deliverable 4.2, report on plant sources of vitamins ......organic-plus d4.2 report on plant sources...

Organic-PLUS D4.2 Report on plant sources of vitamins, antiparisitics and antimicrobics for livestock page 1

Organic-PLUS

Deliverable 4.2, Report on plant sources of vitamins,

antiparasitics and antimicrobics for livestock

Version 1.5, 31 January, 2019

Versions

Version: 1.0 (January 2019) Draft led by Federico Righi

Version: 1.5 (31 January 2019) Text with feedback from all WP4 LIVESTOCK participants.

Funding

This project has received funding from the European Union’s Horizon 2020 research and

innovation programme under grant agreement No [774340]


Project Details:

Programme: H2020, SUSTAINABLE FOOD SECURITY – RESILIENT AND RESOURCE- EFFICIENT

VALUE CHAINS

Call topic: SFS-08-2017, (RIA) Organic inputs – contentious inputs in organic farming

Project Title: Pathways to phase-out contentious inputs from organic agriculture in Europe

Project Acronym: Organic-PLUS

Proposal Number: 774340-2

Lead Partner: Coventry University, Centre for Agroecology, Water and Resilience

Time Frame: 01/05/2018 – 31/04/2022

Authors:

Federico Righi, Rosario Pitino, Giulio Grandi, Afro Quarantelli, Andrea Summer, Judith Conroy,

Sara Burbi, Carmen L. Manuelian, Massimo De Marchi

Deliverable Details:

WP: 4 LIVESTOCK

Task(s): 4.2: Identification of alternatives: Investigation of plant vitamin sources, anti-infective

and immuno-stimulatory properties of plant products

Deliverable Title: D4.2 Report on plant sources of vitamins, antiparasitics and antimicrobics for

livestock

Lead beneficiary: UNIPR

Involved Partners: UNIPD, SLU, OC

Deadline for delivery: month 9, 31/01/2019

Date of delivery: 31/01/2019


Table of Contents

1. Summary ....................................................................................................................... 5 2. Introduction .................................................................................................................. 6 3. Review of natural sources for vitamins on organic livestock farming as potential alternatives to the use of synthetic vitamins .......................................................................... 7

3.1 Methodology ........................................................................................................ 7 3.2 The interest of algae, and olive oil, citrus, wine and carob industries by-products in terms of alternative source to synthetic vitamin ................................................................ 8 3.3 By-products from olive oil industry ....................................................................... 8 3.4 By-products from citrus industry ..........................................................................11 3.5 By-products from wine industry ...........................................................................11 3.6 By-products from carobs ......................................................................................12 3.7 Discussion and Conclusions ..................................................................................12 3.8 References ...........................................................................................................13

4. Review of natural sources of antiparasitics on organic livestock farming ......................19 4.1 Introduction.........................................................................................................19 4.2 Economic and productivity losses from parasitoses ..............................................19 4.3 Methodology .......................................................................................................20 4.4 Bibliometric results ..............................................................................................20 4.5 Reliability of the results on the antiparasitic activity of plant molecules ...............21 4.6 Plants and plant products with antiparasitic effects .............................................23 4.7 Plants and plant products with active compounds against parasites in fish farming 26 4.8 Conclusions..........................................................................................................27 4.9 References ...........................................................................................................27

5. Review of natural sources of antimicrobics on organic livestock farming.......................29 5.1 Introduction.........................................................................................................29 5.2 Phytochemicals compounds classification ............................................................31 5.3 In vitro trials to test antimicrobial capacity of plants and plant-derived products and mechanism of action .................................................................................................31 5.4 In vivo studies ......................................................................................................33 5.5 Antimicrobial capacity of food industry by-products ............................................39 5.6 Conclusions and perspective ................................................................................39 5.7 References ...........................................................................................................40

6. Discussion and conclusion of potential natural sources for vitamins, antiparasitics and antimicrobics for organic livestock farming ...........................................................................52


List of Figures

Figure 4.1. Number of documents by year of publication retrieved from Web of Science database based on the search terms antiparasitic and plants from 1985 to January 2019 .................. 20

Figure 4.2. Number of documents by country of publication retrieved from Web of Science database based on the search terms antiparasitic and plants from 1985 to January 2019 ... 21

Figure 4.3. Number of documents by field of publication retrieved from Web of Science database based on the search terms antiparasitic and plants from 1985 to January 2019 .................. 21

Figure 5.1. Classification of the phytochemical compounds ................................................. 31

List of Tables

Table 3.1. Example of natural sources of vitamin E and C, and polyphenols ........................... 8

Table 3.2. Summary of the finding of the use of olive oil industry by-products in aquaculture and livestock.............................................................................................................................. 10

Table 4.1. Reasons that lead to inconsistency on the bibliographic research about plants and plant product as antiparasitic in livestock ............................................................................ 22

Table 4.2. Main phytochemical compounds and their effects on parasites from the literature 23

Table 4.3. Indian indigenous plants that presented antiparasitic capacity, and specifically anthelmintic activity ........................................................................................................... 24

Table 4.4. Plant derived anthelmintic medications for human and animal use in Nordic countries.. ........................................................................................................................... 24

Table 4.5. Plants with anti-ectoparasites properties ............................................................ 25

Table 4.6. Plants with anti- protozoans, myxozoans and helminths (monogeneans) properties in fish farming......................................................................................................................... 26

Table 5.1. Properties that any candidate to natural antimicrobial should follow .................. 29

Table 5.2. In vitro studies of plant derived compound against pathogens causing bovine mastitis ........................................................................................................................................... 32

Table 5.3. Main pathogens/disorders for which antimicrobials are mostly used in poultry .. 37


1. Summary

Organic production and labelling of organic products in the European Union are regulated by the Regulation (EU) 2018/848 of May 30, 2018. Although the principles for organic farming seem easy to follow, the regulation is comprised of several exceptions since in certain instances a compromise has to be taken to ensure animal health and welfare. To help in fully achieve the principles of organic livestock farming, effective alternatives to the use of synthetic vitamins, anti-infective and immune-stimulators, and bedding materials have to be examined and developed. A literature review on potential plant alternatives to the use of synthetic vitamins, antibiotics and antiparasitics has being conducted to support further activities within the Organic-PLUS project, notably the selection of components to be tested in in vitro studies.

Vitamins are organic compounds essential for animal health and performance. Conventional farming systems allow the use of synthetic vitamins to fulfil animals’ requirements because they are cheaper and usually more stable than those from natural sources. The European regulation for organic livestock production indicates that vitamins in animal feed should correspond to those naturally occurring in feed stuff, with some exceptions to assure animal health. They also allow the use of algae and food industry by-products, which are relevant sources of vitamins, pro-vitamins and antioxidant compounds. Economically relevant by-products in the Mediterranean area are those from the olive oil, citrus, wine, and carob food industries. Based on scientific evidence from several research trials, the use of these products is a reasonable alternative to the use of synthetic vitamins to assure the adequate vitamin intake in livestock and ameliorate animals’ oxidative status. However, there is very little information on the vitamin content characterisation of these products and very few studies have evaluated their impact on livestock performance and products such as milk, meat and eggs.

In general, parasites affecting livestock can be classified as endoparasites (protozoa and helminths) and ectoparasites (arthropods). An alternative to treat parasitosis could be the use of plants and plant products, whose application is nowadays still mainly based on traditional and local empirical knowledge. This alternative could help reducing the development of antiparasitic resistance and provide new treatment methods for organic farming. This report summarises the scientific evidence related to plants and plant products that have been tested in clinical and experimental controlled studies to control ecto- and endoparasites in livestock, reporting on availability of standardized procedures for production and testing of plant-based antiparasitic compounds, as well a listing of some of the studied plants and plant-derived products used against endoparasites, ectoparasites and fish parasites.

Antimicrobials represent one of the most important discoveries, both in human and animal health. However, the diffusion of multi-resistant microorganisms and the development of alternative livestock farming systems such as organic farming has led to the need of exploiting new alternative and natural compounds, minimally processed, from plants to replace synthetic molecules. The wide group of phytochemicals has been recently investigated mostly to replace the use of antimicrobial-growth promoters in swine and poultry. Among them, essential oils have showed very interesting antimicrobial properties against many pathogenic bacteria. Several in vitro studies have been published on essential oils and other plant extracts. There is still a need for more information on these alternatives before using them on daily basis at farm level; however, they appear as very promising tools to contribute to reducing antimicrobial-resistance and at the same time to meet food safety requirements without compromising animal health and welfare.

The purpose of this document is two-fold: it summarises the knowledge available on potential plant alternatives to the use of synthetic vitamins, antibiotics and antiparasitics; and it reports on the selected phytochemical compounds that are going to be studied in the following in vitro experiments.


2. Introduction

Organic production and labelling of organic products in the European Union are regulated by the Regulation (EU) 2018/848 of 30 May 2018, where organic production is defined as “the use, including during the conversion period […], of production methods that comply with this Regulation at all stages of production, preparation and distribution” (Regulation (EU)2018/848).

A literature review on potential plant alternatives to the use of synthetic vitamins and immunostimulators, antibiotics and antiparasitics has being conducted to support further activities within the Organic-PLUS project, notably the selection of components to be tested in in vitro studies. A detailed introduction is included in each topic section. Moreover, a factsheet of each topic has been presented as D4.3 and tailored for stakeholder uptake.


3. Review of natural sources for vitamins on organic livestock farming as potential alternatives to the use of synthetic vitamins

Vitamins are organic compounds needed in small amounts (grams) per day that are essential for the efficient use of other nutrients and for many metabolic processes. The most important classification of these micronutrients is based on their solubility, being vitamins A, D, E and K fat soluble and vitamins C and B complex group (thiamine, riboflavin, pyridoxine, pantothenic acid, niacin, biotin, folic acid and vitamin B12) water soluble. Vitamins that must be included in the diet are considered as dietary essential, but the microbiota of some animal species is able to synthetize some of them at the adequate level, so the host animal can absorb them. Thus, vitamin nutritional requirements differ among species. For instance, due to the rumen microbiota, healthy adult cattle are able to synthesise adequate amounts of vitamin C and many B vitamins, fulfilling cow’s requirements for those vitamins (Weiss, 2017).

The origin of vitamins that can be used in organic livestock production is regulated by Regulation (EU) 2018/848 of May 30, 2018, that stipulates that the animal diet supplementation with vitamins should be done with vitamins derived from raw materials occurring naturally in feedstuffs. However, the regulation allows the use of synthetic vitamins identical to natural vitamins for monograstrics; and for ruminants, the use of synthetic vitamins A, D, and E identical to natural vitamins with prior authorisation of the Member States, based on the assessment of the possibility for organic ruminants to obtain the necessary quantities of the said vitamins through their feed rations. For instance, pasture and grass and legume silages are animal feed that provide provitamin A (β-carotene) and vitamin E, but their availability for organic farmers is highly location-based. The European Regulation, Annex VIII, lists the food additives that could be used in organic livestock farming, such as extracts from plant and animal origin. Annex V lists the non-organic feed materials that could be used under certain conditions, which include food industry by-products from non-organic production. Important by-products in the Mediterranean region are the ones from the olive oil, citrus, wine, and carob food industries because those are important crops on that region.

The possibility of using food industry by-products opens an important door to the philosophy underlying environmental sustainability and organic farming. It allows to reduce industry waste, to convert low value products into high value ones both from an economical and nutritional point of view , to keep geographical proximity of the farm from where the animal feed is produced, and to contribute to the reduction of the feed to food competition in livestock production (Nonhebel et al., 2015; Valenti et al., 2018).

The evaluation of algae and food industry by-products as sources rich in vitamins will contribute to solving the availability of natural vitamins to be used in organic livestock farming. Therefore, this review summarizes the scientific evidence supporting the capacity of algae, and olive oil, citrus, wine, and carob food industries by-products to act as vitamin sources in livestock.

3.1 Methodology

Scientific evidence on the alternatives to the use of synthetic vitamins has been searched using the specialised browsers www.pubmed.com, www.scopus.com, www.webofscience.com, and www.sciencedirect.com. The keywords and phrases used for the literature research were: ‘Regulation 889/2008’, ‘Regulation 834/2007’, ‘organic farming’, ‘vitamins supplementation in conventional and organic livestock’, ‘vitamins requirements’, ‘feed sources rich of vitamins’, ‘alternative feed sources rich of vitamins’, ‘feeding strategies in organic livestock’, ‘vitamins and antioxidant activity’, ‘bioactive compounds with antioxidant activity’ (phenolic compounds), ‘olive oil by-products’, ‘grape by-products’, ‘citrus by-products’, ‘carob pulp’, ‘tomatoes by-products’, ‘wheat germ oil’, and ‘algae supplementation’. The literature review covered material from 1969 to 2018. First, a selection of the articles was carried out based on the title and the abstract. Then, in-depth reading was done to discard the ones that did not match with the criteria of the review. Finally, only papers that focused on the by-products from olive oil, wine,

http://www.pubmed.com/

http://www.scopus.com/

http://www.webofscience.com/

http://www.sciencedirect.com/


citrus and carob industries have been included, as well as algae supplementation in livestock. From each article the following information has been extracted: alternative source of vitamin used and dose, synthetic vitamin and dose, species (and breed) on which the substance was tested, number of animals, study design, length of the study, samples collected, and main results observed.

