DISSERTATION. Shirron Nicole LeShure. Graduate Program in Animal Sciences. The Ohio State University. Dissertation Committee:

Transcription

1 Use of Naturally Occurring Anthelmintics to Control Parasites in Small Ruminants DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shirron Nicole LeShure Graduate Program in Animal Sciences The Ohio State University 2014 Dissertation Committee: Maurice L. Eastridge, Advisor Jeffrey L. Firkins Steven C. Loerch Normand St-Pierre Sandra Solaiman

2 Copyrighted by Shirron Nicole LeShure 2014

3 Abstract There is a critical need to identify natural anthelmintics for food animal production because of the increased resistance of intestinal parasites to commercial anthelmintics and the inability to use commercial anthelmintics for certified organic food production. Condensed tannins (CT) and flavonoids have been investigated and shown varied efficacy as natural anthelmintics. This research was done to investigate the effects of utilizing by-products of the juice and wine making industries, pomegranate husk (PH) and grape pomace (GP), which both contain these bioactive compounds of interest. An extraction was done on both by-products to determine the concentration of CT available. Pomegranate husk varieties of interest, Parifanka and Desertnyi, contained approximately 1.49 and 2.02% CT on a dry matter (DM) basis, respectively. Grape pomace varieties, Shiraz and Cabernet Sauvignon, contained 4.83 and 3.68% CT, respectively. In vitro batch culture was conducted in a slope ratio design to determine the effects of the by-products on dry matter degradation (DMD) when compared to and mixed with a control, alfalfa hay. Both varieties of GP had lower (P <0.05) DMD at 96 h with greater than 70% dry matter remaining (DMR), however both varieties of PH had similar digestibilities as alfalfa with approximately 40% DMR. There was an inverse response in DMD when GP was mixed with the ground alfalfa hay; as the proportion of ii

4 GP to alfalfa increased, the DMD decreased (P < 0.05). Parifanka PH had a DMD similar to alfalfa and did not have a significant effect (P > 0.10) on DMD in mixed ratios. Desertnyi PH was observed to have slightly better digestibility than alfalfa, and the DMD decreased with increasing alfalfa. In vitro parasitology studies were done on stage three larvae of O. ostetagia using extracts of PH and GP. There were several varieties of PH available, so preliminary studies were done to determine two varieties showing highest efficacy on larvae to use in subsequent studies. The Sogidana and Wonderful varieties were used for the PH, and Shiraz and Cabernet Sauvignon varieties were used for GP. Overall, both PH and GP extracts had approximately twice the number of inactive larvae present (P < 0.05) in the well at 24 h when compared to the control, 32 to 41% inactivity versus 17% inactivity, respectively. Grape pomace extracts had a marginally greater (P < 0.05) efficacy on reducing the viability of the parasites than the pomegranate husk extracts at 24 h when observed at 12.5 mg/ml of crude extract. The PH had a higher extractability than GP was able to reach 50 mg/ml of crude extract. The Wonderful variety of PH had the highest (P < 0.05) efficacy against the parasites when compared to Sogidana at the same concentration and against the control. Two trials were conducted for evaluating the effect of GP on mixed gastrointestinal helminth parasite infestation in growing lambs. The first trial examined the effect of a GP diet (28 g CT/kg DM) against a positive (Cydectin, no CT, normal alfalfa pellet diet) control and a negative control (no CT, normal alfalfa pellet diet). The second trial examined the effects of GP diets containing 25 (GPD1) or 45 (GPD2) g iii

5 CT/kg DM against a negative control. Lambs were assessed for body weight (BW), FAMACHA (anemia eye score), fecal egg count (FEC), average daily gains (ADG), packed cell volume (PCV) and feed intake (FI). In trial 1, there was an increase (P < 0.05) in BW observed each week which would be expected as the lambs grew, irrespective of treatment. As the weeks progressed, the treatments varied in ADG with the Cydectin treatment group having the highest (P < 0.05) ADG by week 3. FI and FAMACHA were not different (P > 0.10) between treatments but FI increased (P < 0.05) weekly. There was no difference (P > 0.10) observed between treatments for PCV until the second week in which the Cydectin treatment had the highest (P < 0.05) PCV at 33.2±1.8% and continued to have the highest (P < 0.05) PCV for the duration of the trial. Cydectin treated lambs also had the lowest (P < 0.05) FEC with there being no differences (P > 0.10) between the negative control and GP treatment groups. I expected to observe a decrease in FEC with the positive control and GP diet compared to the negative control, but this result was not observed. Body weight also increased (P < 0.05) with time in trial 2. ADG was higher (P < 0.05) for GPD1 during the first week when compared to the control and other GP diet. After the first week, the negative control had the highest (P < 0.05) ADG but by week 3 there was no difference (P > 0.10) observed between treatments. PCV also increased (P < 0.05) over time with there being no differences (P > 0.10) between treatments. There were no differences (P > 0.10) observed between treatments for FEC. FAMACHA scores and FI increased (P < 0.05) over time but there were no differences (P > 0.10) between treatments. iv

6 An egg hatch and larval development study was done on feces from parasitized lambs from different farming practices (organic versus conventional) in the presence or absence of GP extract (38 g CT/kg DM) to evaluate the effects of GP on egg hatchability and larval development. The GP treatment showed a 100% inhibition (P < 0.05) of egg hatch into developing larvae when compared to the control distilled water treatment. The data from the research conducted has shown that GP from the wine industry and PH have efficacy against larval helminth stages of GIP and GP also has efficacy against egg hatchability and larval development. The PH and GP could potentially have practical application in becoming a natural anthelmintic for small ruminants, but more in depth studies are needed to verify and finalize application methods. v

7 Acknowledgments I would first like to acknowledge and thank all of my committee members individually for their invaluable help and support. I would like to show my appreciation to my advisor, Dr. Maurice Eastridge, for seeing and hearing the passion I had for research and taking a risk on a dream that became this research project. I give my sincerest thanks to Dr. Steve Loerch for his awesome support, guidance, and wonderful vision for animal nutrition research. He was my rock in a storm of chaos when I did not know how to bring my thoughts or visions to fruition. I would also like to thank Dr. Jeff Firkins for helping me better understand the complex world of rumen microbiology and assisting in the design of my dry matter degradation study. I cannot thank Dr. Normand St-Pierre enough for his assistance and patience in helping with statistically analyzing the data. Without his help, I do not know how I would have survived this project and analyzing the data. My struggle with statistics is real and Dr. St-Pierre was my light in the darkness. I must also show my gratitude to Dr. Sandra Solaiman for being such a warm, sweet spirit. Her support and advice uplifted me in times where I had almost given up hope. Secondly, I would like to extend many thanks to the support staff and research team at the OARDC Beef and Sheep center. Without the help of everyone, including the fellow graduate students who came out to assist on collection days, I would not have been vi

8 able to do the animal trials so efficiently. They all truly made experiences out at the farm fun and amazing. I do not think I would have enjoyed my time and gained as much useful on-farm knowledge as I did without them. I would not have been able to accomplish my in vitro parasite experiments without the support, guidance, and teachings I received from everyone at USDA-ARS Beltsville, MD. I would like to send many thanks to Dr. Donald McLellan for funding and Ms. Tracey Troutman for assisting in my placement in the Animal Parasitic Diseases Laboratory at the Beltsville Agricultural Research Center. Sincere thanks to Dr. Dante Zarlenga, who was so influential in helping develop and supervise my in vitro trial and parasite work. I greatly appreciate him challenging me mentally to think outside the box on many issues that arose during the project. I would not have been able to get as far or have as much knowledge about parasitology lab work if it had not been for Debbie Hebert. Her invaluable knowledge helped me tremendously in developing and executing my ideas for the parasitology studies. I am truly grateful to have had my grants approved and funded by OARDC SEEDS graduate student grants and NCR-SARE to do my research. I would also like to thank the Department of Animal Sciences for my graduate assistantship. Last but not least, I would like to thank my family and close friends for their continued support in believing in me to make it this far. I am the first on both sides of my family to obtain my doctorate and without the support I received from them, I would have struggled to continue my education. There are times when stress and frustrations became overwhelming, and I wanted to quit so I could pursue other dreams of going to culinary vii

9 school and open a restaurant, but my family, especially my wonderful mother, kept me going. A sincere thanks to everyone involved in the process of my dreams to become a doctor becoming reality. You all are truly a blessing. viii

10 Vita May Hoover High School B.S. Animal Science, Tuskegee University M.S. Chemistry, Tuskegee University 2010 to present...graduate Teaching Associate, Department of Animal Science, The Ohio State University Fields of Study Major Field: Animal Sciences Specialization: Ruminant Nutrition ix

11 Table of Contents Abstract... ii Acknowledgments... vi Vita... viii List of Tables... xiii List of Figures... xv Chapter 1: Introduction... 1 Preliminary Research... 2 Literature Review... 6 Gastrointestinal parasites... 6 Natural products used in medicine... 8 Tannins: Overview... 9 Tannins: Effects on ruminants and gastrointestinal parasites Pomegranates: Overview Pomegranates: Effects on animals Grape pomace: Overview x

12 Grape pomace: Effects on animals Chapter 2: Effects of pomegranate husk and grape pomace on dry matter degradation in vitro Abstract Introduction Materials and Methods Plants source and analysis In vitro dry matter degradation Statistical analysis Results and Discussion References Table and Figures Chapter 3: Effects of pomegranate husk and grape pomace extracts on developmental life stages of helminth parasites in vitro Abstract Introduction Materials and Methods Extraction of plant material Larval assay xi

13 Adult helminth assay (preliminary trial) Statistical analysis Results and Discussion References Table and Figures Chapter 4: Effects of wine grape pomace on gastrointestinal parasitism in naturally infested grazing lambs Abstract Introduction Materials and Methods Animal diet Animal trials Statistical analysis Results and Discussion Animal diet Trial Trial References Table and Figures xii

14 Chapter 5: Effect of wine grape pomace on egg hatchability and larval development of helminth parasites: Organic versus conventional farming methods Abstract Introduction Materials and Methods Extraction Fecal culture Statistical analysis Results and Discussion References General Conclusions References Appendix A: Cumberland Valley Analytical Services, Inc Procedure References Appendix B: FAMACHA Eye Score Chart xiii

15 List of Tables Table 1. Nutrient composition (DM basis) of alfalfa, grape pomace, and pomegranate husk Table 2. Estimated percentage of the DM in the A pool (readily digestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) Table 3. Estimated percentage of the DM in the B pool (potentially digestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) Table 4. Estimated percentage of the DM in the C pool (potentially indigestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) Table 5. Estimation of k (%/h, rate of degradation) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) Table 6. Estimation for the effective digestibility for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) xiv

16 Table 7. Effect of grape pomace and pomegranate husk extracts on activity (% active) of L 3 O. ostetagia Table 8. Effects of pomegranate husk extracts on activity of adult N. brasiliensis Table 9. Composition (DM basis) of diets in Trial 1 and Trial 2 fed to lambs as a control or experimental diet Table 10. Body weight (BW), average daily gain (ADG), packed cell volume (PCV), FAMACHA, and fecal egg counts (FEC) by week and overall for lambs treated with a negative control, Cydectin or Grape pomace pelleted feed in Trial Table 11. Body weight (BW), average daily gain (ADG), packed cell volume (PCV), FAMACHA, and fecal egg counts (FEC) by week and overall for lambs treated with a negative control, 0.9 kg grape pomace pellet (GPD1), or 1.5 kg grape pomace pellet (GPD2) diet in Trial xv

