45, 64, 78, 111, 125, 134–136, 144 Prunus communis 5 Pseudomonas 69, 104, 105 Pulsatilla koreana 46 Putrescin 25 Pyrimidine derivatives 65 Pyrrolidin 25

Q Quillaja saponaria 49, 58 Quisqualis indica 46

R Rats 28, 43, 73, 103, 110 Residues 1, 2, 15, 30, 65, 75, 98, 115, 117 Resin 116 Rhizome 6 Ribes nigrum 46, 71 Ronidazole 116 Root 49, 149 Rosaceae 1 Rosemary 3, 6, 12, 23, 99, 160, 161 Rumen fermentation 115, 124, 126, 130–132, 134, 137, 138, 144 Ruminant 126, 143 Rutaceae 1, 6

S Sabinene 13 Saffron 12, 160 Safrole 4, 13, 119 Sage 4, 12, 22, 99, 160, 161 Salmon 151, 154 Salmonella 48, 69, 138 Salmonella enteritidis 53

Index 179 Salmonella typhimurium 49, 68, 104, 149 Salvia officinalis 4, 7, 99 Sandalwood 3, 6 Santalaceale 1 Satureja douglasii 7 Saururaceae 1, 6 Scatol 26 Sea bream 151 Seasoning 8 Seaweed 49 Sedative 3, 11 Seeds 6 Sesame 12 Sesquiterpenes 2, 3 Shelf-life 15 Shell 72, 157, 159, 161–163, 165 Shigella dysenteria 104 Shrimp 147, 150, 152, 158 Solanaceae 1, 6, 43 Sophora flavescens 39, 46, 70 Sow 49, 50, 87–96 Sparus aurata 151, 154 Spearmint 4, 6 Spermidin 25 Spermin 25 Spices 1, 12, 20, 21, 24, 28–30, 43, 62, 67, 73–75, 98, 103, 105, 112, 124, 149 Staphylococcus 69, 104, 124, 138, 144 Steam distillation 8, 66, 117, 149 Stem cells 41 Streptococcus bovis 125 Streptococcus iniae 151, 154 Sucrase 43 Sulfur 3 Sulphonamides 64 Swine 10, 15, 40, 45, 47, 49, 51, 88, 150, 158 Sylimarin 23 Syzygium aromaticum 4, 71, 129

T Taste 1, 11, 14, 15, 107 T cells 44 Temperature 1, 7, 49, 117, 125, 157 Terpenes 3, 98, 118 Terpenoids 2, 68, 113, 115, 118 Terpinene-4-ol 13 Thyme 20, 47–50, 67, 69, 70, 98, 100–103, 130, 134, 149, 151, 158–162, 166

180 Index Thymol 13, 15, 25, 30, 47, 49, 51, 67–69, 71, 98, 99, 101, 102, 115, 121–123, 126, 128–130, 132, 133, 149, 152, 159, 161–163 Thymus vulgaris 4, 50, 51, 71, 99, 130, 164 Tilapia 150, 151, 154 TNF-a 40 Toltrazuril 72 Toxins 20, 42, 63 Trachyspermum ammi 4 Triazinetriones 65 Trichomes 6, 116 Trypsin 28, 103 Turkeys 40, 41, 70, 116 Turmeric 12 Turnover 41, 43, 55 Turpentine 113 Tylosin 116 Tyrosine 118, 119

U Ulmus macrocarpa 46 Urease 28, 38

V Vaccine 40, 46, 71, 80 Vanilla 7, 12 Vanillin 4, 126, 128, 132 Vetiver 9 Vibrio harveyii 152 Villi 26, 27, 42 Virginiamycin 116 Virulence 40, 44 Vitamin E 162, 164 Volatile fatty acids (VFA) 26, 129–134, 136, 138

W Weaning 20, 22, 25, 27, 47, 49, 50, 87–89, 92, 96 Weight gain 15, 64, 94, 102, 150, 151 Wintergreen 5, 6 Withdrawal 15, 116 Wormwood 4

Index 181

Y Yield 7, 105, 112, 116, 117, 120, 143 Yolk 157, 159,–164 Yucca schidigera 28, 140

Z Zingiberaceae 1