3.2 The interest of algae, and olive oil, citrus, wine and carob industries by-products in terms of alternative source to synthetic vitamin

Regarding vitamin and vitamin precursors contain, algae include α-tocopherol, β-carotene, niacin, thiamine, and ascorbic acid (Sieburth and Jensen, 1969), and by-products mainly contain ascorbic acid, carotenoids and Vitamin E. However, little was found regarding a detailed characterisation of the vitamin and vitamin precursors, and studies conducted with algae supplementation in livestock were focused on the effect of minerals rather than of vitamins. Among algae, A. nodosum is of a great interest.

Moreover, products rich in polyphenols (Table 1) can simulate the antioxidant activity of Vitamin A, E and C, and enhance the activity of those vitamins. Polyphenols are secondary plant metabolites synthesized by plants to provide protection against invasive pathogens, such as bacteria, viruses and fungi, and to prevent damage to DNA and photosynthetic apparatus due to ultraviolet radiation (Gessner et al., 2017). Plant polyphenols are classified as flavonoids and non-flavonoids according to the number of phenol rings and the structural elements that bind these rings to one another (Tangney and Rasmussen, 2013). Polyphenols are reducing agents, and along with other dietary reducing agents such as vitamin C, vitamin E and carotenoids, they protect the body's tissues against oxidative stress. Thus, in human diets, consuming foods rich in these bioactive compounds, such as green tea, dark chocolate, wholegrain cereals, sesame seeds and many fruits and vegetables, may be beneficial for the enhancement of vitamin E status and health.

Table 3.1. Example of natural sources of vitamin E and C, and polyphenols. The classification of natural

polyphenols was adapted from Ebrahimi and Schluesener (2012). Compound Source

Vitamins C Citrus by-products

E Grains, wheat germ oil

Polyphenols Anthocyanins Grapes, raspberries, strawberries, aubergine skin

Flavanols Onion, grape pomace, apple skin, berries, black grapes, tea, broccoli

Flavones Broccoli, apple skin, lemon, olive, fruit skin, parsley

Flavanones Orange juice

Phenolic acids White grapes, olive, apple, tomato, pear, cherry, grain, cabbage, asparagus

Lignans Sesame seed oil

3.3 By-products from olive oil industry

The use of olive oil by-products for energy production and for the extraction of bioactive compounds has received great attention, whereas their use as animal feed has been less studied (Berbel and Posadillo, 2018).

3.3.1 The crop and its transformation

Olive tree (Olea europaea L.) groves are a key sector in the Mediterranean area, being Spain


(31%), Greece (13%) and Italy (12%) the largest world producers (21 × 106 tons in 2017; FAOSTAT, 2019; accessed 23 Jan 2019). The majority of the olives harvested is used to produce oil. Olives groves have also an important function in terms of hydrological and landscape preservation (Loumou and Giourga, 2003). About one third of the olive groves are from organic production (“DG Agriculture and Rural Development”, EU, 2015). Olive oil is obtained by crushing the olives, followed by the aqueous extraction of oil and then centrifugation to separate the oil from the aqueous phase (Yao et al., 2016). By-products of this process are: olive oil mill wastewater (OMWW), which includes olive pomace (OP), olive leaves (OL) and olive stones (OS), and olive tree pruning biomass (OTPB) which also includes OL. The production of OL from pruning has been estimated to be 25 kg per olive tree, that can increase if we consider the OL collected at the oil mill which is a 5% of the weight of harvested olives (Delgado-Pertíñez et al., 2000). The OP is a solid heterogeneous mixture of olive skin, pulp, woody endocarps and seeds which proportion depend on the olive variety and oil extraction method, and represents approximately 35% (weight/weight) of the processed olives. Its chemical composition indicates a small amount of olive oil, a high content of crude fibre and sugars, and moderate values of crude protein and fatty acids, chiefly oleic (C18:1n − 9) and other C2–C7 fatty acids (Karantonis et al., 2008). Olive stones are mainly used for energy production. Limitations of the use of olive oil by-products for animal feeding are: very little available protein, phytosterols presence, and high energy content that could reduce animals’ total feed intake.

3.3.2 As a vitamin source

A summary of the findings on the use of olive oil industry by-products in aquaculture and livestock is displayed in Table 2. Olive oil is known to contain substantial amounts of carotenoids and vitamin E (Aggoun et al., 2016). Nasopoulou and Zabetakis (2013) reviewed the evidence of olive by-products as feed for aquaculture and livestock concluding that a moderate consumption does not affect growth and improves the fatty acids (FA) profile of animal products by reducing saturated acids and increasing unsaturated components both in meat and milk (Rodríguez et al., 2008). Moreover, levels of OP below 10% in ruminants’ diets reduced feeding costs and improved milk composition without impacting milk yield (Molina-Alcaide et al., 2010). In rabbits, it has been reported that the supplementation with OP inhibited atherosclerosis development due to specific antagonists of the platelet activating factor (Tsantila et al., 2010). Diet supplementation with dried stoned OP in dairy water buffalos increased the total tocopherols, retinol and hydroxytyrosol in milk without affecting gross milk composition and animal performance (Terramoccia et al., 2013). In dairy cattle, this supplementation did not reveal detrimental effects on animal performance, milk production, milk gross composition and milk rennetability (Zilio et al., 2015). On the other hand, performance was negatively affected (i.e., reduced the feed intake, growth rate, carcass weight and dressing out percentage) in rabbits when the diet was supplemented with 5% of dried stoned OP (Dal Bosco et al., 2012). Moreover, the meat presented greater mono- than poly-unsaturated fatty acids and greater oxidative processes (Dal Bosco et al., 2012). Diets supplemented with stoned olive cake in dairy cattle did not affect milk fat content, nor daily milk fat production, but decreased feed efficiency (Cibik and Keles, 2016). In lambs, Mele et al. (2014) reported that the supplementation with stoned olive cake did not affect average feed intake and growth performance, but they observed a greater concentration of c9-18:1 in the neutral lipids of the carcass suggesting an intensive biohydrogenation of dietary c9-18:1. In dairy ewes, stoned olive cake supplementation increased the unsaturated/saturated fatty acid ratio and decreased the atherogenic and thrombogenic indices in milk (Chiofalo et al., 2004; Abbeddou et al., 2011; Vargas-Bello-Pérez et al., 2013). In lambs, supplementation with olive cake (crude, treated with NAOH or with urea) increased feed digestibility (Owaimer et al., 2004). Awawdeh and Obeidat (2013) did not observe any effect on nutrient intake and digestibility in lambs supplemented with olive cake (sundried or acid-treated), but they reported a greater growth rate, final body weight and total body weight gain. The use of OS for rabbits and birds (Alba et al., 1997; Carraro et al., 2005; Sanz Sampelayo et al., 2007), as well as olive soap stocks for ewes and Iberian pigs (Lara et al., 2014),


revealed changes in the fatty acid profile with a relative increase in unsaturated fatty acid. The use of raw OL has been recently tested in rabbits (Mattioli et al., 2018), revealing that this by-product does not alter productive performance. In fish, Alesci et al., 2014 have reported a reduction of the negative consequences of steatosis when feeding Carassius auratus with a hypercholesterolemic diet and OMWW. Dietary supplementation with OMWW showed an improvement of the redox status in broilers (Gerasopoulos et al., 2015; Papadopoulou et al., 2017) and increased the antioxidant mechanisms in piglets (Gerasopoulos et al., 2015).

Table 3.2. Summary of the findings on the use of olive oil industry by-products in aquaculture and livestock.

By-product Species Effect Reference

Olive Pomace Aquaculture and livestock

No effect on growth. Reduce saturated fatty acid proportion in meat and milk.

(Nasopoulou and Zabetakis, 2013)

Ruminants Reduce feed cost. No effect milk yield Improve milk composition.

(Molina-Alcaide et al., 2010)

Rabbits Inhibit atherosclerosis development (Tsantila et al., 2007)

Dried stoned olive pomace

Water buffalos No effect on animal performance and gross milk composition. Increased the total tocopherols, retinol and hydroxytyrosol in milk.

(Terramoccia et al., 2013)

Dairy cattle No effect on animal productive performance and gross milk composition and rennetability.

(Zilio et al., 2014).

Rabbits Reduce feed intake, growth rate, carcass weight and dressing out percentage. Increase monounsaturated fatty acids in meat. Increase oxidative processes.

(Dal Bosco et al., 2012)

Stoned olive cake

Dairy cattle No effect on milk fat content or daily milk fat production. Decrease feed efficiency.

(Cibik and Keles, 2016)

Lambs No effect on average feed intake and growth performance. Greater concentration of c9-18:1 in the neutral lipids of the carcass.

(Mele et al., 2014)

Dairy ewes Increase unsaturated/saturated fatty acid ratio in milk. Decrease the atherogenic and thrombogenic indices in milk.

(Chiofalo et al., 2004; Abbeddou et al., 2011; Vargas-Bello-Pérez et al., 2013)

Olive cake Lambs Increased digestibility of DM, CP, EE, NFE and TDN.

(Owaimer et al., 2014)

Lambs Increase growth rate, final body weight and total body weight gain. No effect on feed conversion ratio and dressing percentages.

(Awawdeh and Obeidat, 2013)

Olive stones Rabbits and birds Reduce saturated fatty acid proportion. (Alba et al., 1997; Carraro et al., 2005; Sanz Sampelayo et al., 2007)

Olive soap Ewes and Iberian pigs

Reduce saturated fatty acid proportion. (Lara et al., 2014)

Olive leaves Rabbits No effect in animal performance. (Mattioli et al., 2018)


Olive oil mill wastewater

Fish Reduce negative consequences of steatosis.

(Alesci et al., 2014)

Broilers and pigs Improve redox status. (Gerasopoulos et al., 2015; Papadopoulou et al., 2017)

3.4 By-products from citrus industry

The potential use of citrus by-products is related to the fact that Citrus fruits (Rutaceae family) are an important source of ascorbic acid (Vitamin C) and carotenoids (Vitamin A precursors) as well as other antioxidants such as flavonoids and other phenolic compounds (Abeysinghe et al., 2007; Rapisarda et al., 2008; Ghasemi et al., 2009).


Citrus (Citrus spp.) is one of the most abundant fruit crops with a world production of 147 × 106 tons in 2017, being China (27%), Brazil (14%) and India (8%) the greatest world producers (FAOSTAT, 2019). About 18% of the citrus world production is in Mediterranean countries (FAOSTAT, 2019). After the mechanical extraction of the juice from the fruit, the remains are a residue named citrus pulp and comprised of peel (flavedo and albedo), pulp (juice sac residue), pith (membranes and cores) and seeds (Bampidis and Robinson, 2006). This by-product contains a high amount of pectin and soluble carbohydrates, high energy and low crude protein (7%) (Fegeros et al., 1995).


Lambs supplemented with dried citrus pulp showed an improvement of the meat fatty acid composition (Lanza et al., 2015), a better meat oxidative stability (Gravador et al., 2014; Inserra et al., 2014) and an increase of antioxidant status of muscle due to the increase of α-tocopherol (Luciano et al., 2017). Furthermore, Villarreal et al. (2006) reported that tropical beef cattle fed with pelleted citrus pulp improved forage intake, digestion and ruminal pH.

3.5 By-products from wine industry

The potential use of wine industry by-products in organic livestock is due to their wide range of polyphenols (Bonilla et al., 1999; Alonso et al., 2002; Torres et al., 2002). The polyphenols described in those by-products are: anthocyanins, catechins, flavanols, alcohols, stilbenes, benzoic (gallic, protocatechuic, 4-hydroxybenzoic) and cinnamic (p-coumaric) acids (Chedea et al., 2014, 2017;Chedea et al., 2018).


Grape (Vitis spp.) is one of the most valued fruits in the world (García-Lomillo and González-SanJosé, 2017) based on hectares cultivated and economic value (Torregrosa et al., 2015). Italy, France and Spain represent 24% of world grape production (74 × 106 tons in 2017), being these three countries among the 5 largest producers in the world (FAOSTAT, 2019). The main by-products are collected during destemming (stems), grape crushing, and pressing (skins, seeds, and lees). Grape pomace consists mainly of peels, stems, and seeds, and accounts for about 20% of the weight of the grape processed into wine (Llobera and Cañellas, 2007).