17 List of Figures Figure 1. Classification of tannins: a) gallotannin, b) ellagitannin, c) complex tannin, and d) condensed tannin (Khanbabaee and van Ree, 2001)... 9 xvi

18 Chapter 1: Introduction Much emphasis is being placed on decreasing the use of synthetic drugs in food animal production, and there are a limited number of drugs approved by the Food and Drug Administration for some of the minor species (e.g., goats and sheep). Yet, intestinal parasites are among the primary health risks to the growth and survival of small ruminants. In addition, there is a need on further reducing the need of agricultural animals on human-edible foods while increasing the utilization of recycled agricultural waste or by-products. This project investigates improving animal health and production with use of bioactive based products, which, at the same time, might have social and environmental benefits. By using the by-products from juice and wine-making industries, the amount of waste is reduced, which would reduce the cost of waste disposal by the respective industries. This research gives an opportunity to provide a value-added component to the fruit industry and to improve the health, growth, and efficiency of production of small ruminants. The discoveries made from this research provide an opportunity for internal parasite control of animals in organic production practices. These aspects will aid in providing for a more efficient and affordable food system and improving economic viability of food production systems that can lead to economic benefits for both the plant and animal industries, while reducing dependency on chemical 1

19 anthelmintics to assist in improving animal health and productivity. Utilizing by-products of the juicing and wine making industry is an area of relevance to Ohio, which ranks 10 th in grape production, with about 5,700 tons annually harvested, and for small ruminants, with Ohio ranking 13 th in sheep production (state with largest production east of the Mississippi River) with about 128,000 head. Ohio also has about 59,000 head of goats, and farmers are showing growing interest in meat goat production. This project has provided an opportunity for the researchers and farmers/producers to work together to gain experiences that can be taken from the lab and incorporated directly into farm practices. Preliminary Research A study was conducted at Tuskegee University to verify the presence of specific bio-active compounds, condensed tannins (CT) and pelletierine, in pomegranate husk, which is a by-product of the pomegranate juicing industry (LeShure, 2010). Both these compounds have historically shown efficacy against helminth parasites in animals. The pomegranate root bark was used as one of the controls and commercial pelletierine, to identify the presence of pelletierine in the husk samples. Investigating the presence of hydrolysable tannins was not one of the main focuses of this study since the presence of these tannins in husk and root bark already has been established in other research (Seibold, 1884; British Pharmacopoeia Codex, 1911; Gil et al., 2000; Li et al., 2006). In the study by LeShure (2010) small amounts of pelletierine were present in pomegranate husk (PH) and root bark samples, using the commercial pelletierine as a standard. A positive result from a ninhydrin assay of the two dimensional thin layer chromatography 2

20 (2-D TLC) done on extracts of the husk and root bark samples for maximum separation of compounds also indicated the presence of pelletierine in the samples. The presence of CT was confirmed from the positive result using a vanillin/hcl assay of the 2-D TLC. A preliminary study (Min et al., 2009) also was done on the parasite motility of third stage Haemonchus contortus larvae in the presence of crude CT extract from PH. This experiment was an in vitro simulation to test how the extract would inhibit the passage of the larvae into the gastrointestinal lining. The results of this study showed that the extract had an inhibition rate close to 80% at 150 mg/ml of crude PH extract and the commercial pelletierine standard had an inhibition rate at 90% at 100 μg/ml. A CT butanol/hcl assay also was done on the crude CT extracts of the PH to obtain the concentration of CT. The results showed pomegranate husk contained, on average, 5.94% CT (DM basis). A preliminary study was done at The Ohio State University to determine the concentration of CT in grape skins. Red and black grapes were bought from a local market, and the skins were harvested. The skins were dried, powdered, and stored in a dark, dry place until extraction could be performed. The dried skins were extracted with 70% acetone, and extracts were subjected to a CT butanol/hcl assay. The red grapes contained 4.14% CT (DM basis) and black grapes contained 3.95% CT (DM basis), which was comparable to the literature that reported an average of 4% tannin content (Lu and Yeap Foo, 1999). This also strengthened the idea that GP could have a similar potential benefit to being a natural anthelmintic as the PH. 3

21 These experiments have shown the promise of PH extracts and GP as a natural anthelmintic with the presence of the bioactive compounds, including tannins. The indications of CT being present in the PH and GP were especially useful due to CT having both direct and indirect effects on gastrointestinal parasites (GIP). This research is part of the newly growing interest within the scientific community for determining the potential direct impacts of using pomegranate and grape by-product extracts on parasites and sheep. Preliminary research conducted at Tuskegee University showed there is efficacy of PH extracts in vitro against helminth parasites. There were very limited data available, however, to ensure that there would be no negative ruminal effects or to demonstrate in vivo efficacy of either pomegranate or grape by-products. There has been quite a bit of work done to support the use of several plant sources containing condensed tannins, flavonoids, and flavonols as natural sources of anthelmintics with regard to small ruminants; however, extensive information was also lacking within the scientific literature on the potential direct impacts of using pomegranate and grape by-product extracts, which also have these bioactive compounds, on internal parasites of sheep. The goal of this research project was to determine effects of these extracts on ruminal DM degradation and against helminth parasites both outside and within the host animal. The overall hypotheses of this research were that GP and PH would significantly reduce the viability of helminth parasites and that GP would reduce parasite burden in sheep without causing detrimental effects on DM degradation. Relative to the overall hypothesis, experiments were conducted with the following objectives: 4

22 Objective 1. Extract and quantify the naturally occurring bioactive compounds in PH and GP and determine the effects of these bioactive compounds on ruminal dry matter degradation. Hypothesis 1. PH and GP extracts will not compromise the ruminal DM degradation. Objective 2. Evaluate the effects of PH and GP extracts on reducing parasite viability in vitro and to evaluate if extracts will be detrimental to certain life stages of helminth parasites. Hypothesis 2. PH and GP extracts will reduce the viability of helminth parasites and will cause eggs to not develop into larval stages or will cause increased morbidity and mortality in larvae. Objective 3. Evaluate the effects of GP to reduce the parasite load of naturally infested grazing lambs and evaluate if GP will have comparable efficacy to commercial anthelmintics. Hypothesis 3. GP will reduce the viability of intestinal parasites against a control (no treatment; negative control) and will have similar effectiveness as a commercial anthelmintic (positive control). This program has provided an opportunity for the graduate student investigator to perform discovery research that will potentially have major impacts in agricultural communities. Implementation of successful results from this research could improve animal health, reduce dependence on commercial drugs, lower environmental risks by 5

23 reducing plant wastes, and improve economic sustainability for both the plant and animal industries. Literature Review Gastrointestinal Parasites Gastrointestinal parasitism has become one of the most devastating diseases in ruminant animals (Min and Hart, 2003). The most relevant gastrointestinal parasites (GIP) to small ruminants are from the order Strongylida, superfamily Trichostrongyloidea (Balic et al., 2000; Zajac, 2006). The most important of these parasites is Haemonchus contortus because of its high reproduction rates and the fact this helminth parasite feeds on blood, causing the host animal to become severely anemic with large numbers of parasites (Zajac, 2006). The life cycle is generally the same in all species of the Trichostrongyloidea family. The adult female in the abomasum or small intestine produces eggs that are passed out in the feces. The eggs start to develop in the fecal matter, which provides protection from various environmental effects and food for the first few larval stages. The first stage larvae (L 1 ) hatch out of the egg and feed on bacteria and organic matter. The larvae goes through two developmental stages (L 2 and L 3 ) after the first stage from which it sheds its outer covering, also known as exsheathment, until it reaches the third stage of development. The third stage larva (L 3 ) retains its outer coat as a protective cuticle and migrates out of the fecal matter onto grass leaves. The larvae can move horizontally and vertically up blades of grass by way of moisture accumulation on the grass leaves. Once the larvae get mid-way or to the top of forages, they can be ingested by grazing ruminants (Zajac, 2006) and can develop into fourth larval (L 4 ) and adult stages (L 5 ) once they reach the abomasum or small intestines. 6

24 The larvae can also migrate into the abomasum wall or between gastric pits and arrest development (Balic et al., 2000; Zajac, 2006). In some cases, the larvae remain latent through the winter months and emerge out of the tissue in the early spring to develop into L 4 and L 5 in order to allow the eggs produced from the adult stage to have more of a chance to survive in the pasture (Zajac, 2006). Control of ruminant GIP over the last two and a half decades has been accomplished through the use of commercial anthelmintics; however, it has become challenging due to increasing resistance of parasites to common anthelmintics, with some parasites becoming multi-drug resistant (Aas, 2003; Min and Hart, 2003; Fleming et al., 2006). Resistance has become a problem worldwide, not just in the United States. It has been seen in Europe, South Africa, South America, and Australia (Fleming et al., 2006). There is a vast amount of variability in the genome of helminth parasites. When anthelmintics are administered and a small population survives, those are the parasites that have resistant genes. With such genetic diversity of nematode populations, random mutations can arise. Parasites that have high reproduction rates can rapidly pass resistance to the next generation produced (Fleming et al., 2006; Papadopoulos, 2008). Multidrug resistance has been attributed to multiple gene mutations, such as specific mutations in a P-glycoprotein (Kerboeuf et al., 2008) or in generalized mutations that affect the receptor binding site(s) where the drugs work, or in differences in enzymes, transport mechanisms, or metabolism of anthelmintics (Papadopoulos, 2008). This resistance to chemically synthesized anthelmintics has lead researchers to look for alternatives for controlling GIP. Natural anthelmintics with biologically active 7

25 compounds, such as tannins, polyphenols, and flavonoids, are becoming more common and present an opportunity to overcome these challenges (Aas, 2003; Min and Hart, 2003). Natural products used in medicine Natural products are those chemical compounds derived from living organisms, plants, animals, and insects that have biologically active compounds. The study of natural products for medicinal purposes is the investigation of their structure, formation, use, and purpose in the organism. In the U.S. and elsewhere, before the invention of specific pharmaceuticals, herbal medicine was used to treat many different illnesses. In recent years, there has been a resurgence of interest in herbal remedies. In 1998, there was over $12 billion in sales involving natural cures and/or treatments with a 10% increase in growth expected per year ( Natural products, 2009). Using natural products in agriculture has been around for centuries on other continents, such as Asia and Africa (Githiori et al., 2006). There have even been a select number of plants with anthelmintic activity that have been added to the 1953 and 1965 British pharmacopeia. Plants and seeds such as garlic, onion, mint, walnuts, dill, parsley or tobacco have been used to treat agricultural animals suffering from GIP (Githiori et al., 2006). Utilizing natural products has become a growing interest more recently due to the increasing resistance among GIP in animals. Several researchers have started looking at bioactive compounds in certain plants as a way of controlling GIP in agricultural animals. One of the bioactive compounds of interest being investigated as a natural way to control GIP is the use of tannins (Min and Hart, 2003). There are several ways to classify tannins, but the majority 8

26 of them fall under either condensed or hydrolysable tannins (Figure 1) (Khanbabaee and van Ree, 2001). a b c d Figure 1. Classification of tannins: a) gallotannin, b) ellagitannin, c) complex tannin, and d) condensed tannin (Khanbabaee and van Ree, 2001) Tannins: Overview Tannins are naturally occurring compounds in plants that are water-soluble and can precipitate proteins out of aqueous solutions. They can be classified as hydrolysable or condensed tannins. Condensed tannins (CT, d) (or proanthocyanidins) are polymers of flavon-3-ols and are comprised of a group of polyhydroxyflavan-3-ol oligomers and polymers linked via C4-C6 or C4-C8 bonds. In plants, CT are derived from catechin or epicatechin (Schofield et al., 2001; Romani et al., 2006). Hydrolysable tannins (HT) consist of a carbohydrate moiety or a glucose core in which the hydroxyl groups are esterified with gallic acid (a) (gallotannins) or with hexahydroxydiphenic acid (b) (ellagitannins) (Mangan, 1988; Mueller-Harvey, 2001). HT can be easily hydrolyzed by acids, bases, hot water, or enzymes (Hoste et al., 2006). Gallotannins (a) are tannins in which galloyl units or their meta-depsidic derivatives are bound to diverse polyol-, 9

27 catechin-, or triterpenoid units. Ellagitannins (b) are those tannins in which at least two galloyl units are C-C coupled to each other and do not contain glycosidically linked catechin units. Complex tannins (c) are tannins in which a catechin unit is bound glycosidically to a gallotannin or an ellagitannin unit (Khanbabaee and van Ree, 2001). Presumably, CT are found in most vascular plants (Schofield et al., 2001; Romani et al., 2006), but there are contradictions as to what the percentage of either type of tannin is in the pomegranate. In some studies, the majority of tannins found in the pomegranate were the HT (e.g., ellagitannins and gallotannins; British Pharmacopoeia Codex, 1911; Gil et al., 2000), whereas in other studies, the tannin content is generalized and simply labeled as tannic acid equivalents or total phenols (Lloyd, 1897 and Singh et al., 2002). In some reports, the presence of both types is noted (Murthy et al., 2002; Li et al., 2006). There is literary evidence lacking as to the exact concentration(s) of CT in pomegranate husk varieties. Tannins: Effect on ruminants and gastrointestinal parasites CT have beneficial effects on ruminants by having the potential to help control anthelmintic-resistant GIP, as long as the CT is given in moderation (25 to 40 g/kg DM) (Nguyen et al., 2005). The CT has been shown to decrease FEC in small ruminants and to boost overall health (Mangan, 1988; Nunez-Hernandez et al., 1991; Athanasiadou et al., 2001; Nguygen et al., 2005). The direct effect of CT on GIP is mediated through CTparasite interactions that affect the physiological functioning of the GIP by directly interfering with parasite egg hatching and prohibiting development to infective larval stages (Nguyen et al., 2005; Kerboeuf et al., 2008). Hydrolysable tannins (HT) are 10

28 potentially harmful by having the ability to be further metabolized into compounds, such as polygallols, that are toxic to ruminants (Min and Hart, 2003). The HT also can potentially inhibit rumen fermentation, which is essential for complex carbohydrate fermentation in ruminants (Mangan, 1988). The CT in feed protects plant proteins from microbial degradation in the rumen, making the rumen-undegraded protein (RUP) available for digestion and absorption in the small intestine (Nguyen et al., 2005). This increase in RUP would benefit protein nutrition while not affecting ruminal fermentation in small ruminants (Nunez-Hernandez et al., 1991; Athanasiadou et al., 2001). The ideal concentration of CT is generally between 20 to 40 g/kg DM, at which level the CT may bind with the dietary proteins during mastication and neutral ph of the saliva, and protect the protein from microbial attack in the rumen (Nguyen et al., 2005). The protein-tannin complex is bound by simple H-bonding. This H-bonding can be reversed or counteracted by polyethylene glycol, which was used to determine that tannins were responsible for the action on protein protection (Mangan, 1988; Nunez-Hernandez et al., 1991). Once the protein and tannins are bonded together in the mouth and forestomach, the complex is stable in ph ranging from 4 to 7, but outside of this ph range, the complex is readily dissociated. This would mean that the complex would escape the rumen fermentation (ph~5 to 7) and protein would be readily dissociated and digested in the abomasum s gastric secretions (ph~2.5) and digested by the pancreatic secretions in the small intestine (ph~8 to 9), allowing increased absorption of essential amino acids in the small intestine (Mangan, 1988). This absorption of RUP in the small intestine would allow for more protein 11

29 availability to be partitioned to the immune system, allowing for host resilience and resistance to helminth parasites (Balic et al., 2000; Hoste et al., 2005; Hoste et al., 2006). This extra protein can be used by the innate immune system to help in proliferating mast cells to help in eliminating the parasite. The mast cells release products that stimulate gastrointestinal nerves to increase water secretion and stimulate muscle contraction that serves as a washer/sweeper mechanism to purge the host gastrointestinal tract of parasites from the lumen and epithelial wall (McKay and Bienenstock, 1994). The ability to assist in increasing the protein availability in the animal to aid in host-regulated immunity alludes to CT being an alternative strategy to control parasitism in small ruminants. Having CT at an ideal concentration has been shown to increase wool growth, body mass, milk production, and amount of protein in the milk, and it does not affect ruminal fermentation or digestive kinetics in ruminants (Nunez-Hernandez et al., 1991; Athanasiadou et al., 2001). Increased protein availability has been considered responsible for enhanced immunological responses towards GIP (Anthanasiadou et al., 2001). In contrast, if CT are given in excess amounts (> 85 g/kg DM) to ruminant animals, they can prove harmful. The free CT (CT not bonded to any proteins as complexes) react, inactivating microbial enzymes. This results in reduced rumen fermentation of readily fermentable carbohydrate and hemicelluloses (Nguyen et al., 2005). Overall, the correct concentration of CT strengthens the consideration of being an alternative strategy to control parasitism in small ruminants. 12

30 CT also can directly affect helminths by adhering to the proline/hydroxyprolinerich cuticle of certain developmental stages (L 3 to adult) and not allowing exsheathment, which will not allow the larvae in infective stages to release itself from its cuticle to feed or grow, causing consequential starvation and death (Thompson and Geary, 1995; Bahuaud et al., 2006). Bahuaud et al. (2006) witnessed this phenomenon when they studied the effects of four tanniferous plant extracts on the exsheathment of third stage larvae. Their studies showed that pine tree and heather extracts caused a significant delay in exsheathment, with chestnut extract having the greatest efficacy of not allowing the exsheathment process to occur at all. A study conducted by Novobilský et al. (2011) investigated the role of CT on inhibiting exsheathment and feeding patterns on the larvae of cattle nematodes. In their study, they examined the use of extracts from three tannincontaining plants, Onobrychis viciifolia (Sainfoin), Lotus peducaulatus (Greater Birdsfoot Trefoil), and Lotus corniculatus (Birdsfoot Trefoil), at three concentrations of 600, 1200 and 2400 µg/ml. All three extracts were observed having a dose-dependent response of significantly inhibiting larval feeding of first stage larvae. They also observed the same dose-dependent response from all three extracts on the delayed exsheathment of third stage larvae. The authors confirmed the role of CT as the main constituent responsible for these results by also examining the effects of these extracts after the addition of polyvinylpolypyrrolidone, a known tannin inhibitor. This activity of CT on altering the exsheathment process is another way in which it is being considered an antiparasitic bioactive compound. 13

31 Hur et al. (2005) conducted a study in Korea on the effects of feeding CTcontaining plants to goats infested naturally with coccidian oocysts and reported that feeding fresh pine needles (40 g CT/day/goat) and oak leaves (40 g CT/day/goat) in combination with lucerne chaff had rapid anticoccidial effects in goats, which was indicated by a sharp decrease in eggs per gram (EPG) of oocysts in feces. Two days after feeding the CT-containing diet, the EPG number declined by an average of 42%; by day six, there was a 76.5% decline; and by day 10, the EPG number had decreased by 94% (Hur et al., 2005). Another type of compound along with CT that has shown activity towards helminths is flavonoids. Some tannin complexes are considered flavonoids or derivatives of flavonoids. Most flavonoids present in foodstuffs are bound to sugars as β-glucosides. The binding of these flavonoids to sugars makes them soluble in water, but they cannot be absorbed into the body without the hydrolysis of the β-glucoside by microorganisms in the gut (Kerboeuf et al., 2008). Ruminant microbes that have an affinity to sugars could break down these bonds to allow the flavonoids to be available for absorption in the gut to help with immunity to parasites due to the indirect and direct effects flavonoids have toward both protozoa and helmintic parasites. Some flavonoids have been found to interact on the host-parasite interaction of protozoa, whereas others disturb protozoan development and metabolism, and can limit the resistance to other drugs (Kerboeuf et al., 2008). Flavonoids have been shown to change the activity of several enzymes and/or metabolic processes, such as the nitric oxide pathway, in the parasite that can lead to temporary paralysis and potential elimination (Kerboeuf et al., 2008). Flavan-3-ols, the 14

32 monomer units of CT, and their galloyl derivatives were examined in a study by Molan et al. (2003) against the helminth parasite Trichostrongylus colubriformis. Their assay showed the flavan-3-olgallate compounds had an efficacy of 100% inhibition of egg hatchability at concentrations of 1 mg/ml against T. colubriformis parasite eggs and effectively inhibited L 3 larval migration at concentrations of 500 µg/ml. The extracts showing the most efficacies against these parasites were the flavan-3-ol galloyl derivatives, with epigallocatchin (a CT) being the most active in both egg hatch and larval development inhibition assays (Molan et al., 2003). Multidrug resistance (MDR) is an increasing socioeconomic concern. Due to the increase to search for less toxic inhibitors to help in overcoming this resistance, flavonones, flavonols, and biflavonoid groups have shown to be a potential source to help with MDR by being able to inhibit a mutated gene, a P-glycoprotein, which has been isolated as the source of resistance in protozoa exhibiting MDR. Flavonoids have been shown to effect helminthes by changing the activity of several enzymes and/or metabolic processes which increase the nitric oxide production in the parasite. The increase in nitric oxide production leads to paralysis and putative vermifugal activity by acting on the neurotransmitters at the neuromuscular junction and causing myoinhibition in helminth parasites (Kerboeuf et al., 2008). This action allows the helminthes to be paralyzed long enough to be purged from the animal s gastrointestinal tract. The CT that are polymers of flavon-3-ols (Schofield et al., 2001; Romani et al., 2006) have inhibitory effects on both hatching and larval development on helminthes (Kerboeuf et al., 2008). 15