Recently, several studies have highlighted the antioxidant activity of grape pomace in vivo e in vitro (Cotoras et al., 2014; Goutzourelas et al., 2015; Vergara-Salinas et al., 2015; Chedea et al., 2018; Rasines-Perea et al., 2018). Research on animal diet supplemented with grape pomace has mainly focused on chickens (Goni et al., 2007; Brenes et al., 2008; Viveros et al., 2011; Aditya et al., 2018), showing the potential of grape pomace, in combination with Vitamin E, to reduce lipid oxidation of meat, during refrigerated storage and to increase liver α-tocopherol


concentration.

3.6 By-products from carobs

The potential use of carob pulp for animal feeding is linked to its fatty acid profile (Ayaz et al., 2009) and its strong antioxidant capacity due to the phenolic compounds (Kumazawa et al., 2002; Owen et al., 2003; Papagiannopoulos et al., 2004; Rtibi et al., 2017; Ydjedd et al., 2017; Stavrou et al., 2018). However, an important limitation for its use in animal feeding is the high content of condensed tannins, which are anti-nutritional factors.


Carob tree (Ceratonia siliqua L.) is a typical crop in the Mediterranean area. Carobs world production in 2017 was 0.14 × 106 tons, being the largest producers Portugal (31%), Italy (21%), and Morocco (16%) (FAOSTAT, 2019). The processing procedure of the pods produces the carob pulp as a by-product (Vekiari et al., 2011). The carob pulp presents high sugars levels (such as sucrose), and low protein and fat content (Ayaz et al., 2009). In addition, its fatty acid profile includes essential fatty acids for animal nutrition such as linoleic and α-linolenic acids (Ayaz et al., 2009).


Diet supplementation with carob pulp has been investigated in ruminants (Priolo et al., 2000; Silanikove et al., 2006; Kotrotsios et al., 2012; Abu Hafsa et al., 2017). It has also been evaluated in pigs (Inserra et al., 2015) and lambs (Gravador et al., 2015), showing an increase of polyunsaturated fatty acids in the muscle, including rumenic acid, and a reduction in saturated fatty acids and n-6/n-3 ratio.

3.7 Discussion and Conclusions

The possibility to use synthetic vitamins for organic livestock production allowed by the European regulation is based on the current knowledge and availability of natural vitamins trough additives or feeds that could ensure anima health and welfare. This review provides evidence of the lack of information regarding the use of algae and food industry by-products to fill this gap, which will contribute to a greater implementation of organic farming principles. The bibliographic search highlighted that food industry by-products have been investigated primarily for their potential as energy production, rather than for their use as animal feed or diet additive. Even though there is very little information on vitamin characterization in those products and its impact on animal performance and produce, it seems that algae and olive oil, citrus, wine and carob food industries by-products are a feasible alternative to the use of synthetic vitamins to ensure adequate vitamin intake in livestock.

The literature review showed that several by-products that can be used in organic production as natural sources of vitamins; for instance, grape pomace has the potential to be one of the best by-products because it provides good amounts of pro-vitamins and high amounts of polyphenols, exerting a strengthening and saving effect on dietary vitamins. This product should therefore be tested in poultry, dairy and beef cattle organic production to provide further evidence of the benefits of it use in animal feed. Furthermore, taking into account the fact that in the grape/wine sector, there is a trend to move to organic production, it is reasonable to expect that in the future these by-products could be easier to source. Similar considerations could be made also for olive oil and citrus by-products.

Finally, the use of organic by-products in organic livestock farming seems to be an interesting solution also from a sustainability point of view, in an integrated value chain perspective with minimum transport of raw materials. One of the aims of the Organic-PLUS project is to test these by-products for livestock feeding and to provide scientific evidence that it is possible to use them as a natural source of vitamins without compromising productive performance and food quality traits. It appears probable that the use of such kind of by-products will be a satisfactory solution. Based on the literature findings, it appears that the main sources of by-products and alternatives


to synthetic vitamins are from Mediterranean countries. This probably depends on the large amount of these by-products historically available, that in the lasts 40 years drove the research towards their potential utilization in animal feeding. Very limited information about the use of other by-products is available in central and north European countries. Only a few papers reported information on the possibility of using more Nordic plant products, like apple pomace and hops/brewery by-products or polyphenols-rich plants like lavender in animal feeding, but information in their in vivo vitamin-like effects is lacking. These raw materials however could be of interest and need to be further investigated.

3.8 References

Abbeddou, S., Rischkowsky, B., Richter, E. K., Hess, H. D., & Kreuzer, M. (2011). Modification of milk fatty acid composition by feeding forages and agro-industrial byproducts from dry areas to Awassi sheep. Journal of Dairy Science, 94(9), 4657–4668. https://doi.org/10.3168/jds.2011-4154

Abeysinghe, D. C., Li, X., Sun, C., Zhang, W., Zhou, C., & Chen, K. (2007). Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species. Food Chemistry, 104(4), 1338–1344. https://doi.org/10.1016/j.foodchem.2007.01.047

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4. Review of natural sources of antiparasitics on organic livestock farming

4.1 Introduction

Parasites that affect livestock may live on the skin or layers (external parasites or ectoparasites) or in the digestive tract, liver, lungs, kidneys or elsewhere in the body (internal parasites or endoparasites). Ectoparasites can be insects or arachnids (mites and ticks), whilst endoparasites can be either microscopic organisms known as protozoa or larger parasites known as metazoan, worms or helminths. Infestation with both ecto- and endoparasites has detrimental effects on animals’ performance, production and health. Ectoparasites can also act as vectors for several vector-borne pathogens of viral, bacterial or parasitic nature. Systematic control of parasites in conventional farming is based on synthetic drugs, which has increased drug resistance and it is not allowed in organic farming. Nevertheless, plants and plant-derived products for both endoparasites and ectoparasites treatment are in fact part of the traditional medicine culture, and were the only way to treat animals until mid-20th century (French, 2018).

Several plants described as able to expel parasites appeared in medieval herbals and in 17th–19th century printed books (French, 2018). Nowadays, the use plants to treat livestock diseases is still ongoing in small extensive farms and by pastoralists around the world. Small farmers in northern Europe still rely on old “traditional” pastures rich in medicinal herbs and legumes for the perceived anthelmintic qualities of specific wild plants (French, 2018). This traditional and socio-cultural knowledge is being documented by the ethnoveterinary medicine. The ethnoveterinary medicine comprises the traditional management of veterinary diseases, their remedies, and the spiritual elements associated with the healing procedures practiced by a local community (Mathias, 2004).

According to French (2018), plants naturally produce over 60,000 chemical compounds to deter herbivores, to destroy microbial pathogens, and to communicate with other organisms like pollinators (Wink 2010). Based on this evidence, exploiting the diversity and bioactivity of plant secondary metabolites may be a viable alternative to treat ecto- and endoparasites. Some of the plants and plant products used around the world and documented by ethnoveterinary medicine have been tested in clinical trials or experimental controlled studies. However, the testing procedures have been in general heterogeneous, which sometimes generated confusing results.

One of the reasons for the limited use of plants and plant products as antiparasitic substances is due to the fact that some promising candidates can show negative (antinutritional, toxic) and positive (immunomodulatory) side effects. Even if not yet clearly characterised, the majority of compounds with anthelmintic properties from forages and plants are plant secondary metabolites (PSM). It is mandatory to identify active compounds from plants in order to avoid the already known toxic effects from whole plant administration (Athanasiadou et al., 2007). While the negative effect on the host is largely unknown, a small number of studies is available regarding hosts’ performance and in some cases clinical toxicity is reported. The positive side effect related to the administration of plants with antiparasitic effect is immunostimulation, related to the increase specific effector cells.

Therefore, this review aimed to summarize the scientific results regarding the use of plants and plant products and their potential to control ecto- and endoparasites in livestock.

4.2 Economic and productivity losses from parasitoses

According to a review from Armour and Gettinby (1982), attempts to quantify the production effects of the para-physiological changes related to parasitic infections have been carried out under both experimental and field conditions. Documents on these trials are available starting from the middle of the 20th century. Key findings are summarised below.

Regarding experimental monospecific infections, the reduction in live weight gain was directly proportional to the numbers of larvae administered of groups of 10 calves infected with Ostertagia spp. A significant reduction of 20-39%, reflected by a decrease in staple lengths and


wool fibre diameter properties, was observed Australian Merino sheep infected with Fasciola. In heifers infected with Fasciola, a delayed first oestrus by at least 30 days and a reduction of milk yield (9% less milk) in the following first calving were observed. Moreover, Pattison et al. (1979) in Northern England reported a decrease by 13.1 kg of mean body weight from service to weaning of infected animals of the litter of 10 sows infected with larvae of Oesophagostomum; while controls gained 11.3 kg. Moreover, infected sows’ litters averaged 11.3 piglets born, while an average of 12.8 were born to the controls. The birth weights of the piglets from the infected dams were 15% less than those from the controls and subsequently at weaning piglets from infected sows were significantly lighter (10.5%) of those from infected sows, and demonstrated also a lower feed efficiency.

Regarding experimental combined infections, concurrent infections of abomasal and intestinal trichostrongyle in lambs were more pronounced than they would have been predicted from the results obtained following monospecific infections, with wool growth reduced by 66% versus only 25% and 10% in the individual infections.

4.3 Methodology

Following French (2018) methodology, the literature research has been done on www.webofscience.com. The keywords were: ‘plants’ and ‘antiparasitic’. We considered papers published in all fields throughout all years available in the database (1985 – 2019). A total of 523 publications has been included.

4.4 Bibliometric results

The number of publications on plants used to treat livestock parasites infections has doubled in the past ten years (Figure 4.1), from 15 documents in 2008 to 70 documents in 2018. As suggested by French (2018), this increase reflects the issue of increasing resistance of parasites to the conventional synthetic antiparisitics.

Figure 4.1. Number of documents by year of publication retrieved from Web of Science database based on the search terms ‘antiparasitic’ and ‘plants’ from 1985 to January 2019.

The countries that have published more documents related to this topic are USA (90 documents), Brazil (89 documents), France (55 documents) and India (43 documents; Figure 4.2). Research on antiparasitics based on plants and plant products has been conducted in 4 continents. The majority of the documents fall within the field of pharmacology, chemistry, plant science, parasitology, and veterinary sciences (Figure 4.3).

http://www.webofscience.com/


Figure 4.2. Number of documents by country of publication retrieved from Web of Science database based on the search terms ‘antiparasitic’ and ‘plants’ from 1985 to January 2019.

Figure 4.3. Number of documents by field of publication retrieved from Web of Science database based on the search terms ‘antiparasitic’ and ‘plants’ from 1985 to January 2019.

4.5 Reliability of the results on the antiparasitic activity of plant molecules

The scientific community requires that the antiparasitic capacity of plants and plant products described in ethnoveterinary and medicinal reports is demonstrated through test procedures performed under controlled experimental conditions. However, the literature research has highlighted a wide variability of methodologies employed to validate and quantify such plant activity and the lack of measures to minimise experimental variability, leading to a general inconsistency between studies results. Some of the reasons generating this inconsistency are displayed in Table 1. Moreover, there is currently a lot of controversy on the choice of a methodology as accepted standardised way to evaluate the activity and the role of medicinal plants for parasite control (Athanasiadou et al., 2007).


Table 4.1. Reasons that lead to inconsistency on the bibliographic research about plants and plant product as antiparasitic in livestock.

Issue

Misinterpretation of the results of the trials due to lack of scientific knowledge

In vitro trials without simultaneous in vivo experimentation

Plants’ properties tested only in one animal species

Identification of the active compound and dose tested (lack of)

Variability between plant products

Variability of the protocols of collection and storage of the plant material prior to its use

Greater efficiency of extracts than using whole plants

Bioavailability and interaction with nutritional value when using of whole plants

Host/parasite/compound interaction

Parasite niche

4.5.1 Efficacy testing of anthelmintics

Parasitologists have recommended that threshold points similar to those set, for example, for commercial anthelmintics should be established, below which the efficacy of medicinal plants for parasite control should not be considered. Another suggestion is to justify their use based on the economic threshold, as it relates to efficacy. In this direction, Athanasiadou et al. (2007) emphasize the need for incorporating additional measurements in ongoing research related to performance measurements, indicators of immunity and behavioural observations. The inclusion of these three aspects would lead to a more holistic investigation of the properties of plants with antiparasitic activity, and this knowledge could drive their potential exploitation in livestock farming.