33 There are some by-products of the juicing industry that have shown promise as natural anthelmintics due to their bioactive compounds, such as tannins. The two byproducts of interest currently being investigated are PH and GP. Pomegranates: Overview The 2007 Census of Agriculture showed that there were 599 farms growing pomegranates in the U. S. on 24,517 acres, with only 12,103 acres bearing production. In 2010, California s San Joaquin Valley established that there were 22,000 acres growing pomegranates ( Pomegranate Profile, 2010). The recent focus in research on the pomegranate has been on its antioxidant properties. Gil et al. (2000) found that commercial pomegranate juice had antioxidant activity three times higher than that of red wine and/or green tea (Gil et al., 2000). In an evaluation of the antioxidant properties of the husk (outer skin) extract versus the pulp (inside of fruit), the PH extracts had more potential to be a health supplement because the husk had twice the concentration of natural antioxidants (Li et al., 2006). Singh et al. (2002) conducted studies on the antioxidant activity of the husk and seed extracts using various in vitro models, such as β-carotene linoleate and 1,1- diphenyl-2-picryl hydrazyl (DPPH). They found that a methanol extract of the husk at 50 ppm showed the highest antioxidant activity versus the seed extract at 100 ppm. This extract was selected for testing of its effects on lipid peroxidation, hydroxyl radical scavenging activity, and human low-density lipoprotein (LDL) oxidation (Singh et al., 2002). 16

34 Singh et al. (2002) later performed a study using the methanol husk extract in vivo on male albino rats of the Wistar strain, giving them treatments of carbon tetrachloride (CCl 4 ) with and without the pre-treatment of the extract. When giving the rats a single dose of CCl 4 at 2.0 g/kg of body weight (BW), they observed reduction in the levels of catalase, superoxide dismutase (SOD), and peroxidase by 81, 49, and 89%, respectively; however, the lipid peroxidation increased nearly 3-fold. When the rats were given the pretreatment of a methanolic extract of PH at 50 mg/kg BW (in terms of catechin equivalents) before the CCl 4, they found that the extract caused preservation of catalase, peroxidase, and SOD to values comparable with the control values; whereas, lipid peroxidation was brought back by 54% as compared to control (Murthy et al., 2002). In addition to these recent research studies on the pomegranate, there are historical uses of the pomegranate that have been applied as worming methods (Seibold, 1884; Lloyd, 1897; British Pharmacopoeia Codex, 1911; Wibaut and Holstein, 1957). The anthelmintic properties of the root and husk of the pomegranate were well known among many of the ancient civilizations (Lloyd, 1897). The Chinese were acquainted with the anthelmintic property of the root; some Roman authors recommended the fruit husk; and the Arabian writers maintained that the root-bark was very effective, specifically against tapeworms (Lloyd, 1897). In the early 1800s, English physicians started treating patients with this plant-derived remedy and some pharmacologists in the late 1800 s started publishing how to make the remedy taste more appealing so that it could be taken more effectively (Seibold, 1884; Lloyd, 1897). The 17

35 uses of the PH and root bark as medicinal treatments were recognized in the U.S. Pharmacopeia in In 1878 and 1880, Tanret discovered several alkaloids in the root-bark of the pomegranate tree, the most prominent of which was a nitrogen-containing compound called pelletierine. Pelletierine was the alkaloid thought to be responsible for the anthelmintic properties of the root (Lloyd, 1897). The other alkaloids present in the root bark are isopelletierine, methylpelletierine, and pseudopelletierine. Wibaut and Holstein (1957) proved that pelletierine was the alkaloid most responsible for the anthelmintic properties by experimenting with the compound on liver fluke and reported the hydrochloric salt of pelletierine was active against the parasite, even in concentrations as low as 1/16,000 to 1/32,000 in aqueous solutions. In a 1/10,000 aqueous hydrochloric salt solution of pelletierine, tapeworm lost power of movement in about 6 minutes, and by 10 minutes of immersion in the solution, the worm was killed (British Pharmacopoeia Codex, 1911). The amount of pelletierine found in the root bark was as much as 3.5% but, on average, ranges from 0.5 to 1.0% (Lloyd, 1897 and British Pharmacopoeia Codex, 1911). The presence of this natural anthelmintic holds promise as a natural anthelmintic for humans but has not yet been investigated in any agricultural animals. Pomegranates: Effects on animals A preliminary study was done at Assuit University in conjunction with Tuskegee University to test the effectiveness of the PH on coccidian infested goats (Huessin, 2005). They used eight goats, with two naturally infested with coccidian oocysts and the other 18

36 six un-infested. Over a period of eight weeks, they monitored the goats red blood cell count, weight gain, and the number of oocysts excreted in the feces (i.e., EPG). They reported that an infested goat fed up to 25% PH supplemented ration was able to maintain as steady a weight gain as that of an uninfested goat. If fed up to a 50% PH supplemented ration, the goat was actually able to increase weight gain. The only drawback to the experiment was that the FEC were not decreasing as originally expected. They rationalized that there might be a different mode of action occurring to increase the health of the animal other than decreasing the amount of EPG. In a study conducted on the dietary effects of pomegranate seed oil on immune function in mice, Yamasaki et al. (2006) reported that concentrations as low as 0.12% pomegranate seed oil in the diet significantly increased IgG and IgM. IgG and IgM are some of the first level of defense immunoglobins to help fight infestation (Yamasaki et al., 2006); however, there was no significant increase in IgA levels. The IgA is the first line of defense for infectious agents entering the mucosal lining in the intestine; IgA works by attaching itself to the mucosal epithelium (Aas, 2003). Amin et al. (2009) conducted a study that tested water extracts of 20 indigenous plants, including pomegranate, on the GIP Haemonchus spp., Trichostrongylus spp., Cooperia spp., Oesophangostomum spp., Trichuris spp., and Strongyloides spp. They collected the GIP directly from the abomasums of 1200 randomly infested cattle and tested water extracts at concentrations of 25, 50, 100 mg/ml in petri dishes containing 50 adult worms. Their results showed that pomegranate leaf water extract had effectiveness on the motility of the adult worms of 30, 52, and 86% at concentrations 25, 50, and

37 mg/ml, respectively (Amin et al., 2009). This study showed promise that pomegranate could potentially be used to help minimize parasite burden in the gastrointestinal tract. Shabtay et al. (2008) conducted a study utilizing pomegranate peels as a nutritiverich byproduct that could be used to serve as a potential disease preventative measure in cattle and to help with improvement of beef cattle products. They observed that ensiled pomegranate peels, when used as an ad lib dietary supplement, increased the overall feed intake of bull calves and tended to increased weight gain, while also increasing α- tocopherol blood plasma concentrations. This increase in α-tocopherol in the plasma was directly associated with increased vitamin E availability to assist in improving the health status of the calves. They concluded from their study that including ensiled pomegranate peels in the diet of bull calves would add nutritive value while increasing antioxidant capacity of the diet, therefore promoting favorable health benefits to feedlot beef cattle. Other researchers have found that certain levels of tannins present in the pomegranate also may be beneficial toward animal health. The highest amounts of tannins were present in the husk of the fruit and the bark of the root, ranging from 19 to 28% (Lloyd, 1897; British Pharmacopoeia Codex, 1911). Grape pomace: Overview The by-product of the pomegranate juicing industry, PH, is not the only byproduct that could possibly show promise as a natural anthelmintic. Ohio grape industry contains > 1,500 acres of vineyards producing on average 1.5 to 4.0 tons/acre of fruit since 2004 ( Ohio Grape, 2010). GP is a by-product of the juicing and wine industry that consists of mainly seeds, skin, and pulp (~18 to 20 kg/100 kg of grapes). 20

38 Traditionally, uses of GP have been largely restricted to land applications, such as an organic way to add nutrients back into the soil or as a heavy metal absorbent (Arvanitoyannis et al., 2006; Spanghero et al., 2009). GP has not had a lot of consideration as an animal feed for ruminants due to the low nutritional value of most but not all pomace. The GP pulp has been shown to contain (g/ kg DM): 906 organic matter, 392 neutral detergent fiber, 364 acid detergent fiber, 105 crude protein with the addition of 224 crude protein bound to acid detergent fiber, and 226 lignin. Starch and free sugars (i.e., sucrose, glucose, and fructose) were determined, but values were less than the detection levels of 5 mg/kg DM for starch and less than the detection levels of 2 mg/kg DM for each sugar (Spanghero et al., 2009). Lu and Yeap Foo (1999) examined the isolations and identification of a range of polyphenols in GP using a wide range of grape cultivars. Their study revealed that both HT and CT were present in the GP in concentrations averaging up to 4% of total dried samples (Lu and Yeap Foo, 1999). Some of the same HT and CT in grape pomace also are found in the pomegranate. These tannins include gallic acid, anthocyanins, catechin, epicatechin, and procyanidin and flavonoids, which were the most abundant polyphenol in the GP extracts (Lu and Yeap Foo, 1999). Grape pomace: Effects on animals A study was conducted by Famuyiwa and Ough (1982) to investigate the possibilities of wine grape pomace as a potential animal feed. They examined the in vitro dry matter digestibilities of three varieties of GP: Cabernet Sauvignon, Gerwurtztraminer, and Tinta Madeira, using rumen fluid from cattle, sheep and goats. Cabernet Sauvignon 21

39 pomace had the highest digestibility of the GP varieties at 38.6% in cattle, 35.1% in goats, and 36.6% in sheep; in contrast, Tinta Madeira had the lowest digestibility of 25.9% in cattle and sheep and 26.9% in goats. The pomace with the highest total phenolic content had the lowest digestibility among the varieties examined. They also examined mixing the pomace with alfalfa or sudan grass hay to determine if there was a feed interaction. Cabernet Sauvignon had roughly 4% tannic acid equivalent with only a slight decrease in digestibility of 1.6%, therefore concluding that the tannin level in that variety of pomace did not significantly lower the digestibility of hay or have a feed interaction when mixed at 50% of the diet. They concluded that seedless GP from varieties like Cabernet Sauvignon, had potential to be included in feed formulation of ruminants. Villalba et al. (2006) conducted a study examining the preference of lambs to foods that differed temporally and spatially in nutrients and toxins. They fed two biochemically different feeds, one with tannins (included 30% GP and 10% quebracho tannin; 2.4 Mcal/kg, 8% crude protein) and one without tannin (3.0 Mcal/kg, 15% crude protein). They noticed during the trial, both groups of lambs preferred the feed without tannins, but the lambs that were conditioned previously to the tannins always ate more tannin containing feed than lambs not conditioned. When the feed not containing tannins was offered less, lambs preconditioned to tannins spent more time at eating both feeds instead of just tannin containing feeds. This suggested that, even though lambs could be conditioned to eat feeds high in tannins, they still preferred feeds without tannins when given a preference. This result was not surprising because most animals will not prefer to eat more astringent, toxic feeds over those with higher nutrient value and palatability. 22

40 Baumgärtel et al. (2007) also conducted a study feeding GP to sheep but examined the differences in digestibility and energy of fresh GP from either white or red wine varieties. White GP had higher metabolizable energy due to higher sugar content but had a lower crude protein digestibility due to increased CT content than that of the red GP. Red GP had lower DM and organic matter (OM) digestibilities. The Red GP having lower DMD and OMD was thought to be due to higher lignin and fiber contents of the red wine grapes than that of the white wine grape variety. Even thought the white GP had a higher CT content, the higher sugar content of the pomace was thought to be the cause of increased digestibility due to these sugars being readily fermented by rumen microbes. Their overall conclusion to the study was that GP can vary drastically in the feeding value for ruminants depending on the way the grapes are processed. This conclusion was formed due to red wine grapes being processed by pre-fermenting the skins with the juice, unlike white wine varieties which are not processed with skins present so more compounds are left in the pomace, such as the sugars. This paper did not specify what varieties of grapes were used for each white wine or red wine pomace. Knowing that nutrient value and chemical composition can vary based on variety, this information would have been beneficial in evaluating the important applications of this paper. There have been several other researchers that have examined adding GP to feeds on the effects on digestibility and degradability of various digestive parameters. Besharati and Taghizadeh (2009) fed lambs four diets that gradually replaced alfalfa in the diets (0, 150, 300, or 450 g dried GP/kg of alfalfa DM). They observed decreasing DM and crude 23

41 protein (CP) digestibilities with increasing GP in the diet. The researchers accredited the observed results to increasing tannins in the diet that decreased protein availability to rumen microbes. This unavailability of nutrients to the microbes also resulted in volatile fatty acid and rumen ammonia concentrations being lower than in alfalfa diets. Abarghuei et al. (2010) conducted a similar study only with three different diets: one was a control diet with alfalfa hay, the second with GP and a third using GP with the addition of polyethylene glycol (PEG). They also noticed the decrease in CP digestibility, in addition to decreased OM and neutral detergent fiber (NDF) digestibilities that improved when adding PEG to the diet. There was also a reduction in rumen parameters (ph, ammonia, cellulolytic and proteolytic bacteria populations and protozoa numbers) and microbial yield of animals fed on diets with GP when compared to the control diet. In both studies, Besharati and Taghizadeh (2009) and Abarghuei et al. (2010), fed over the recommended amount of tannins (> 50 g/kg DM) in their experimental diets, so we would expect detrimental effects to rumen digestion and microbial populations. On the other hand, Bahrami et al. (2010) conducted a study feeding dried GP at 0, 5, 10, 15, and 20% of the diet and observed that the lambs on diets containing 5 to 10% dried GP greatly improved their growth performance when compared to the other treatments. They noted DM, CP, OM, and NDF digestibilities increased significantly with the addition of 5 or 10% dried GP to the diet and concluded that adding up to 10% GP to the diet would actually be useful in increasing weight gains in lambs without negative effects on performance. There appears to be a beneficial effect for small ruminant animals and producers when GP is added to animal diets within optimal concentrations. 24

42 Chapter 2: Effects of pomegranate husk and grape pomace on dry matter digestibility in vitro Abstract Agriculture feed industries have historically utilized agricultural by-products as animal feed for ruminants as non-competitive food sources for these animals. Pomegranate husk (PH) and grape pomace (GP), by-products of juicing and wine making industries, are being considered as potential feedstuffs. This research goal was to ensure that when these by-products can be added to diets without causing decreases in dry matter degradation (DMD) and degradability kinetics of the feed. A batch culture in vitro dry matter degradability study was conducted at 0, 6, 12, 24, 48, and 96 h using 100% Cabernet Sauvignon or Shiraz GP, 100% alfalfa, 100% Parifanka or Desertnyi PH, or mixtures of alfalfa and GP or PH varieties at 25, 50, or 75%. When GP was mixed with alfalfa, both GP varieties decreased in DMD with increasing inclusion of the treatment (P < 0.05). The GP varieties also had higher proportions (P < 0.05) of potentially indigestible DM when compared to all other treatments. Both PH varieties had little to no effect on DMD (P > 0.10). The PH varieties had comparable DMD to that of alfalfa, except for 50:50 Parifanka treatment that had a higher DMD (P < 0.05). Adding GP to the diet at concentrations greater than 25% can decrease DMD, whereas PH has less risk 25

43 for decreasing DMD, perhaps because of its high concentration of non-fiber carbohydrates. Introduction Ruminants have utilized agricultural by-products as animal feed for centuries. This utilization of agricultural by-products has allowed for decreased dependence of farm animals on human-edible crops and grains and adds value to waste products that would otherwise be used for landfills (Mirzaei-Aghsaghali et al., 2011). Pomegranate husk (PH) and grape pomace (GP) are by-products of the juicing and wine-making industries that have been used in the past few years for animal feed. Pomegranate husk has been receiving extra attention because of its various benefits including increasing immune function and antioxidant activity (Gil et al., 2000; Singh et al., 2002; Li et al., 2006; Yamasaki et al., 2006). The antioxidant activity of the peel decreased risk of several diseases and mortality and had a positive correlation with decreasing stress and illness in cattle (Shabtay et al., 2008). Shabtay et al. (2008) showed that including PH extracts up to 4% in the diet of lactating cows increased dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF) digestibilities and increased milk and energy-corrected milk yields. Wine GP has also been investigated as potential animal feed. Addition of the pomace up to 4% of the diet only slightly decreased DM digestibility (1.6%) when mixed with alfalfa or sudan grass hay (Famuyiwa and Ough 1982). Bahrami et al. (2010) conducted a study adding GP to the diet of lambs and noted inclusion of 5 to 10% grape pomace increased the digestibility of DM, CP, organic matter, and NDF. Their 26

44 conclusions were that the addition of GP to the diet at 10% would be beneficial in increasing weight gain in growing male lambs without detrimental effects on health. There have been several studies that have shown benefits of adding PH and GP to ruminant feeds, but they both contain bioactive products, such as condensed tannins (CT), that have observed negative effects on digestion (Nguyen et al., 2005; Baumgärtel et al., 2007; Besharati and Taghizadeh 2009; Abarghuei et al., 2010). The inclusion of CT over a certain threshold decreased rumen function and altered microbial populations of cellulytic, proteolytic, and fiberlytic microbes (Smith et al., 2005; Jami et al., 2012). Other than containing CT, PH and GP also have flavonoids that have both have efficacy as anthelmintics. Condensed tannins have consistently decreased gastrointestinal parasites (GIP) in ruminant animals (McKay and Bienenstock, 1994; Thompson and Geary, 1995; Hur et al., 2005; Bahuaud et al., 2006; Novobilský et al., 2011) and flavonoids have efficacy for decreasing parasites, even those showing multidrug resistance (Molan et al., 2003; Kerboeuf et al., 2008). The overall aim of this project was to evaluate the effects of PH and GP on DM digestibility to ensure no detrimental effects will be observed so that later studies can be conducted on utilizing these byproducts as potential natural anthelmintics. Dry matter degradation by rumen microbes is one of the beneficial functions of ruminant digestion, given that all other feedstuffs consumed by the animal can be degraded, absorbed, and utilized further down the GI tract. Because the presence of CT in feedstuffs has been reported to have detrimental effects on rumen microbes that lead to decreased DMD, this study was done to evaluated if the concentration of CT in PH and GP would alter rumen 27

45 microorganisms, microbial populations, or change dry matter degradation kinetic negatively that could then lead to undesirable conditions for the animal. We hypothesized that PH and GP would not detrimentally compromise the dry matter degradation in the rumen by evaluating PH and GP in an in vitro dry matter degradation study. Materials and Methods Plant source preparation and analysis Pomegranate husks were obtained from a grove at USDA ARS University of California, Davis. Wine GP was obtained from a local Ohio Vineyard (Chalet DeBonné Vineyard, Madison, OH). Plant material was dried to 90% DM and ground to pass through a 1-mm sieve to ensure maximum surface area for extraction purposes and to use raw material for in vitro fermentation studies. A crude tannin extraction was done on PH and GP using 70% aqueous acetone plus 1 g/l ascorbic acid to obtain most extractable compounds with minimal oxidation of sensitive constituents (Hagerman et al., 2000). A CT analysis was performed on samples using a modified butanol/hcl procedure (Terrill et al., 1992; Jackson et al., 1996) to determine the concentration of CT in the PH and GP and to decide which two varieties from each plant source would be used for subsequent study. Cabernet Sauvignon and Shiraz varieties were used for GP samples, Parifanka and Desertnyi varieties were used for PH samples, and ground alfalfa pellets were used as a control. Samples of alfalfa, both GP varieties, and a mixture of PH varieties were sent to Cumberland Valley Analytical Services (Hagertown, MD) for nutritional analysis and procedural references are provided in Appendix A. Due to limited amount of PH sample 28

46 needed for analysis, a mixture of all varieties was used to provide enough material to the lab for the procedures. In vitro dry matter degradation study Rumen fluid and solids were obtained from two cannulated Jersey cows prior to morning feeding at the Waterman Dairy Farm in Columbus, OH (Animal Use Protocol #2010A ) for the in vitro batch culture experiment. Experimentation was done using a modified procedure described by Piwonka and Firkins (1993). Liquids and solids were obtained from cannulated cows and blended to get a complete innocula with fiber/particle-associated microbes. The experimentation was performed as a randomized complete block design with blocks being time using a 4 x 4 factorial arrangement of concentration and plant source plus an overall control (alfalfa). The concentrations were based on utilizing a slope ratio technique of mixing the ground PH or GP varieties with ground alfalfa pellets. This technique was used to account for the extra fiber from the PH and GP that may interfere with determining degradation over time of those treatments. The treatments were in ratios of 100:0, 25:75, 50:50, or 75:25% for alfalfa:ph or GP varieties. The treatment and substrate totaling 0.5 g were incubated in triplicate and the entire experiment was repeated twice. All treatments were incubated for 0, 6, 12, 24, 48, or 96 h in a shaking water bath at 39 C. After incubation, samples were analyzed for DM remaining (AOAC, 1980). Statistical Analysis PROC NLIN procedure of SAS 9.3 (SAS Inst., Cary, IN) was used to estimate the degradation kinetics from the DM data using the model % DMR = C + Be -kt, where DMR 29

47 is the DM remaining, C is the potentially indigestible DM, B is the potentially digestible DM, k is the rate of DM degradation, and t is time. These estimates were analyzed in the PROC MIXED procedure of SAS 9.3 with LSMEANS to determine treatment estimates using a different model per kinetics term of A, B, C, effective digestibility (ED), or k = treatment. A and ED were calculated from the following formulas: A = 100 B C and ED = A + B (k d /k d +k p ), where k d was the rate of DM degradation and k p was the passage rate at 0.05/h. Linear, quadratic, and cubic effects were determined utilizing polynomial orthogonal contrasts for equally spaced treatments and the contrasts were used to determine DMD treatment differences. Data were significant at P < 0.05 and tendency at 0.05 < P < Results and Discussion Table 1 shows the nutrient composition of the alfalfa, GP and PH used in the trial. Alfalfa and GP had similar DM (92 to 94%), but PH was lower at 83.7%. Alfalfa had the highest crude protein at 21.6% with GP and PH having 12.1 and 3.30%, respectively. PH had the lowest CP and fiber but had the highest energy and non-fiber carbohydrates values. The concentrations of CT (DM basis) in the samples were 1.49, 2.02, 3.68 and 4.83% for Parifanka PH, Desertnyi PH, Cabernet Sauvignon GP, and Shiraz GP, respectively. Alfalfa has no detectable CT present. The estimated DM degradation kinetics are provided in Tables 2, 3, 4, 5 and 6 for the A pool, B pool, C pool, k, and ED, respectively. Both PH treatments contained more (P < 0.05) readily degradable DM (A pool) than the other treatments, even when mixed with alfalfa. The PH varieties had a linear increase (P < 0.05) in the A pool as the portion 30