A more general and comprehensive procedure for the evaluation of anthelmintic properties of plants has been created by French (Oxford University) that suggested the ‘best practices’ that could be helpful in standardizing plant bioactivity evaluation and in increasing test reproducibility. These ‘best practices’ describe how to perform the main phases of the evaluation process, including preparation of plant material, fractionation, assay selection, and in vivo tests: - Preparation of plant material: plants should be collected at the same time of day and dried

or rather lyophilized, trying to avoid damage to plant metabolites damage during the treatment (e.g., terpenes can be lost during oven drying). At least 4 replicates of each plant from each site are needed to have a representative sample.

- Plant material can undergo fractionation, meaning the extraction of metabolites using a variety of solvents -e.g., ethanol, acetone, chloroform, methanol, water- capable to determine the release of molecules based on polarity and hydrophilicity. It should be considered that even if extraction and purification of secondary metabolites allows for the identification of highly active components, testing these molecules as individual entities could mask the role of synergy between molecules in determining the effects of plant products on parasites.

- Choice of the assay to employ is another critical step on the testing process. Minimal Inhibitory Concentration (MIC) and Lethal Concentration (LC) values of a crude extract on helminths are generally determined using the agar dilution assay and broth dilution assay. Agar assay is possible -a petri dish is seeded with nematodes and E. coli (their food source) and then exposed to a plant extract -, but this procedure is not adequate for plant products like e.g. essential oils that do not move through agar easily, leading to false negatives. Broth dilution assays is generally performed on 96-well microtiter plates filled with a nematode growth medium, nematodes (e.g., 10–50 L-4 stage adults) and the crude extract under assessment. It allows screenings for parasites and for compounds. In practice, many compounds can be assayed against nematodes in different life stages (e.g. eggs, larvae, adults) at once and the system can be semi- automated, allowing also the detection of


parasite motility if a camera attached to an inverted microscope is available. - When testing plant products, in vitro results should be confirmed in vivo. Monitoring the in

vivo effects can involve slaughtering of the animals, but most of the time only a continuous monitoring of faecal egg counts or the determination of the number of eggs and/or larvae in soil cores and on grass samples is necessary depending on treatment type, to evaluate changes in the abundance of parasites where animals are grazing.

4.5.2 Efficacy testing anti-ectoparasites

In 2005 the European Medicine Agency made available a document containing the guidelines (EMEA, 2004) on specific efficacy requirements for ectoparasiticides in cattle. In the document they presented: general requirements (introduction, general principles -protocols, assessment of efficacy-, persistent efficacy trials, efficacy field trials), parasites species-specific information (dose determination/dose confirmation studies, persistent efficacy trials, efficacy field trials), and an appendix with calculation formulae (Abbott formula 1 and 2) for the evaluation.

4.6 Plants and plant products with antiparasitic effects

Based on the data contained in the USDA phytochemicals database (USDA, 2019), the top five plants with the highest number of anthelmintic compounds are Achillea millefolium, Dryopteris filix-mas, Peumus boldus, Rosmarinus officinalis, and Salvia officinalis. However, many wild legumes found in British meadows (e.g. Lathyrus pratensis, Vicia cracca), as well as many indigenous plants recently evaluated for anthelmintic qualities are absent, highlighting the need for a continuous update in this topic (French, 2018).

Various authors in different geographic regions have reviewed the literature on the antiparasitic plant products in the attempt to provide some clarity on the topic. The main results regarding the phytochemical compounds and their effects on parasites is reported in Table 4.2. Table 4.2. Main phytochemical compounds and their effects on parasites from the literature.

Phytochemical Effect

Saponins Affect permeability of parasitic cell membrane; vacuolization and disintegration of teguments

Benzyl isothiocyanate Inhibition of energy metabolism, affects motor activity of the parasites

Cysteine proteinases (i.e. papain, chymopapain)

Digestion of nematode cuticle

Isoflavones Inhibition of the glycolytic enzymes, interference with Ca2+ homeostasis

Artemisin Damage of macromolecules of parasites’ tissues through the generation of oxidative stress

Phenolic compounds Interfere with energy generation mechanism and with the glycoprotein of the cell surface of the parasites

Tannins Interfere with energy generation in helminths; binding to the free proteins of the gastrointestinal tract of the host animal or to helminth cuticle glycoprotein (leading to death).

Alkaloids Antioxidants, paralysis; inhibition of ribosomal protein biosynthesis (emetine from Rubiaceae)

4.6.1 Plants containing anthelmintic antiparasitic compounds

Several Indian indigenous plants presented antiparasitic capacity, and specifically anthelmintic activity (Table 4.3; Bauri et al, 2015). In Nordic countries the ancient, rich and diverse history of plant-derived anthelmintic medications for human and animal use has also been reviewed; however, at that time evidence was almost entirely anecdotal (Table 4.4; Waller


et al., 2001). Moreover, according to Waller et al. (2001), the action of tanniferous plants against internal parasites could be explained by the increased availability of digestible protein to the animal or by a direct anthelmintic effect on resident worm populations or on free-living stages in animals. In Serbia, this has been reported in traditional anthelmintic treatments, especially nematocides, the use of White mugwort (Artemisia absinthium L., Asteraceae) and Black mugwort (Artemisia vulgaris L., Asteraceae) (Davidovic et al., 2012). The same authors indicated that decoction of the rhizome of genuione brachen s, Male Fern (Dryopteris filixmas L., Aspidiaceae) represents one of the strongest natural drugs to control tapeworms (Taenia saginata, Taenia solium) and flukes (Fasciola hepatica) through the active compound filicin and filmarone, that is toxic for the worms, and oleorescin, that paralyzes parasite musculature preventing their adhesion. Table 4.3. Indian indigenous plants that presented antiparasitic capacity, and specifically anthelmintic activity. Shown are: plant scientific name, active compound and parasite against whom has been tested to be active for (adapted from Bauri et al., 2015).

Plant Active compound Active against

Zingiber officinale Zingiberene, bisabolene, gingerols, shogaols

H. contortus, mosquitoes

Nigella sativa Thimoquinone, dithimoquinone- cymen α-pinane

Tapeworms, hookworms, nodular worms, schistosomes; H. contortus (+ ivermectin)

Piper longum Piperine Gigantocotyle explanatum, A. lumbricoides

Flemingia procumbens Genistein Raillietina, Fasciolopsis, Fasciola hepatica

Melia azedarach Mliacaprin, scopoletin, meliartenin H. contortus; T. solium; ticks, lice and mosquitoes

Ocimum sanctum Eugenol, β-caryophyllene, Urosilic acid

A. galli, C. elegans; mosquitoes

Azadirachta indica Azadirachtine F. gigantica; Railletina, H. contortus, Trichostrongylus spp., ticks

Calotropis procera Calotropin, calactin. Oesophagostomum columbianum; Bunostomum trigonocephalum; Ostertagia, Nematodirus, Dictyocaulus, Teania, Ascaris and Fasciola. H. contortus (latex). Coccidia (Eimeria tenella)

Artemisia annua Artemisinin, quercetin F. hepatica, GI nematodes, Plasmodium, Babesia, Leishmania, coccidia, Toxoplasma, Neospora

Pongamia pinnata Karanjin, pongapin, kanjone, pongaglabrone, diketone pongamol, fatty acids (palmitic, linoleic…)

Setaria

Achyranthes aspera Triterpenoids and saponins Paramphistomum, ticks

Moringa oleifera Tannins, flavonoids, triterpenoids, saponins and alkaloids

Filarial parasites, mosquitoes

Table 4.4. Plant derived anthelmintic medications for human and animal use in Nordic countries. Shown are: plant scientific name or group, active compound if known and parasite against whom has been tested to be active for (adapted from Waller et al., 2001).

Class of plants or plants Active compound Active against Lichens and ferns. Dryopteris filix-mas (aspidium)

Filicic acid Tapeworms


Trees and shrubs (e.g Salix) Acetilsalicilic acid Anthelminthic

Herbaceous plants (e.g. Chenopodium ambroisoides (wormseed or goosefoot)

Ascaridiol Ascaris spp.

Carum carvi (caraway), Thymus spp (thyme), Mentha spp (mint)

Trichostronglyus sp. Larvae, in vitro studies

Artemisia sp. Santonin (sesquiterpene lactone)

Ascaris, Enterobius; tapeworm infections

Tanacetum vulgare Thujon Trichostrongylus; Ostertagia circumcincta (in vitro)

Daucus carota, Brassica spp, Allium spp.

Used as antiparasitics in many countries

Cucurbitaceae fam. (pumpkin and cucumber seeds in particular)

Cucurbitine (the amino acid 3 amino 3 carboxy pyrolidin),

Tapeworm

Nicotiana rustica Nicotine In nematocide preparations for ruminants in mid-1950s

Pasture plants: Hedysarum coronarium (sulla) and Lotus pedunculatus (lotus major)

Proanthocyanidins (condensed tannins, a poorly defined group of compounds)

Reduce worm burdens in grazing lambs by up to 50% In vitro and in vivo nematocidal effects

4.6.2 Plants and plants products containing active compounds against ectoparasites

According to the review of Alemu and Kemal (2015) from Africa, ectoparasites or external parasites are “parasites, with few exceptions, that live or burrow into the surface of their host’s epidermis”. They generally acquire blood meals from their host without penetrating entirely in their host. Animals can undergo infestations from different ectoparasites. For the control of ectoparasites, at the moment, farmers rely mainly on chemical acaricides and repellents that help in controlling ticks and limit animal production losses. However, resistant ticks are now found with growing frequency and this is likely to be related to an increased application of acaricides.

In places where small scale farms are the largest proportion of livestock enterprises – like in Africa and in other developing countries- ethnoveterinary plant use for ectoparasites control is very important, even if it is often forced by the financial impossibility to purchase synthetic acaricides. The use of plants or plant-based products for the control of arthropod ectoparasites in livestock is in fact widespread among small scale livestock keepers in Africa and the plant species used for this purpose can vary with the community considered.

Because of the importance of ectoparasites, especially ticks, both from a medical and economical point of view, it is urgent to screen some ethnoveterinary plants that have acaricidal properties and could be used widely. Evidence of some activity against ectoparasites has been reported for the following plants displayed in Table 4.5 (Alemu and Kemal, 2015; Davidovic et al., 2012).

Table 4.5. Plants with anti-ectoparasites properties. The Table displayed: plant scientific name, active compound if known and ectoparasite against whom has been tested to be active for (adapted from Alemu and Kemal (2015) and Davidovic et al. (2012)).

Plant Active compound Active against

Calpurnia aurea (Natal laburnum)

Juice, leaves, bark, powdered roots, leaf extracts (?)

Ticks (e.g. Rhipicephalus pulchellus), lice

Otostegia integrifolia Mosquito repellent

Jetropha curcas Ticks (egg eclosion inhibition)

Nicotiana tabacum Lice, ticks (including acaricide effect on R. appendiculatus)

Artemisia absinthium Fly repellent


(white mugwort), A. vulgaris (black mugwort)

Helleborus L., Ranunculaceae (Stinking hellebore), Veratrum album L., Liliaceae (hellebore), Nicotiana tabacum L., Solanaceae (Tobacco)

Three plants boiled cooked together

Lice, mite infestation (mange)

4.7 Plants and plant products with active compounds against parasites in fish farming

A further research area that should be considered when studying the use of plant as alternative to synthetic antiparasitics, is the involvement of plant products in fish farming. This topic is reviewed by Wunderlich et al. (2017) in a document highlighting the advantages of the use of plant extracts as an alternative treatment against parasites in aquaculture, as well as the environmental impacts of conventional treatments used in fish farming systems. Information is available on plant compounds active against the most economically important parasite species in fish farming, namely protozoans, myxozoans and helminths (monogeneans) (Table 4.6). Monogeneans (e.g. Dactylogyrus spp. and salmon fluke Gyrodactylus salaris) and protozoans (e.g. Ich Ichthyophthirius multifiliis and Trichodina spp.) are very common ectoparasites living on the gills of freshwater and marine fish. In particular, essential oils, extracts and isolated substances from plants might be important alternatives to oral and immersion treatment against parasites in aquaculture and can play also immunostimulant activity covering a phototherapeutic role against infections. Table 4.6. Plants with anti- protozoans, myxozoans and helminths (monogeneans) properties in fish farming. The Table displayed: parasite against whom has been tested to be active for, the fish host species and plant scientific name (adapted from Wunderlich et al., 2012).