48 of PH increased in the samples, whereas Cabernet Sauvignon GP tended to have a linear decrease (P = 0.06) and Shiraz GP numerically followed a similar trend (P = 0.11) as the Cabernet Sauvignon GP as the portion of GP increased in the samples (Table 2). The higher A pool for PH samples is not surprising when the non-fiber carbohydrates in the samples were twice to nearly three times as much than the GP or alfalfa samples. These carbohydrates would be readily degradable by rumen microbes to be used for energy for their growth. In all samples, expect Parifanka PH (P > 0.10), there was a linear decrease (P < 0.05) in the B pool with greater inclusion of PH or GP in the samples. Shiraz GP had a cubic or skewed decrease (P < 0.05) with Cabernet Sauvignon having a tendency (P = 0.10) for a cubic effect due to the rapid drop in the B pool at inclusion of 100% GP (Table 3). The decrease observed in the GP for the B pool is most likely due to the low degradability associated with GP. GP is a pre-fermented by-product of the wine-making industry so there would be an expectation of a smaller B pool available for DM degradation by rumen microbes. The decrease in B pool for PH is also expected when there is a smaller amount of NDF present in PH samples at 13.5% NDF (DM basis) compared to 38.2% for alfalfa, 54.2% for Shiraz GP, and 43.1% for Cabernet Sauvignon GP (Table 1). The GP varieties contained higher (P < 0.05) portions of indigestible DM (C pool) than all other treatments and the C pool linearly increased (P < 0.01) with greater inclusion in the samples. There was no significant effect (P > 0.10) observed with the Parifanka PH, but a cubic effect (P < 0.05) with a tendency (P = 0.10) for a quadratic 31

49 effect was observed for Desertnyi PH. There was an increase in the C pool as Desertnyi PH was added to the sample until it was included at 75% in which the C pool decreased (Table 4). There is not a clear explanation as to why a skewed increase in the C pool was observed, except the possibility that the C pool was over estimated in SAS from the DMD data. Calculated values of the C pool for Desertnyi PH show that the C pool should have followed a more linear decrease as Desertnyi PH was included in the sample at 25% increments (25% DPH %, 50% DPH 39.7%, 75% DPH 39.3%) instead of the estimated values from SAS. There was a cubic effect (P < 0.05) observed for Cabernet GP and Parifanka PH with a tendency (P = 0.06) for Desertnyi PH to have a cubic effect. The cubic or skewed effect of treatment was observed by increases in the rate with 25% inclusion of PH or GP in the sample that decreased drastically with greater inclusion but increased slightly by 100% of PH or GP (Table 5). The ED values give a better idea of the effects of adding PH or GP to the samples by allowing an estimation of the percent digestibility in the rumen assuming a constant passage rate of 5%/h (Table 6). GP had a negative associative effect on alfalfa DMD that tended to have a linear decrease (P = 0.07) in DMD for Shiraz GP and DMD was linearly decreased (P < 0.05) for Cabernet Sauvignon GP, but when comparing with calculated values for the ratios (data not shown) the alfalfa inclusion added a positive associative effect to GP samples when included up to 50% of the sample for Shiraz GP only. On the other hand, PH had an overall positive associate effect on alfalfa DMD that tended to increase linearly (P = 0.09) and cubically (P = 0.06) for Desertnyi PH and Parifanka PH 32

50 had a numerically similar trend (P = 0.12) for increasing DMD linearly and cubically (Table 6). GP had lower degradability observed by lower A and B pools and higher C pool than the other samples, but this was likely due to high levels of lignin associated with the fiber present in the skin, seeds, and stems (Baumgärtel et al., 2007). The Cabernet Sauvignon GP was reported to have an average DM digestibility of 36.8% and to only slightly decrease DM digestibility of alfalfa by 1.6% when Cabernet Sauvignon GP was added at 50% of the diet (Famuyiwa and Ough, 1982), but our research does not support that conclusion. The DM degradability of Cabernet Sauvignon GP was far less than 36.8% (21.3%), and at 50:50 with alfalfa, DM degradability was still lower at 28.6%. In a later study done by Famuyiwa and Ough (1990), they reported the lower digestibility of Cabernet Sauvignon GP as it related to alfalfa/grain mixtures was due to an increased percentage of indigestible cell wall material in the GP samples. They observed Cabernet Sauvignon GP had a cell wall digestibility of 4.1% compared to the 33.6% cell wall digestibility of alfalfa/grain samples (Famuyiwa and Ough, 1990). This would support why there was a decrease in DM degradability with greater inclusion of the GP in samples observed in the ED values (Table 6). On the other hand, Besharati and Taghizadeh (2009) observed that DM digestibility decreased with increasing GP in diets mixed with alfalfa. The results from our study support their findings. Adding more than 20% GP to a ruminant diet may indeed increase some detrimental effects on DM digestibility, but supplementing at low 33

51 amounts, such as 5 to 10 % GP in the diet, can lead to beneficial effects (Bahrami et al., 2010). The PH samples had similar to higher DM digestibility compared to alfalfa and helped to support the results of Shabatay et al. (2008) whereby PH did not alter DM digestibility negatively when incorporated with other feedstuffs for ruminants. Our hypothesis that the DM digestibility would not be negatively compromised, was proven for the PH only but was rejected for GP at concentrations tested. With the inclusion of alfalfa with GP (up to 50%) in diets, GP could assist in countering some of the decreased degradability observed in this trial. References Abarghuei, M. J., Y. Rouzbehan, and D. Alipour. The influence of the grape pomace on the ruminal parameters of sheep. Livestock Science 2010, 132: AOAC Official methods of analysis of the AOAC. 13 th ed., Association of Official Analytical Chemists, Arlington, Va. Bahuaud, D., C. Martinez-Ortiz De Montellano, S. Chauveau, F. Prevot, F. Torres- Acosta, I. Fouraste, and H. Hoste. Effects of four tanniferous plant extracts on the in vitro exsheathment of third-stage larvae of parasitic nematodes. Parasitology 2006, 132(4): Baumgärtel, T., H. Kluth, K. Epperlein, and M. Reodehutscord. A note on digestibility and energy value for sheep of different grape pomace. Small Rum. Res. 2007, 67: Besharati, M., A. Taghizadeh. Evaluation of dried grape by-product as a tanniniferous tropical feedstuff. Ani. Feed Sci Tech. 2009, 152: Famuyiwa, O. and C. S. Ough. Grape pomace: possibilities as animal feed. Am. J. Enol. Vitic.1982, 33(1): Famuyiwa, O. and C. S. Ough. Effect of structural constituents of cell wall on the digestibility of grape pomace. J. Agric. Food Chem. 1990, 38:

52 Gil, M. I., F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft, and A. A. Kader. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agric. Food Chem. 2000, 48: Hur, S. N., A. L. Molan, J. O. Cha. Effects of feeding condensed tannin-containing plants on natural coccidian infestation in goats. Asian-Australasian J. Anim. Sci. 2005, 18, no. 19: Jackson, F. S., W. McNabb, T. N. Barry, Y. L. Foo, and J. S. Peters. The condensed tannin content of a range of subtropical and temperate forages and the reactivity of condensed tannin with ribulose-1, 5-bis-phosphate carboxylase (Rubisco) protein. J. Sci. Food Agric. 1996a. 72: Jami, E., A. Shabtay, M. Nikbachat, E. Yosef, J. Miron, and I. Mizrahi. Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population. J. Dairy Sci. 2012, 95 (10): Kerboeuf, D., M. Riou, and F. Guégnard. Flavonoids and related compounds in parasitic disease control. Mini-Reviews in Medicinal Chemistry. 2008, 8: Li, Y., C. Guo, J. Yang, J. Wei, J. Xu, and S. Cheng. Evaluation of antioxidant properties of pomegranate husk extract in comparison with pomegranate pulp extract. Food Chem. 2006, 96: McKay, D. M. and J. Bienenstock. The interaction between mast cells and nerves in the gastrointestinal tract. Immunology Today 1994, 15: Mirzaei-Aghsaghali, A., N. Maheri-Sis, H. Mansouri, M. E. Razeghi, A. Mirza- Aghazadeh, H. Cheraghi and A. Aghajanzadeh-Golshani. Evaluating potential nutritive value of pomegranate processing by-products for ruminants using in vitro gas production technique. J Agric Biol Sci. 2011, 6(6): Molan, A. L., L. P. Meagher, P. A. Spencer, and S. Sivakumaran. Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis. Int J Parasitol 2003, 33: Möller, J. Determination of crude protein (kjeldhal nitrogen) in animal feed, forage (plant tissue), grain and oil seeds using block digestion with copper catalyst and steam distillation into boric acid. Application note AN FOSS Analytical AB, FOSS tecator, Höganäs, Sweden. Nguyen, T. M., D. V. Binh, and E. R. Orskov. Effects of foliages containing condensed tannins and on gastrointestinal parasites. Anim. Feed Sci. Tech. 2005, 121:

53 Novobilský, A., I. Mueller-Harvey, S. M. Thamsborg. Condensed tannins act against cattle nematodes. Vet. Parasitol. 2011, 182: Piwonka, E. J. and J. L. Firkins. Effect of glucose on fiber digestion and particleassociated carboxymethyl cellulose activity in vitro. J Dairy Sci. 1993, 76:129 SAS User s Guide: Statistics, Version 6 Edition SAS Inst., Inc. Cary, NC Shabtay, A., H. Eitam, Y. Tadmor, A. Orlov, A. Meir, P. Weinber, Z. G. Weinburg, Y. Chen, A. Brosh, I. Izhaki, and Z. Kerem. Nutritive and antioxidant potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed. J Agric Food Chem 2008, 56: Singh, R. P., K. N. C. Murthy, and G. K. Jayaprakasha. Studies on the antioxidant activity of pomegranate (Punica granatum) husk and seed extracts using in vitro models. J. Agric. Food Chem. 2002, 50: Smith, A. H., E. Zoetendal and R. L. Mackie. Bacterial mechanisms to overcome inhibitory effects of dietary tannins. Microb Ecol 2005, 50: Terrill, T. H., A. M. Rowan, G. B. Douglas, and T. N. Barry. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 1992, 58: Thompson, D. P. and T. G. Geary. The structure and function of helminth surfaces. In Biochemistry and Molecular Biology of Parasites. Marr, J. J. and Muller, M. eds. 1995, Academic Press; p Van Soest, P. J. van, J. B. Robertson, and B. A. Lewis. Methods for dietary fiber, neutral detergent fiber and nonstructural polysaccharides in relation to animal nutrition. J Dairy Sci 1991, 74(10): Yamasaki, M., T. Kitagawa, N. Koyanagi, H. Imamura, H. Tachibana, and K. Yamada. Dietary effects of pomegranate seed oil on immune function and lipid metabolism in mice. Nutrition. 2006, 22:

54 Tables and Figures Table 1. Nutrient composition (DM basis) of alfalfa, grape pomace, and pomegranate husk Cabernet Analyzed Shiraz Grape Alfalfa Sauvignon composition Pomace Grape Pomace Pomegranate Husk 1 Dry Matter (%) Crude Protein (%) Soluble Protein (%) Acid Detergent Fiber (%) Neutral Detergent Fiber (%) Lignin (%) N/A Non-fiber Carbohydrates (%) Crude Fat (%) TDN (%) Ash (%) Calcium (%) Phosphorus (%) Magnesium (%) Potassium (%) Sodium (%) Iron (ppm) Manganese (ppm) Zinc (ppm) Copper (ppm) Pomegranate husk is a mixture of 11 varieties including Parifanka and Desertnyi; Mixture was due to insufficient sample needed for the analysis so all varieties were combined to accommodate this issue. 37

55 Table 2. Estimated percentage of the DM in the A pool (readily digestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) % Alfalfa Contrast SEM Linear Quadratic Cubic SGP CSGP PPH < DPH Contrast significant at P < 0.05 Table 3. Estimated percentage of the DM in the B pool (potentially digestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) % Alfalfa Contrast SEM Linear Quadratic Cubic SGP CSGP < PPH DPH Contrast significant at P < 0.05 Table 4. Estimated percentage of the DM in the C pool (potentially indigestible portion) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) % Alfalfa Contrast SEM Linear Quadratic Cubic SGP < CSGP < PPH DPH Contrast significant at P <

56 Table 5. Estimation of k (%/h, rate of degradation) for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) % Alfalfa Contrast SEM Linear Quadratic Cubic SGP CSGP PPH DPH Contrast significant at P < 0.05 Table 6. Estimation for the effective digestibility for alfalfa, Shiraz grape pomace (SGP), Cabernet Sauvignon grape pomace (CSGP), Parifanka pomegranate husk (PPH) and Desertnyi pomegranate husk (DPH) % Alfalfa Contrast SEM Linear Quadratic Cubic SGP CSGP PPH DPH Contrast significant at P <

57 Chapter 3: Effects of pomegranate husk and grape pomace extracts on developmental life stages of helminth parasites in vitro Abstract Trichostrongyle nematodes are becoming a great concern due to them being the major cause of production losses in small ruminants. There are only a limited number of FDA approved drugs available for use in small ruminants, and nematodes are quickly becoming resistant. Due to the interest in chemical anthelmintic alternatives, natural products for medicinal purposes in agriculture have started to gain research interest. This study examined the effects of differing concentrations of extracts from two varieties of pomegranate husk (PH, Wonderful and Sogidana varieties at 12.5, 25, and 50 mg/ml) and two varieties of wine grape pomace (GP, Shiraz and Cabernet Sauvignon varieties at 6.25 and 12.5 mg/ml) on third stage larvae of Ostertagia ostertagi. The effects of PH extracts on adult Nippostrongylus brasiliensis were also investigated in a preliminary study. Larval assays conducted on L 3 O. ostetagia showed that the Wonderful variety PH had the greatest (P < 0.05) efficacy at 50 mg/ml when compared to the GP treatments across concentrations. The greatest efficacy was observed after 4 h exposure of the larvae to the extracts (P < 0.01), but there was no additional efficacy observed at 24 h (P > 0.10). Preliminary research conducted with N. brasiliensis revealed potential efficacy of 40

58 PH extracts to decrease activity of adult parasites, but further in depth studies need to be conducted for confirmation of results. Overall, PH and GP extracts have a potential to help decrease parasite burden in ruminants. Introduction The first study was conducted to examine the effects of GP and PH varieties on DM degradation in vitro to determine if there would be a significantly negative effect of those substrates on DM degradation kinetics. This second study was designed to examine the effects of GP and PH extracts on ruminant gastrointestinal parasites to determine if the extracts would have an effect on reducing viability of harmful gastrointestinal parasites in small ruminants. Trichostrongyle nematodes, which are helminth parasites that affect the gastrointestinal tract, are one of the major causes of production losses in pasture raised ruminants (Hoste et al., 2006). This is especially the case in small ruminants because there are very limited number of drugs acceptable for use in these animals, and a high proportion of the helminth parasites are becoming resistant to current drugs used (Fleming et al., 2006; Hoste et al., 2006). There has been an increase in the desire to utilize more natural ways to expel the parasites because of increasing cost of use for chemical anthelmintics, growing concerns with chemical residues left in animal products from ruminant animals, and increasing resistance to chemical therapies. Using natural products in agriculture has been around for centuries on other continents, such as Asia and Africa (Githiori et al., 2006). Most of the active compounds in plant natural products 41

59 are from secondary metabolites which the plant uses as a defense mechanism against disease and injury (Mueller-Harvey and McAllen, 1992; Githiori et al., 2006). There are several of these secondary plant metabolites and other compounds, such as alkaloids, flavonoids, and condensed tannins, that have shown efficacy against helminth parasites (Athanasiadou et al., 2001; Min and Hart, 2003; Kerboeuf et al., 2008). These compounds of interest also have been found in PH and GP (Lloyd, 1897; Lu and Foo, 1999; Li et al., 2006). Amin et al. (2009) showed that pomegranate leaf water extract had effectiveness on decreasing the motility of the adult worms by 30, 52, and 86% at concentrations 25, 50, and 100 mg/ml, respectively. This study showed promise that pomegranate parts could potentially be used to help minimize worm burden in the gastrointestinal tract, but there has been little to no work done to investigate the GP anthelmintic effect or other extensive studies done to further examine PH as a potential natural anthelmintic. This research addresses this gap in knowledge by investigating the effects of the extracts of both PH and GP extracts on exsheathed L 3 larvae to understand how the extracts affect the larvae once they have entered the host animal. We also investigated the effects of these extracts on some adult helminth parasites. The main hypothesis is that the extracts from the PH and GP decrease larval viability, while increasing death in both stages when compared to the negative control of no treatment. Materials and Methods Extraction of plant material Two varieties each of PH and GP were used. Wonderful and Sogidana varieties were used for PH (USDA-ARS University of California, Davis). Cabernet Sauvignon and 42

60 Shiraz wine grape varieties were used for GP (Debonne Vineyard, Madison, Ohio). The varieties of pomegranate were chosen due to commercial availability of Wonderful cultivars and previous anthelmintic activity of Sogidana cultivar based on preliminary data collected. The grape varieties were chosen due to seasonal limitations. Each of the plant samples were dried at 55 C and ground to pass through a 1-mm sieve. Twenty five grams of each sample was extracted over 24 h with 100 ml of 70% aqueous ethanol with 1 g/l ascorbic acid to enhance extractability and decrease oxidation of sensitive compounds (Hagerman et al., 2000). Excess dried samples were kept in a desiccator at room temperature until needed and extraction stock solutions were kept at 2 to 4 C until needed. Aliquots (1 ml) of each extract were dried using a micro-rotary evaporator. Residues were weighed to determine extract concentrations of stock solutions and dried extracts were reconstituted in a phosphate buffered saline solution (PBS, 0.05 M NaCl, 5% dimethylsulfoxide, and 1 g/l ascorbic acid). Pomegranate husk extraction samples were diluted to concentrations of 6.25, 12.5, 25, or 50 mg/ml; in contrast, GP extraction samples were diluted to 6.25 or 12.5 mg/ml due to decreased extractability and solubility to achieve higher concentrations. The stock solutions of the PH extracts were 60 to 65 mg crude extract/ml, whereas the GP extracts were only extractable at concentrations ranging between 18 to 25 mg crude extract/ml. Larval Assay Third stage O. ostetagia larvae were collected from fresh fecal culture and double baermannized to get a clean, viable stock culture. Larvae were exsheathed using a modified procedure from von Samson-Himmelstjerna et al. (1998). Larvae were placed in 43

61 0.2% sodium hypochlorite in saline for 15 min at 37 C. The larvae were then washed three times in warm saline (37 C), and the exsheathment process was verified microscopically. Approximately 100 larvae per well were added in a 96-well plate containing various concentrations of plant extracts. The treatment structure was a completely randomized block design with a 10x4 factorial arrangement of treatments by variety concentration and time plus a control. The replication of plate was considered as a block. Plates were incubated at 39 C for 0, 2, 4, and 24 h. Larvae were visually assessed using an inverted microscope at a given time interval to determine the number of dead (no activity; larvae completely straight), sick (minimal to no activity; larvae slightly curled or noticeable slow muscle movement), or alive (very active movement or tightly curled). Dead and sick larvae were categorized as inactive, but alive larvae were categorized as active. The larval assay was replicated five times (i.e. five blocks) using five wells per concentration per extract variety. Adult helminth assay (preliminary trial) Adult N. brasiliensis worms were collected from mice per the protocol described in Camberis et al. (2003). The selection of this species of adult parasite used was due to the perceived similarity to ruminant Trichostrongyle nematodes (Githori et al., 2006) and the ease of rapid propagation. Cultivated adult worms were kept at 37 C in RPMI-1640 media (Sigma-Aldrich Co., St. Louis, MO) containing 1% antibiotic streptomycin/penicillin at a ph = 7.2. For the assay, only PH extracts were utilized due to limited supply of GP extract. The treatment structure was a completely randomized design with a 2x2 factorial arrangement of treatments for PH extract and concentration 44

62 plus a control. Both varieties of PH extracts were used at 15 and 30 mg/ml after 50% dilution of concentrated extracts with RPMI antibiotic media solution in 24-well plates with the control being just RPMI-1640+antibiotic media solution. Approximately 100 adult worms were incubated in extract overnight at 37 C in 5% CO 2 jacketed incubator, then observed and counted microscopically for motility, morbidity, and mortality. Plates were done in duplicate with four wells per concentration per variety. Statistical Analysis For the both larval and adult helminth assay, data from each variety at a given concentration were analyzed as individual treatments. Treatments from the larval assay were analyzed with repeated measures of time using PROC MIXED procedure and heterogeneous compound symmetry covariance structure in SAS 9.3 (SAS Inst., Inc. Cary, NC). The five experimental replicates/blocks for the larval assay were analyzed as a random effect, while treatment and time were analyzed as fixed effects. The PROC MIXED procedure of SAS was used without repeated measure for the adult helminth assay because there was only one time point used (~24 h). For the adult helminth preliminary trial, the data were averaged from the quadruplicate measurements per concentration. The two replications of the adult helminth assay were analyzed as a random effect, while treatment was analyzed as a fixed effect. Treatment differences for both larval and adult helminth assays were compared using Fisher s LSD by analyzing data with LSMEANS using the DIFF option in SAS. Data were reported significant at P < 0.05 with tendencies reported P 0.10 and P > 0.10 was considered non-significant. 45

63 Results and Discussion Treatment, time, and the interaction were significant (P < 0.01) in the larval assay, so the data were analyzed for differences by treatment within a time point (Table 7). At the beginning of the assay, both pomegranate varieties were different (P < 0.05) from the wine grape pomace except for the lowest concentration of Shiraz. Both the Wonderful and Sogidana varieties started with lower activity than GP varieties or the control which may suggest there is an initial paralysis or death that occurs within minutes of contact with the extracts. By 2 h, this difference between extracts became more obvious with the Shiraz GP having the highest activity and both varieties of PH at 50 mg/ml having the lowest activity (P < 0.05). The GP treatments, regardless of concentration, were not different (P > 0.10) from each other. The PH treatments for both varieties at 12.5 mg/ml were not different (P > 0.10) from the Cabernet Sauvignon GP at concentrations of 6.25 and 12.5 mg/ml. All treatments were lower (P < 0.01) in activity than the control at 2 h. At 4 h, the Shiraz GP continued to have the highest activity (P < 0.05) among the extracts at 38%, and there was no difference observed between concentrations; whereas, the Cabernet Sauvignon had a lower activity (P < 0.05) and was the most effective among the GP varieties, regardless of concentration. There was no difference (P > 0.10) observed between the Cabernet Sauvignon GP and the PH varieties at 12.5 and 25 mg/ml. The PH varieties at 50 mg/ml had the lowest activity (P < 0.05) and best efficacy, but there was no difference (P > 0.10) between PH varieties. All extracts continued to be different (P < 0.01) from the control, which only decreased in activity 16.6% within 24 h. There was no difference (P > 0.10) in activity of extracts 46