Active against Host species Plant

Protozoans

Myxosporeans - Origanum1

I. multifiliis

Colossoma macropomum (tambaqui)

Lippia alba (bushy matagrass)1

Fresh and marine farmed fish Magnolia officinalis2; Sophora alopecuroides2

Carassius auratus Eclipta prostrate (false daisy), Lycium chinense (Chinese matrimony vine), Ophiopogon bodinieri3; Trichosanthes kirilowii (Chinese cucumber)3

Poecilia latipinna (sailfin molly). Allium sativum (garlic), Matricaria chamomilla (chamomile)3

Mixozoan

Polysporoplasma sparis Myxobolus sp.,

Sparus aurata (Gilthead sea bream); Puntazzo puntazzo (sharpsnout sea bream)

Origanum1

Myxobolus sp., Puntazzo puntazzo (sharpsnout sea bream)

Origanum minutiflorum (spartan oregano)1

Enteromyxum leei

Sparus aurata (Gilthead sea bream)

several essential oils1

Achillea millefolium (milenrama milfoil), Betula alba (silver birch), Calendula officinalis (marigold), Cerasus sativa (sweet chestnuts), Crategus monogyna (hawthorn), Equissetum arvensis (horsetail), Hypericum perforatum (st. johnswort), M. chamomilla, Mentha piperita


(peppermint), Ocinum basilicum, Prunus spinosus (blackthorn), Rosacanina (dogrose), Sambucus nigra (elder), Thymus serpillum (wild thyme), Tilia sp., Vaccinium myrtilus (bilberry) and Viola tricolor (Johnny Jump up)4

Helmints

Cichlidogyrus tilapiae, C. thurstonae, C. halli; Scutogyrus longicornis

Lippia sidoides (pepper rosemary)1; M. piperita1

Anti-parasite effect Colossoma macropomum (tambaqui)

Ocimum gratissimum (clove basil)1

Anacanthorus spathulatus,Notozothecium janauachensis and Mymarothecium boegeri

Colossoma macropomum (tambaqui)

L. alba1

Parasites Heterobranchus longifilis (vundu) Artemisia annua3

Dactylogyrus intermedius C. auratus (goldfish); Semen aesculi; Radix Bupleuri chinensis (schisandra fruit)3; Dryopteris crassirhizoma (thick stemmed wood fern), Kochia scoparia (kochia) and Polygala tenuifolia (yuan zhi)3; Cinnamomum cassia (cinnamon), Lindera aggregata (evergreen lindera) and Pseudolarix kaempferi3

D. vastator C. auratus (goldfish); Euphorbia fischeriana (Lang‐Du)3

Gyrodactylus turnbulli and Dactylogyrus sp.) (decreased movement); Capillaria sp.

Cyprinus carpio (common carp); A. sativum3

Dactylogyrus spp C. auratus (goldfish); Ginkgo biloba (ginkgo) and Dioscorea zingiberensis (yellow ginger)3

1Essential Oil 2Methanol extract 3 Plant extract 4Acqueous, methanol extract

4.8 Conclusions

The number of publications focused on the study of the potential use of plants and plant-derived products as antiparasitics has increased in recent years. This witnesses the growing interest into developing new, alternative approaches to the control of parasites in veterinary medicine. Despite the large amount of published studies, there is still no consensus around the procedures to test the efficacy of plant-derived antiparasitic substances. For this reason, in most cases it is not possible to compare different studies or to rely on the use of plants or plant products as antiparasitics in practice. Even if the economic role of antiparasites has been widely demonstrated, there is still a need to test plants and plant-derived products in a way that will allow these products to be applied and introduced in the routine of livestock farming.

4.9 References

Armour, J., & Gettinby, G. (1982). A critical review of the evaluations of production effects of helminth disease and mismanagement on livestock production. In : Proceedings of the 3rd International Symposium on Veterinary Epidemiology and Economics - ISVEE 3: Veterinary Epidemiology and Economics, Proceedings of the 3rd International Symposium, Arlington, Virginia, USA (1982), pp 164-172.


Athanasiadou, S., Githiori, J., & Kyriazakis, I. 2007. Medicinal Plants for Helminth Parasite Control: Facts and Fiction. Animal 1 (9): 1392–1400. https://doi.org/10.1017/S1751731107000730.

Bauri, R. K., Tigga, M. N., & Kullu, S. S. 2015. A Review on Use of Medicinal Plants to Control Parasites. Indian Journal of Natural Products and Resources 6 (4): 268–77. https://doi.org/10.1371/journal.pone.0147934.

Davidovic, V., Todorovic, J., Stojanovic, B., & Relic, R. 2012. Plant Usage in Protecting the Farm Animal Health. Biotechnology in Animal Husbandry 28 (1): 87–98. https://doi.org/10.2298/BAH1201087D.

French, K. E. 2018. Plant-Based Solutions to Global Livestock Anthelmintic Resistance. Ethnobiology Letters 9 (2): 110. https://doi.org/10.14237/ebl.9.2.2018.980.

Alemu, S., & Kemal, J. 2015. The properties of selected medicinal plants against Bovicola ovis and Amblyomma varigatum : A Review. European Journal of Applied Science, 7(6):277-290. https://doi.org/10.5829/idosi.ejas.2015.7.6.101174.

Mathias, E. 2004. Ethno veterinary medicine: harnessing its potential. Veterinary Bulletin 74(8):27–37.

EMEA. 2004. Guideline on specific efficacy requirements for ectoparasiticides in cattle agreed by efficacy working party adoption by CVMP (EMEA/CVMP/625/03/Final).

USDA. 2019. https://www.ecfr.gov/cgi-bin/text-idx?c=ecfr&SID=9874504b6f1025eb0e6b67cadf9d3b40&rgn=div6&view=text&node=7:3.1.1.9.32.7&idno=7 (accessed 1.16.19).

Waller, P. J., Bernes, G., Thamsborg, S. M., Sukura, A., Richter, S. H., Ingebrigtsen, K., & Hoglund, J. 2001. Plants as De-Worming Agents of Livestock in the Nordic Countries : Historical Perspective, Popular. Acta Veterinaria Scandinavica. Supplementum 42 (1): 31–44. https://doi.org/10.1186/1751-0147-42-31.

Wink, M. 2010. Introduction. In Functions and Biotechnology of Plant Secondary Metabolites, edited by M.Wink, pp. 1–20. Blackwell Publishing Ltd., Oxford, UK.

Wunderlich, A. C., Guimarães, A. C., & Takeara, R. 2017. World’s Largest Science, Technology & Medicine Open Access Book Publisher Plant-Derived Compounds as an Alternative Treatment Against Parasites in Fish Farming : A Review. Natural Remedies in the Fight against Parasites, 246. https://doi.org/10.5772/67668.


5. Review of natural sources of antimicrobics on organic livestock farming

5.1 Introduction

Phytochemicals are non-nutritive plant-derived compounds (Kamboh et al., 2018) which

have an important role in defencing plants against infections, pests, stressful conditions, and

physical damage (Cowan, 1999). They are mainly plant cell secondary metabolites, being the

most important the polyphenols (phenolic acids, flavonoids, stilbenes, phenolic alcohols, and

lignans),(Abbas et al., 2017). Some examples of those polyphenols are alkaloids, tannins,

cyanogenic glycosides, flavanones, quinones, saponins, coumarins, and terpenoids ((Robbins,

2003; Analele, 2007). The composition and concentration of the polyphenols is very

heterogeneous, indeed the plant, parts of the plant, geographical origin, harvesting season,

environmental factors, storage conditions, and processing techniques influence these due

features (Gadde et al., 2017) which deeply affect their activity. Since the dawn of time, plant-

derived compounds have found applications for flavouring food, but also for traditional

medicine (Gyawali et al., 2014). In some countries, such as in South-America, Africa, Asia, the

use of herbs in livestock for treatments is widespread, as confirmed by some recent reviews

exploiting individual ethnoveterinary treatments (Chinsembu et al., 2014; Verma, 2014; Pasca

et al., 2017; Syakalima et al., 2018).

Phytochemicals, along with probiotics, prebiotics, organic acids, antimicrobial peptides,

bacteriophages and hyperimmune belong to a group of substances known usually as antibiotic

alternatives. An ideal alternative to an antibiotic should have the same beneficial effects of

antibiotic growth promoters, therefore it should guarantee optimum animal performances, and

increase nutrient availability (Lillehoj et al., 2018). Moreover, any candidate as natural

antimicrobial should be cheap, available, secure, safe, bioavailable, effective, metabolisable,

and biodegradable (Table 5.1; Cheng et al., 2014). On this matter, food industry by-products

such as peels, seeds, husks and kernels have shown to be promising sources for polyphenols,

tannins and flavonoids and other bioactive components with antimicrobial activity

(Balasundram et al., 2006; Tiwari et al., 2015).

Table 5.1. Properties that any candidate to natural antimicrobial should follow (Cheng et al., 2014).

Properties for a natural antimicrobial

non-toxic or no side effects on animals

short term of residues

be stable in the feed and animal gastrointestinal tract

low environmental impact

without influences on palatability

without disturbing physiological intestinal flora

efficacious against pathogenic bacteria

enhance the body resistance to diseases

ameliorate feed efficiency and enhance animal growth

good compatibility

without contributing the development of antimicrobial-resistance

In livestock, antimicrobial resistance has been demonstrated to be a great threat to public

health worldwide (Da Costa et al., 2013; Agga et al., 2015; Marquardt and Li, 2018). As a matter of fact, it has been suggested that the use of antimicrobials in livestock production can promote bacterial resistance in treated animals (O’Brien, 2002). Thus, the interest of using herbal products in livestock for treating illnesses and promoting growth has been increasing in the last years. According to Viegi et al. (2003), cattle, horses, sheep, goats and pigs represent about 31%,


14%, 17%, 17% and 7%, respectively, of the animals treated with herbal remedies, followed by poultry (9%), dogs (5%) and rabbits (4%) (Viegi et al.,2003). Moreover, an increasing amount of evidence regarding the efficacy of herbal remedies, in terms of animal performances and nutrient, in livestock have been reported in recent years (Dalle Zotte et al., 2016). When compared to antibiotics or inorganic chemicals, phytochemicals and plants-derived compounds are overall less toxic and free of unwanted residues (Falcão-e-Cunha et al., 2007). They present antimicrobial properties and are active against pathogenic micro-organisms and spoilage microbes (Hayek et al., 2013). In addition, they can protect against oxidant activity of free radicals (Lee et al., 2017) and act as growth promoters (Falcao-E-Cunha et al., 2007; Cowan, 1999; Omonijo et al., 2018; Valenzuela-Grijalva et al., 2017) because they are active at intestinal level through antioxidant, antimicrobial, prebiotic and anti-inflammatory mechanisms (Valenzuela-Grijalva et al., 2017). The growth promoter effect of these chemical compounds has awakened the interest of several sectors (i.e. ruminants, swine and poultry industries) to use phytochemicals as natural growth promoters (Lillehoj et al., 2018) due to the ban on the use of antimicrobial promoters by the European Union (Diarra and Malouin, 2014). Moreover, EMA (European Medicine Agency) and EFSA (European Food Safety Authority) recently concluded that these compounds are effective in promoting growth in chickens, but that efficacy depends, at least to some degree, on the part of the plant used (EMA and EFSA Joint Scientific Opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union, and the resulting impacts on food safety, 2016).

Most of the available scientific studies published on plant-derived antimicrobials in the last decade involve the antimicrobial or antioxidant activity of herbs, spices, and their compounds (Tajkarimi et al., 2010; Gyawali et al., 2014; Negi, 2012; Redondo-Cuevas et al., 2017). Polyphenols isolated from fruits, vegetables, green and black teas, herbs, roots, spices, propolis, beer and red wine have shown their potential in terms of health promotion in livestock. Great attention has been deserved to thyme, oregano, rosemary, marjoram, yarrow, ginger, green tea, black cumin, coriander and cinnamon; especially in poultry industry for their potential application as antibiotics growth promoter (AGP) alternatives (Gadde et al., 2017). Furthermore, some alternatives to antimicrobials (e.g sugar cane extract, aniseed extract, chestnut wood extract, Forsythia suspensa, Portulaca oleracea extract), when tested as growth promoters, did not negatively affect animals’ performance parameters in contrast with other phytochemicals from grape pomace, cranberry fruit extract, Macleaya cordata extract, garlic powder, grape seed extract, and yucca extract (Gadde et al., 2017). Plant and plant products with high concentration of phytochemical compounds can be fed in solid, dried and ground form or as extracts (crude or concentrated). In livestock studies, phytochemicals are usually fed as essential oils (volatile lipophilic substances obtained by cold extraction or steam/alcohol distillation) and less as oleoresins (extracts derived by non-aqueous solvents) depending on the process used to derive the active ingredients (Gadde et al., 2017).

From a classification perspective, essential oils are usually classified into two major classes of compounds: terpenes (e.g, carvacrol and thymol) and phenylpropenes (e.g., cinnamaldehyde and eugenol). This oils are aromatic, volatile and extracted from plants components such as seeds, flowers, leaves, buds, twigs, herbs, bark, wood, fruits and roots (Brenes and Roura, 2010). In animal nutrition, carvacrol, thymol, citral, eugenol and cinnamaldeyde are the most used essential oils (Omonijio et al., 2018).

Therefore, the use of phytochemicals as natural antimicrobials should be encouraged and considered as a potential tool to reduce antimicrobial resistance in livestock, as well as because of its potential use in organic farming to treat animal diseases and promote growth. Several scientific reviews described the antimicrobial activity of phytochemical compounds reporting the main plants/herbs/spices they originate from (Cowan, 1999; Abdallah, 2011; Papatsiros, 2013; Atanasov et al., 2015; Chandra et al., 2017; Saeed et al., 2017). Thus, this section summarised the current scientific literature regarding the use of plants and plant products as natural antimicrobials in livestock.


5.2 Phytochemicals compounds classification

Phytochemical is a term used to indicate a wide and heterogenous group of compounds, which have different chemical properties and, therefore, different antimicrobial activities. A classification of the phytochemical compounds in several plants is displayed in Figure 5.1 (Bolling et al., 2011).

Figure 5.1. Classification of the phytochemical compounds (Bolling et al., 2011).

5.3 In vitro trials to test antimicrobial capacity of plants and plant-derived products and mechanism of action

The minimum inhibitory concentration (MIC) is a widely used laboratory parameter that

measures in vitro the minimum concentration of an antimicrobial product needed to inhibit the

growth of a specific microorganism. The MIC varies according to the bacteria species and strains

tested and the laboratory conditions. Omonjio et al. (2018) have recently published a review

reporting MIC values of commonly used essential oils on several swine bacterial pathogens.

Several studies have determined the MIC of many pathogenic bacteria which have importance

both from a zoonotic and zootechnical perspective, such as E. coli O157: H7 and Brachyspira

spp., respectively (Si et al., 2006; Navarrete et al., 2010; Vande Maele et al., 2016). Along with

the MIC determination, it is important to study the pharmacokinetics of the proposed products

before recommending their use as an alternative to conventional antimicrobials and to calculate

the withdrawal period. For instance, the pharmacokinetic of garlic, thymol and carvacrol has

been studied by Sharon et al. (2018), indicating a partial absorption from milk or skin into body

of thymol and carvacrol administered at intra-mammary level or by topical administration.

S. typhimurium growth was inhibited in vitro by tannins, even if no effect on the excretion of

the bacteria in an infection model in pigs was found (Van Parys et al., 2010). Another plant-

derived source, Quebracho (Schinopsis lorentzii) raw extract, has showed bacteriostatic effect

on Salmonella Enteritidis in vitro, and when used in an experimental infection model in broilers

led to a reduction of excretion of the bacteria (Redondo et al., 2014). In a study by Prosdocimo

et al., (2010), quebracho showed antimicrobial activity against Salmonella Enteriditis and S.

gallinarum in vitro (Prosdócimo et al., 2010).

Quorum sensing is referred to a regulatory system that depends on population density in

bacteria and that regulates gene expression in response to cell density through accumulation of


diffusible signalling molecules (de Kievit and Iglewski, 2000). Numerous in vitro studies and

studies on food microbiology (Kerekes et al., 2013; Alvarez et al., 2014) have shown that various

essential oils can disrupt the quorum sensing of pathogenic bacteria (Bjarnsholt et al., 2005;

Choo et al., 2006; Zhou et al., 2013), hence reducing their aggressiveness. Furthermore, in

aquaculture, it has also been reported that cinnamaldehyde and its derivatives are effective

against Vibrio harveyi in brine shrimp (Niu et al., 2006; Brackman et al., 2008). Another study

had evidenced that carvacrol and eugenol inhibit specific virulence determinants in

Pectobacteria through interrupting the quorum sensing machinery and directly inhibit AHL (N-

acyl-homoserine lactones) production, perhaps through direct interaction with Expl/ExpR

proteins (Joshi et al., 2016).

Regarding pathogens causing bovine mastitis, several in vitro studies have been conducted

showing inhibitory effect (Table 5.2). In addition, a project named “Non-Antibiotic Alternatives

for Bovine Mastitis Therapy” (LNC12-343) conducted by the Michigan State University (2012-

2016) demonstrated that essential oils from oregano, carvacrol, thymol, and trans-

cinnamaldehyde presented good antimicrobial activity against pathogens isolated form

mammary glands of cows with clinical mastitis; but, taking into account the calculated MIC, the

infused fluid should reach a concentration of essential oils between 50 and 100%, which is too

irritant for the epithelium of mammary gland. Moreover, they reported that Manuka honey

showed too high MIC for E. coli, Staphylococcus and Streptococcus to be considered effective.

Table 5.2. In vitro studies of plant derived compound against pathogens causing bovine mastitis.

Bacterial species Plant derived compounds Reference

Staphylococcus xylosus, S. intermedius, S. chromogenes, S. hyicus, S. aureus, Vibrio fluvialis, Serratia liquefaciens, Escherichia coli, Lactococcus lactis ssp. lactis, Enterobacter intermedius, Bacillus cereus, Yersinia ruckeri, Aeromonas hydrophila/caviae, Kytococcus sedentarius

Mentha pulegium, Nepeta cataria, Melissa officinalis; Agastache foeniculum; Lavandula angustifolia; Origanum vulgare; Althaea officinalis; Plantago lanceolata; Artemisia absinthium; Populus nigra; Evernia prunastri

(Pasca et al., 2017)

Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli

Trans-cinnamaldehyde, eugenol, carvacrol, and thymol

(Baskaran et al., 2009)

Staphylococcus aureus (ATCC and clinical isolate), Staphylococcus epidermidis, Streptococcus agalactiae, and Streptococcus dysgalactiae

Diterpenoids: manool, ent-kaurenoic acid, and ent-copalic acid

(Fonseca et al., 2013)

E. coli, Staphylococcus spp. Caryocar brasiliense Camb. (Caryocaraceae), Annona crassiflora Mart. (Annonaceae), S. brasiliensis Engl. (Anacardiaceae), Piptadenia viridiflora (Kunth) Benth. (Fabaceae), Serjania lethalis A.St.-Hil. (Sapindaceae), Casearia sylvestris (Flacourtiaceae), and Ximenia Americana L. (Olacaceae). Aqueous extract from Caryocar brasiliense from Schinopsis brasiliensis

(Izabella et al., 2018)

Staphylococcus spp. 3 alkaloids: benzylisoquinoline alkaloids (1) 5'-Hydroxythalidasine, (2) Thalrugosaminine and (3)

(Mushtaq et al., 2016)

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O-Methylthalicberine from Thalictrum minus roots

Polyherbal formula called Ya-SaMarn-Phlae composed by Curcuma longa, Areca catechu, Oryza sativa and Garcinia mangostana

(Chusri et al., 2017)

Streptoccocus uberis Limonene and Minthostachys verticillate essential oil

(Montironi, et al., 2016)

S. aureus and CNS (Coagulasi-negative Staphylococci)

L. orientalis leaf extracts (Ökmen et al., 2017)

The main target of carvacrol and thymol essential oils is the biosynthetic machinery of

bacterial cell wall, because these oils are able to synthetize bacterial cell wall and to disrupt it

(Faleiro M.L., 2011; Yap et al., 2014). Another important property of these essential oils is

represented by their lipophilic structure which lets them to get into the bacterial membranes

(Omonjio et al., 2018) causing membrane cells permeability disruption, followed by loss of

cellular components and eventually cell death (Windisch et al., 2008; Brenes and Roura, 2010;

Solórzano-Santos and Miranda-Novales, 2012; O’Bryan et al., 2015;). Gram-positive bacteria are

less tolerant than Gram-negative ones to cell permeability disruption (Brenes and Roura, 2010).

Furthermore, although thymol and carvacrol have both antimicrobial activity, this can be

different because of the different position of some functional groups, as the antimicrobial

activity is related to the hydroxyl group of the phenolic terpenoids and the presence of

delocalized electrons (Lambert et al., 2001; Ultee et al., 2002).

According to Thapa et al. (2015), it is crucial to remark that efficacy of essential oils against

bacterial species in the mixed bacterial population may differ from that of the pure culture

(Thapa et al., 2015). Most of essential oils interactions in the animal gastrointestinal tract are

not well known. In fact, some of these bioactive compounds are generally sensitive to acidity

and digestive enzymes during passage through the stomach, being metabolized before reaching

their optimum site of action (Schindler et al., 2001; Michiels, 2009; Thapa et al., 2015; Zhang et

al., 2017). That suggested the need to protect essential oil forms from gastric absorption.

5.4 In vivo studies

5.4.1 Swine: plants and plant products antimicrobial capacity

The ban of AGP use in swine production in Europe in 2006 has caused the need to exploit

alternative to in-feed antibiotics. This aspect of the swine industry is tremendously crucial for

the long-term sustainability and profitability of swine production (Omonijo et al., 2018). Any

potential alternative to AGP should possess specific features: safe to the public, cost-effective

in swine production, environmentally friendly. Consequently, a single alternative could not exist,

because of the multiple requirements of an ideal alternative. Additional studies about the

efficacy, cost-effectiveness and safety of essential oils in swine production are required. This is

because the potential side effects, regulatory concerns and potential interaction with other feed

ingredients, such as fats, are still not well-known (Lambert et al., 2001; Friedman 2002).

Suckling piglets are often affected by enteritis and an important role in the pathogenesis of

this disease is played by primary agents such as enterotoxic colibacillae or other bacteria such

as Clostridium perfringens type A toxin, Clostridium difficile and Enterococcus spp., all of which

need different antimicrobial treatments. Typical of this phase are also locomotory infections,

such as arthritis due to Streptococcus spp. or Staphylococcus spp. that penetrates the body after


injection, mutilation or wounds following biting or housing defects (EMA and EFSA, 2017). On

the other hand, Brachyspira spp. frequently occurs in fattening pigs, along with Lawsonia

intracellularis (EMA and EFSA, 2017). Another important health problem in pigs is gut

inflammation which could be related to pathogens (pathogen infection-associated), to nutrition

(allergen-associated) or to animal management (weaning-associated) (Yang et al., 2015). Gut

inflammation is usually a subclinical disease and as such does not cause full-blown clinical

symptoms. The gut immune system is different from the systemic immune system because it

also has to face the antigens derived from commensal bacteria and food compounds (Pitman

and Blumberg, 2000). Therefore, gut inflammations, even if in a sub-clinical form, can lead to a

considerable hidden economic losses in pig production because they lead to a significant

reduction in growth performance (Omonijo et al., 2018). In adult phases, the gut microbiota is

more stable, more resistant to dietary perturbations and less susceptible to pathogen infections

in comparison to piglets, therefore the modulation of the gut microbiota of young animals helps

in obtaining a healthy microbiota ensuring better animal performances (Omonijo et al., 2018).

Moreover, pigs are often exposed to several stressors such as weaning, malnutrition disease

challenge, heat-stress, in-feed mycotoxin contaminations, transportation and overcrowding

(Martínez-Miró et al., 2016). Generally, stress occurs when the antioxidant system is

overwhelmed by reactive oxygen species (ROS) production leading often to a drop-in

performances, immune activity, and muscle integrity, and seems to increase the risk of stroke

in fast growing pigs. Moreover, stress conditions have been associated to mulberry heart

disease, reduced appetite, diarrhoea, damage of liver tissue, and increased risk of abortion of

pregnant sows (Omonijo et al., 2018); and negative effects impact sow’s milk production,

reproduction and longevity with consequences on piglets’ long-term health and growth as well

(Berchieri-Ronchi et al., 2011; Zhao et al., 2013).

Several experimental studies have been conducted in order to evaluate the use of essential

oils in swine. Oregano supplementation has been already evaluated in several studies showing

contradictory results regarding animal performance. Sows fed with diets containing 1,000 mg/kg

oregano (dried leaf and flower of Origanum vulgare, consisted of 50% cold pressed essential oil

of O. vulgare) had lower annual sow mortality rate, lower sow culling rate, increased farrowing

rate, increased number of liveborn piglets per litter, and decreased stillbirth rate (Allan and

Bilkei, 2005), and in weaned pigs increased weight gain and reduced disease incidence (Sads and

Bilkei, 2003). On the other hand, some authors did not report beneficial effect in sows fed with

oregano essential oil (Ariza-Nieto et al., 2011) and in weaned pigs performance (Neill, Nelssen,

& Tokach, 2006; Nofrarías et al., 2006). Those contradictory results could be explained by the

difference in terms of terpene composition among studies (Cross et al., 2007). In the grower-

finisher period, Cullen et al. (2005) and Janz et al. (2007) obtained higher average daily gain

(ADG), average feed intake (AFI), and feed conversion ratio when including garlic in the diet

compared to the control group (Cullen et al., 2005; Janz et al., 2007). A significant improvement

in ADG and feed conversion ratio has also been reported in pigs from 25 to 105 kg after receiving

a herb mixture (great nettle, garlic, wheat grass) in the diet during the complete period (Grela

et al., 1998).

Feeding pigs with plant extracts seem to improve gut health by modulating gut microbiota

or immunity. Indeed, Manzanilla et al. (2004) and Nofrarias et al. (2006) reported that a mixture

of plant extracts (oregano, cinnamon and Mexican pepper) increased stomach contents and

percentage of dry matter (DM), suggesting an increased gastric retention time, as well as a

reduction in ileal total microbial mass and an increase in Lactobacilli to Enterobacteria ratio

(Manzanilla et al., 2004; Segales et al., 2006). Several studies have suggested that

supplementation with essential oils can increase the Lactobacillus group and decrease E. coli or


total coliforms in piglets (Namking et al., 2004; Li et al., 2012;; Zeng et al., 2015). After

supplementing with 500 mg/kg carvacrol and thymol, Michiels et al. (2010) reported a reduction

of intra-epithelial lymphocytes and an increase of villus height/crypt depth in the distal small

intestine of weaned piglets (Michiels et al., 2010). In another study, a low dose (10 mg/kg) of

capsicum oleoresin, turmeric oleoresin, or garlicon, in weanling pigs challenged with a

pathogenic E. coli, improved intestinal villi height, gut barrier function and integrity and reduced

systemic and gut inflammation (Liu et al., 2014). The same authors also reported reduced viral

load and serum concentrations of inflammatory mediators, and shorter time of fever in

respiratory syndrome virus (PRRSV) infected weanling pigs (Liu et al., 2013). More in detail,

essential oils have been shown to manipulate both Nrf2 and NF-kB transcription factors, leading

to a suppression of inflammation (Kroismayr et al., 2008; Wondrak et al., 2010; Zou et al., 2016).

In particular, cinnamaldehyde (Wondrak et al., 2010) and oregano oil (Zou et al., 2016) increased

the expression and translocation of Nrf2 and prevented the activation of NF-kB. It has also been

demonstrated that essential oils can significantly reduce the expression of the NF-kB, tumour

necrosis factor-a (TNF-a) and the size of Peyer's patches in the intestine of weaned piglets, as

well as the cyclin D1 in the colon, mesenteric lymph nodes and spleen (Kroismayr et al., 2008).

Also, some experimental evidences show that oral administration of essential oils reduces and

limits the development and severity of experimental autoimmune encephalomyelitis (Alberti et

al., 2014). Diets containing essential oils in pigs increased albumin, immunoglobulin A (IgA), IgG,

total antioxidant capacity in plasma and lower faecal score (Zeng et al., 2015). Pigs

supplemented with 100 mg/kg mixture of carvacrol and thymol (1:1) decreased TNF-a mRNA a

marker for weaning associated intestinal oxidative stress (Wei et al., 2017). On the other hand,

oregano essential oil supplementation has caused an increase in systemic oxidative stress in late

gestation and early lactation of sows but contributed to a better performances in offspring (Tan

et al., 2015).

Therefore, all these studies confirmed that plant extracts are of great interest as alternative

to antimicrobials in feed to improve growth performance and health of pigs in different phases

of their productive career.

5.4.2 Poultry: plants and plant products antimicrobial capacity

As in swine industry, herbs, spices and other several plants extracts or essential oils are

currently used as feed additives in poultry industry, and they are perceived as growth promoters

(Zhai et al., 2018). According to EMA and EFSA (2016) the use of antimicrobials in poultry often

occurs immediately before or after transport of day-old chicks in order to avoid potential losses

of productivity. The main pathogens/disorders for which antimicrobials are mostly used are

listed in Table 5.3 (EMA and EFSA, 2016). During laying period any antimicrobial use is banned

by the European Union.

Table 5.3. Main pathogens/disorders for which antimicrobials are mostly used in poultry (EMA and EFSA,

2016).

Specie Disorder Examples

broiler gastrointestinal disorders such as coccidiosis, necrotic enteritis, dysbacteriosis

respiratory diseases infectious bronchitis, Newcastle disease, infectious laryingotracheitis

locomotion-related diseases bacterial arthritis due to e.g. E. coli, Staphylococcus aureus or Enterococcus spp., and secondary bacterial infections connected with tenosynovitis, necrosis of the femur head


septicaemia, omphalitis

laying hens gastrointestinal disorders enteritis caused by E. coli, avian intestinal spirochaetosis

respiratory and locomotion-related diseases

caused by E. coli and Mycoplasma

secondary bacterial infections

connected with red mite infestation; taeniosis (in free range production systems)

turkeys respiratory diseases Ornithobacterium infection

gastrointestinal disorders coccidiosis

In poultry, many reviews on the use of plant-derived compounds have been recently

published, especially regarding essential oils such as thymol (thyme), eugenol (clove) and

carvacrol (oregano) (Diaz-Sanchez et al., 2015; Zeng et al., 2015; Ezzat Abd El-Hack et al., 2016;

Yadav et al., 2016; Valenzuela-Grijalva et al., 2017; Suresh et al., 2018; Zhai et al., 2018) due to

their proved antimicrobial activity in vitro essays against E. coli and S. typhimurium (Bassolé and

Juliani, 2012; Hippenstiel et al., 2011) However, antimicrobial activity of a specific essential oil

could vary greatly according to different factors such as dietary aspects, dosage, environment

growth performance level, age of animals, and essential oil composition (Zhai et al., 2018). As a

result, this could make it difficult to compare the efficiency of different plant extracts (Gyawari

and Ibrahim, 2014). For example, carvacrol has been reported to range from trace amounts to

80% in the extracts of Origanum vulgare and from 2% to 11% in Thymus vulgaris. Therefore, it

is necessary to take in consideration the variations in active compounds in plants and plant-

derived products when plant extracts are used as potential alternatives to antibiotic growth

promoters.

Several studies in vivo have confirmed some beneficial effects of feeding plant-derived

compounds on important broilers performance. The following effects have been reported:

increase in body weight (Peng et al., 2016), decrease in pathogen counts (Chang et al., 2016),

improvement of fatty acid profile in egg yolk (Raza et al., 2016), increase serum proteins and

antioxidant activity (Azawqari et al., 2016). However, some reviews have reported no variations

in feed intake or small reductions (Bozkurt et al., 2014; Brenes & Roura, 2010; Franz, 2010;

Hippenstiel et al., 2011). Indeed, increased of dietary level of a blend of thyme and star anise

(Halle et al., 2004; Amad et al., 2011 ) or a cocktail of essential oils (oregano oil, laurel leaf oil,

sage leaf oil, myrtle leaf oil, fennel seed oil, and citrus peel oil (Çabuk et al., 2014) led to a

decrease of daily feed intake in broilers. It has been suggested that this reduction could be

related to the irritating smell of essential oils which could reduce palatability (Zhai et al., 2018).

Although feed preference has not been investigated in poultry as it has been done in swine

nutrition, it seems that poultry might not be sensitive to flavour (Moran, 1982) and more

tolerant when exposed to not too high levels of essential oils than pigs (Zhai et al., 2018).

On the other hand, supplementation with essential oils stimulates digestive

enzyme secretion and stabilizes ecosystem of gastrointestinal microbiota, therefore improving

feed utilization and reducing exposure to growth-depressing disorders related to digestion and

metabolism (Williams and Losa, 2001; Lee et al., 2003; Franz, 2010; Bento et al., 2013; Kurekci

et al., 2013; O’ Bryan et al., 2015). In addition, feeding poultry with plant-derived compounds

modifies microbiota composition ameliorating nutrient digestion, which positively impacts body

weight and weight gain (Hernández et al., 2004; Jamroz et al., 2003). A positive effect on

pancreas and intestinal mucosal digestive enzyme secretion has been reported when using

essential oils in broilers (Jamroz et al., 2006; Jang et al., 2007); along with increasing nutrients

digestibility, even without improving growth performances (Amad et al., 2011; Botsoglou et al.,

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2004; García et al., 2007; Hernández et al., 2004; Lee et al., 2003). It has been suggested that

the lack of positive effects on growth performances in these studies could be eventually due to

poor hygienic conditions under which the trials were conducted (Zhai et al., 2018).

Essential oils, used individually or in combinations, have inhibited the growth of Clostridium

perfringens and E. coli in the hindgut and ameliorated intestinal lesions and weight loss in

comparison to the challenged control birds (Mitsch et al., 2004; Jamroz et al., 2006; Jerzsele et

al., 2012). The use of trans-cinnamaldehyde and eugenol inhibited Salmonella motility and

invasion of avian intestinal epithelial cells due to downregulating motility and invasion genes

(motA, flhC, hilA,hilD, and invF) (Kollanoor-Johny et al., 2012). Essential oils have also shown

antioxidant activity in poultry (Habibi, et al., 2014; Karadas et al., 2014) which is important in

poultry meat due to its high content of polyunsaturated fatty acids. Broilers fed with a blend of

carvacrol, cinnamaldehyde, and capsicum oleoresin also showed an increase of carotenoids and

coenzyme Q10 at hepatic level (Karadas et al., 2014). Malondialdehyde concentration in liver,

duodenal mucosa, and kidney have increased when supplementing broiler diet with

ginger power and thyme oil (Habibi et al., 2014). Studies have also reported an antioxidant

activity exerted by extracts of rosemary, oregano, and sage of the Labiate family in broiler meat

(Windisch et al., 2008; Brenes and Roura, 2010; Franz et al., 2010).

5.4.3 Cattle: plants and plant products antimicrobial capacity

The majority of herbal treatments on ruminants, including dairy cattle, has been conducted

with the aim to test their impact on rumen fermentation processes with emphasis on the

reduction of methane emissions from bacterial metabolic activity (McIntosh et al., 2003;

Newbold et al., 2004; Benchaar et al., 2007; Calsamiglia et al., 2007; Giannenas et al., 2011; Patra

and Yu, 2015; Cobellis et al., 2016). In contrast, very few studies have investigated their

potential use as alternative to antimicrobials in treating and preventing diseases in cattle. These

studies have been focused on the use of plant extracts against some specific bacterial species,

causes of mastitis in dairy cattle, such as Staphylococcus aureus, which is the most occurring

disease in dairy herds worldwide (Mushtaq et al., 2018).

Recently, the use of plant extracts against mastitis has been published by Mushtaq et al.

(2018). The information regarding in vitro studies has been reported in Section 5.3. In vivo

studies testing alternatives to antimicrobials for mastitis are still very limited. In cattle, it has

been demonstrated that Tridax procumbens Linn (aqueous and methanol extracts) has

antimicrobial activity against Staphylococcus aureus strains (Dhanabalan et al., 2008). The

application of a tropical herb spray showed a 70% of cure rate in subclinical mastitic cows and a

greater efficiency of reducing SCC in milk compared to the 60% cure when animals were treated

with synthetic gel Mastilep (Hase et al., 2015). Feeding of Morinda citrifolia juice (100 mL/day

per animal) significantly reduced in the mastitis milk the pH, the total bacterial count and total

protein concentration (Sunder et al., 2013). The oral administration of Ocimum sanctum leaf

powder at 600 mg/kg body weight daily (divided into two doses) for 7 days in dairy cows with at

least one quarter with subclinical mastitis eliminated 69.23% of intramammary infections,

reduced somatic cell count ceruloplasmin concentration, increased phagocytic activity of milk

neutrophils and enhanced lactoperoxidase and myeloperoxidase activities compared to the

control group (Shafi et al, 2016). The supplementation of 200–250 mg/kg body weight of a

polyherbal preparation supplementation (Withania somnifera, Asparagus racemosus, Emblica

officinalis, Ocimum sanctum, Tinospora cordifolia, Tribulus terrestris, and Nigella sativa) reduced

periparturient stress and improved immunity and udder health (Sharma et al., 2014). Moreover,

the study conducted between 2010-2013 by the Department of Animal Sciences of the North

Carolina State University named “Evaluation of Herbal Remedies as Alternatives to Antibiotic

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Therapy in Dairy Cattle” (GS10-094) tested two herbal treatments against mastitis: Phyto-Mast®,

which is a botanical multi-purpose intra-mammary preparation for improvement of milk quality;

and Cinnatube™, a blend of essential oils with the aim to prevent infections during the dry

period. They reported that herbal treatments appeared to be as effective as conventional

antibiotics for treating or preventing mastitis in dairy cattle under specific conditions without

compromising cattle health.

5.4.4 Rabbits: plants and plant products antimicrobial capacity

The research on the use of phytochemicals in rabbit is not as extensive as in swine and

poultry. A recent review on this topic has been carried out by Dalla Zotte et al. (2016). The

supplementation with Digestarom® (300 mg of product; onion, garlic, caraway, fennel, gentian,

melissa, peppermint, anise, oak bark and clove mixture) increased weight gain (+18%) and feed

intake (+14%) in bunnies, and stabilized gut microbiota compared to control group (Krieg et al.,

2009). The inclusion of black cumin seed (15 or 20%; Nigella sativa)) in rabbits feed significantly

increased serum concentrations of antibodies in response to intramuscularly injected serum

bovine albumin, increased survival time after intraperitoneal administration of Pasteurella

multocida, and blood cells of rabbits belonging to the treated group inhibited Staphylococcus

aureus growth on sheep-blood agar plates (El Bagir et al., 2010). The supplementation with

Spirulina (5%) and Thyme (3%) led to a decrease of Clostridium coccoides and Clostridium leptum

in caecal content of rabbits (Vántus et al., 2012). Thyme oil has also been investigated by Placha

et al. (2013) reporting an improvement of the intestinal integrity and the values of total

antioxidant status (TAS) in blood plasma and glutathione peroxidase (GPx) activity in the liver,

and a decrease of malondialdehyde (MDA) concentration in the duodenal tissue when

supplementing the diet with 0.5 g thyme oil/kg DM. Moreover, a tendency to stimulate the

abundance of some microbes with positive effects in the rabbit gut has been observed (Placha

et al., 2013). However, there is still a lack of evidence on this topic in rabbits, therefore the

potential of some herbs and spices as feed additives should be encouraged at different levels.

5.4.5 Aquaculture: plants and plant products antimicrobial capacity

The use of phytochemicals has also been recently reviewed in aquaculture systems in several

papers (Chakraborty and Hancz, 2011; Defoirdt et al., 2011; Murthy and Kiran 2013; Chakraborty

et al., 2014; El-Hack et al., 2016; Jana et al., 2018; Sutili et al., 2018), although data about isolated

compounds as dietary supplements for aquatic species is still limited (Sutili et al., 2018).

Phytochemicals compounds have shown to act in aquaculture as growth promoters, appetite

stimulators, immunomodulatory and antioxidant as well as antiparasitic, antibacterial,

anaesthetic and antistress (da Cunha et al., 2010; Harikrishnan et al., 2011; Saccol et al., 2013;

Silva et al., 2013; Chakraborty et al., 2014; Reverter et al., 2014; de Oliveira Hashimoto et al.,

2016; Sutili et al., 2018).

The health status of fishes is greatly influenced by the intestinal bacterial community and

their metabolites, which are able to modulate immune system and other metabolic functions

(Bento et al., 2013; Lazado and Caipang, 2014). Generally, the gastrointestinal tract of healthy

fish contains saprophytic and pathogenic bacteria species. These microorganisms are able to

increase in number and infect fishes when conditions become favourable. Usually, fish has to

maintain a dynamic microbial equilibrium by defending themselves against these potential

invaders using a range of innate and specific defence mechanisms (Ellis, 2001; Gómez and

Balcázar, 2008). There are very few studies that have shown significant effects of different

essential oils supplemented diets and their constituents on intestinal microbial organisms in fish

compared to other livestock. However, supplementation with essential oils in aquaculture have


shown their potential as health promoters of the gastrointestinal tract in fish in some studies

(Sutili et al., 2018).

A considerable change in the intestinal microbiota has been reported in hybrid tilapia

(Oreochromis spp.) fed during 6 weeks with a commercial mixture product of thymol and

carvacrol (Ran et al., 2016). The addition of Ocimum americanum essential oil and of thymol did

not affect the fish gut microbiota in red drum (Sciaenops ocellatus) and in rainbow trout

(Oncorhynchus mykiss), respectively (Navarrete et al., 2010; Sutili et al., 2016). However, in the

Navarrete et al. (2010) study, the in vitro essay showed that Thymus vulgaris essential oil has

antibacterial activity against fish pathogens and common bacteria isolated from the gut of

healthy salmonid, suggesting the encapsulation of the essential oil to ensure the release of

active essential oil into the fish gut. This was confirmed by Giannenas et al. (2012) who, using

encapsulate thymol in the diet of rainbow trout, reduced gut total anaerobic bacteria and

specifically of Lactobacillus loads compared to the group with encapsulated carvacrol or control

group (Giannenas et al., 2012).

5.5 Antimicrobial capacity of food industry by-products

By-products of fruits and vegetables are potentially good sources of minerals, organic acid,

and phenolics that have a wide range of antimicrobial properties (Chanda and Kaneria, 2011).

According to Gyawali and Ibrahim (2014), several studies confirmed the antimicrobial activity of

some bioactive compounds contained in plant by-products. For instance, phenolics and

flavonoids of pomegranate fruit peels have shown antimicrobial activity against Listeria

monocytogenes, S. aureus, E. coli, and Yersinia enterocolitica, Pseudomonas fluorescens ( Al-

Zoreky, 2009; Agourram et al., 2013). Regarding grape, ethanolic extract of grape pomace at

10% has been shown to inhibit the growth of Enterobacteriaceae, S. aureus, Salmonella, yeasts

and molds in beef patties during 48 h of storage at 4 °C (Sagdic et al., 2011). Instead, grape

pomace extract, according to Ozkan et al. (2004), inhibited the growth spoilage and pathogenic

bacteria including Aeromonas hydrophila, B. cereus, Enterobacter aerogenes, Enterococcus

faecalis, E. coli, E. coli O157:H7, Mycobacterium smegmatis, Proteus vulgaris, Pseudomonas

aeruginosa, Pseudomonas fluorescens, S. Enteritidis, S. Typhimurium, S. aureus, and Yersinia

enterocolitica (Özkan et al., 2004) The antimicrobial properties of olive pomace and olive juice

powder have been also investigated by Friedman et al. (2013) and also confirmed by Cicerale et

al. (Cicerale et al., 2011; Friedman et al,, 2013). In poultry, Supplementation with grape seed

extracts (7.2 g/kg), which are a wine industry by-product, favoured the growth of beneficial

bacteria, in particular it increased bacteria diversity and Lactobacillus populations (Viveros et

al., 2011). In a similar study in poultry, Yerba Mate extracts supported and increased the growth

of Lactobacillus and Pediococcus and had antimicrobial effects against Salmonella and

Campylobacter (Gonzalez-Gil et al. 2014). In other cases, studies are still limited, such as on the

antibacterial activity of coffee and tea by-products.

5.6 Conclusions and perspective

Most of the studies on alternatives to antimicrobials in livestock have focused on bioactive

compounds from plants, such as essential oils. Several positive effects on animal health and

productivity of these secondary metabolites have been showed in different species of food-

producing animals. Nowadays, the spread of multiresistant microorganisms in humans,

environment and animals, requests a major focus on these potential nutritional tools which


could give a contribution to reducing the use of antimicrobials at farm level. Nevertheless, it is

important to overcome limitations in experimental studies, because of influences exerted by

several factors, such as diet and essential oil composition on experiments outcomes.

Furthermore, there is still a lack of knowledge on essential oils metabolism and the scientific

evidence showing the potential link between feeding essential oils and animal health is not solid

yet. There are only a few in vivo studies, in comparison to in vitro ones. Furthermore, the

mechanisms by which essential oils affect gut microflora and gut-associated immune system are

not fully understood yet. Given the important role of nutrition in livestock animals, research on

this topic should have a greater role to play in advancing knowledge in livestock production,

with a more effective strategy in the fight against antimicrobial-resistance.

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SITOGRAPHY

https://www.efsa.europa.eu/en/efsajournal/pub/4666

https://www.southernsare.org/News-and-Media/Press-Releases/Herbal-Treatments-Effective-

in-Treating-or-Preventing-Mastitis-in-Dairy-Cattle

https://projects.sare.org/project-reports/lnc12-343/. Final Report for LNC12-343

https://www.southernsare.org/News-and-Media/Press-Releases/Herbal-Treatments-Effective-in-Treating-or-Preventing-Mastitis-in-Dairy-Cattle

https://www.southernsare.org/News-and-Media/Press-Releases/Herbal-Treatments-Effective-in-Treating-or-Preventing-Mastitis-in-Dairy-Cattle

https://projects.sare.org/project-reports/lnc12-343/


6. Discussion and conclusion of potential natural sources for vitamins, antiparasitics and antimicrobics for organic livestock farming

The possibility to use synthetic vitamins for organic livestock production allowed by the

European regulation is based on the current knowledge and availability of natural vitamins administered through additives or feeds that can guarantee animal health and welfare. This report provides evidence of the lack of information regarding the use of algae and food industry by-products to fill this gap, which will help in the full achievement of the organic farming principles. Very little information on the vitamin content characterization of those products and on their impact on animal performance and derived products is available, which highlights the need for future research on this topic. The main by-product and naturals source of vitamins partially tested in animal feeding are from the Mediterranean countries; this probably depends on the huge amount of these by-products historically available that in the lasts 40 years drove the research towards their potential utilization in animal feeding. Very limited information about the use of other by-products is available in central and north European countries. Based on the literature research developed for this report, the algae and olive oil, citrus, wine and carob food industries by-products are a feasible alternative to the use of synthetic vitamins to assure the adequate vitamin intake in livestock.

The number of publications dedicated to the study of the potential use of plants and plant-derived products as antiparasitics has increased during the last years. This witnesses the growing interest in developing new, alternative approaches to the control of parasites in veterinary medicine. Despite the large amount of published studies, there still is no consensus around the procedures to test the efficacy of plant-derived antiparasitic substances. For this reason, in most cases it is not possible to compare different studies or to rely on the use of plants or plant products as antiparasitics in practice. Even if the economic role of antiparasitcs has been widely demonstrated, there is still a need to test plants and plant-derived products in a way that will allow these products to be applied and introduced in the routine of livestock farming. A good protocol to the evaluation of the efficacy of plants and plant products against helminth should include a detailed description of: preparation of plant material, fractionation, assay selection, and in vivo tests to confirm the in-vitro results in the target species. The USDA phytochemical database indicates that the top five plants with the highest number of anthelmintic compounds are Achillea millefolium, Dryopteris filix-mas, Peumus boldus, Rosmarinus officinalis, and Salvia officinalis. However, many wild legumes and indigenous plants recently evaluated for anthelmintic qualities are not included in this database. Regarding ectopararisites, farmers currently rely mainly on chemical acaricides and repellents that help in controlling ticks; however, in places where small scale farms are the largest proportion of livestock enterprises plants or plant-based products for the control of arthropod ectoparasites on livestock are in fact widespread.

Most of the studies on alternatives to antimicrobials in livestock focused on bioactive

compounds from plants, such as essential oils. Several positive effects on animal health and

productivity of these secondary metabolites have been shown in different species of food-

producing animals. Nowadays, the spread of multiresistant microorganisms in humans,

environment and animals, requests a major focus on these potential nutritional tools which

could give a contribution to reducing the use of antimicrobials at farm level. Nevertheless, it is

important to overcome the limitations in experimental studies, because of influences exerted

by several factors, such as diet and essential oil composition on experiments outcomes.

Furthermore, there is still a lack of knowledge on essential oils metabolism and the scientific

evidence showing the potential link between feeding essential oil and animal health is not solid

yet. In fact, there are still few in vivo studies, in comparison to in vitro ones. Furthermore, the

mechanisms by which essential oils affect gut microflora and gut-associated immune system are

not fully understood yet. Given the important role of nutrition in livestock animals, research on


this topic should have a greater role to play in advancing knowledge in livestock production,

with a more effective strategy in the fight against antimicrobial-resistance.

Based on the literature review presented in this document, the plant or plant products selected to be tested in-vitro are: For natural vitamin source: - grape pomace - citrus pulp - olive pomace - carob pulp - Algae: Ascophyllum nodosum Other possible vitamin sources - apple pomace - hops - Lavandula L. (lavender)

For natural antiparasitic source: - bark extracts (tannins) - Chenopodium ambroisoides

- Carum carvi (caraway) - Thymus spp (thyme), - Mentha spp (mint) - Artemisia sp. - Tanacetum vulgare

For natural antimicrobics source: - Cloves

- Oregano - Thymus spp (thyme) - Cinnamon - Mentha spp (mint) - Rosemary - Allium - Lavandula - Camelia sinensis