64 between 4 and 24 h but there was a tendency for difference (P = 0.08), which was mostly observed in the Cabernet Sauvignon GP, Sogidana PH, and control. There being no difference in 4 or 24 h was indicative that whatever efficacy was going to be observed, it would take place within 4 h of exposure to the extracts. The interaction of treatment by time could be explained that there was greater efficacy of decreasing larvae viability or health with longer exposure (up to 4 h) of the larvae to the extract; however, there was a slight increase in activity by 24 h, which raised the question if there was some resistance occurring to the extracts or what was the actual mechanism to which the extracts were causing decreased activity. Many of the compounds thought to be in the crude extract of both GP and PH (i.e. flavonoids, flavonols, pelletierine, and CT) has been thought to cause paralysis to helminth parasites initially followed by death, if death occurs (Wibaut and Holstein, 1957; Athanasiadou et al., 2001; Aas, 2003; Min and Hart, 2003; Nguyen et al., 2005; Kerboeuf et al., 2008). Table 8 shows the preliminary results from the adult helminth assay conducted on N. brasiliensis. PH decreased activity (P < 0.01) of the adults when compared to the control but there were no differences (P > 0.10) for concentration or variety. But once again, it is not clear whether this was just due to paralysis and effects would be reversed once helminths were no longer in the presence of the extracts or if this decreased activity was due to death of the parasite. The main obstacle to this assay was the viability of the helminths decreased, even in the control well, but still not as drastically as the treatment wells. This was thought to be due to adult helminth parasites being more sensitive to in vitro conditions and not as suitable for studies outside the host. In addition to this, N. 47

65 brasiliensis has a corkscrew or tightly coiled morphology and they like to adhere to each other, so dividing them individually into wells was very labor intensive and caused some of the adults to be lost due to physical damage and were removed from the treatment wells. The larval studies were a good indication that the extracts of both PH and GP could have potential use in decreasing activity of helminth larvae. Both of the PH extracts resulted in the best efficacy at 50 mg/ml. If GP can be extracted to a similar concentration, it is possible for there to be greater efficacy than the pomegranate when such low concentrations still have an effect. For the adult helminth parasite assays, there still needs to be more experimentation done to improve the assays and the parameters need to be further worked out to improve viability of the adults for N. brasiliensis. If the both PH and GP extracts are able to decrease larval viability and PH is able to decrease adult activity, then it may be possible to decrease transmission of the infestation by potentially decreasing infective larval stages and also decreasing infestation by allowing paralyzed or dead adults and larvae to be passed out of the host, but this must be tested in further experimentation. Overall, the PH and GP extracts show promise in helping to reduce helminth infestation for larvae and PH in reducing viability of adult helminths, but more studies need to be conducted for adult parasites. The trial results proved that our hypothesis that the PH and GP extracts would decrease viability and increase death in larval helminth stages when compared to the negative control of no treatment. References Aas, E. A practitioner s perspectives: Traditional tannin-treatment against intestinal parasites in sheep and cattle. Ethnobotany Res. and Apps. 2003, 1:

66 Amin, M. R., M. Mostofa, M. E. Hoque and M. A. Sayed. In vitro anthelmintic efficacy of some indigenous medicinal plants against gastrointestinal nematodes of cattle. J. Bangladesh Agril. Univ. 2009, 7, 1: Athanasiadou, S., I. Kyriazakis, F. Jackson, and R. L. Coop. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies. Vet. Parasitol. 2001, 99: Camberis, M., G. LeGros, and J. Urban Jr. Animal model of Nippostrongylus barasiliensis and Heligomosomoides polygyrus. Current Protocols in Immunology. 2003, Fleming, S. A., T. Craig, R. M. Kaplan, J. E. Miller, C. Navarre, and M. Rings. Anthelmintic resistance of gastrointestinal parasites in small ruminants. J. Vet. Intern. Med. 2006, 20: Gil, M. I., F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft, and A. A. Kader. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agric. Food Chem. 2000, 48: Githiori, J. B., S. Athanasiadou, and S. M. Thamsborg. Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants. Vet. Parasitol. 2006, 139: Hagerman, A., I. Harvey-Mueller, and H. P. S. Makkar. Quantification of tannins in tree foliage Lab manual. FAO/IAEA. 2000, 2-4. Hoste, H., F. Jackson, S. Athanasiadou, S. M. Thamsborg, and S. O. Hoskin. The effects of tannin-rich plants on parasitic nematodes in ruminants. TRENDS in Parasitol. 2006, 22(6): Kerboeuf, D., M. Riou, and F. Guégnard. Flavonoids and related compounds in parasitic disease control. Mini-Reviews in Medicinal Chemistry. 2008, 8: Klimpel, S., F. Abdel-Ghaffar, K. A. S. Al-Rasheid, G. Aksu, K. Fischer, B. Strassen, and H. Mehlhorn. The effects of different plant extracts on nematodes. Parsitol. Res. 2011, 108: Li, Y., C. Guo, J. Yang, J. Wei, J. Xu, and S. Cheng. Evaluation of antioxidant properties of pomegranate husk extract in comparison with pomegranate pulp extract. Food Chem. 2006, 96: Lloyd, J. U. Punica granatum. The Western Druggist. 1897,

67 Lu, Y and L. Yeap Foo. The polyphenol constituents of grape pomace. Food Chem. 1999, 65: 1-8. Min, B. R. and S. P. Hart. Tannins for suppression of internal parasites. J. Anim. Sci. 2003, 81 (E. Suppl. 2): E102-E109. Mueller-Harvey, I and A. B. McAllen. Tannins: their biochemistry and nutritional properties. Adv. Plant Cell Biochem. 1992, 1: Nguyen, T. M., D. V. Binh, and E. R. Orskov. Effects of foliages containing condensed tannins and on gastrointestinal parasites. Anim. Feed Sci. Tech. 2005, 121: SAS/STAT Software User s Guide: Statistics, Version 9.3 Edition SAS Inst., Inc. Cary, NC Wibaut, J. P and U. Holstein. Investigation of the alkaloids of Punica granatum L. Arch. Biochem. Biophys. 1957, 69: von Samson-Himmelstjerna, G., S. Rickling, and T. Schnieder. Improved in vitro cultivation of larvae of bovine lungworm Dictycaulus viviparous. Parasitol. Res. 1998, 84:

68 Table and Figures Table 7. Effect of grape pomace and pomegranate husk extracts on activity (% active) of L 3 O. ostetagia Extract 1 Active (%) (mg/ml) 0 h 2 h 4 h 24 h Control, PBS (+) ± 0.02 a,b,c 65.9 ± 0.04 a 64.7 ± 0.03 a 57.7 ± 0.04 a Shiraz ± 0.02 a 54.8 ± 0.04 b 38.2 ± 0.03 b 35.3 ± 0.04 b ± 0.02 b 56.5 ± 0.04 b 38.5 ± 0.03 b 34.5 ± 0.04 b Cabernet Sauvignon ± 0.02 b 46.7 ± 0.04 b,c 27.5 ± 0.03 a 37.3 ± 0.04 b ± 0.02 a,b 45.9 ± 0.04 b 25.2 ± 0.03 a 35.4 ± 0.04 b Wonderful ± 0.02 a,c 42.1 ± 0.04 c,d 27.8 ± 0.03 c 29.9 ± 0.04 b ± 0.02 a,c 32.6 ± 0.04 d 23.0 ± 0.03 c 22.2 ± 0.04 c ± 0.02 a,c 29.7 ± 0.04 e 16.8 ± 0.03 d 18.0 ± 0.04 c Sogidana ± 0.02 a 40.6 ± 0.04 c,e 27.8 ± 0.03 c 36.8 ± 0.04 b ± 0.02 a,c 36.0 ± 0.04 c,e 20.9 ± 0.03 c,d 27.2 ± 0.04 b ± 0.02 d 29.9 ± 0.04 e 17.3 ± 0.03 d 21.6 ± 0.04 c 1 Shiraz and Cabernet Sauvignon Grape pomace extracts, Wonderful and Sogidana Pomegranate husk extracts 2 PBS(+) Phosphate Buffered Saline, 0.05 M NaCl, and 5% DMSO to increase solubility of dried extracts abcde Means within a column with unlike superscripts are different (P<0.05) Table 8. Effects of pomegranate husk extracts on activity of adult N. brasiliensis Extract (mg/ml) Activity 1 SEM Control a 4.0 Wonderful b b 4.0 Sogidana b b Activity reported as number of active adults counted out of approximately Control consisted of RPMI-1640 with 1%/mL penicillin/streptomycin at ph=7.2 a,b Means within a column with unlike superscripts are different (P<0.05) 51

69 Chapter 4: Effects of wine grape pomace on gastrointestinal parasitism in naturally infested grazing lambs Abstract Resistance to commercial chemical anthelmintics among gastrointestinal parasites is increasing worldwide with small ruminant animals, such as goats and sheep, becoming the most susceptible. There have been several plant secondary metabolites that have shown efficacy against gastrointestinal parasites (GIP), including but not limited to condensed tannins (CT), flavonoids, and certain nitrogen containing compounds. This study was to determine the effects of the CT in wine grape pomace (GP) as a potentially natural anthelmintic against GIP in growing lambs. A special GP diet was formulated to deliver 28 g CT/kg DM in a 0.9-kg dose or 45 g CT/kg DM in a 1.5-kg dose for 5 d. Two lamb trials were conducted over three weeks using 54 growing lambs in each trial. In the first trial, the effects of the 0.9 kg/day GP for 5 d was examined compared to a positive commercial anthelmintic control (Cydectin) and a negative control. The second trial examined the GP diet in a dose titration using a negative (no CT) control, a 0.9-kg dose supplemented with 0.57 kg alfalfa pellet, and a 1.5-kg dose. The lambs were assessed for BW and average daily gain (ADG), packed cell volume (PCV), FAMACHA, and fecal egg count (FEC). In trial 1, BW increased (P < 0.01) as the weeks progressed; the 52

70 positive control had higher PCV levels and lower FEC than the other treatments. There were no differences for FAMACHA or ADG among treatments. In the second trial, BW and PCV increased (P < 0.05) with week, but no differences were observed for treatments on any of the variables. Variation within treatments was high and thus more replication would be needed to observe beneficial significant effects of treatment. There needs to be more animal trial research done before conclusions can be made about whether or not GP should be incorporated in diets of growing lambs to potentially help with decreasing gastrointestinal parasitism. Introduction The first experimentation in this research of exploring the effects on GP on DM degradation in vitro showed that including alfalfa with GP would decrease the negative associative effects of GP in a diet. Our second experiment examined the effects of GP extracts on the GIP larvae and resulted in up to a 50% decrease in viability of O. ostertagia larvae in vitro. This led to the current experiment of examining the effects of CT in GP as a potential way to treat GIP in lambs. The results from the previous experiments led us to believe that inclusion of GP in a growing lamb diet, in which the lambs were naturally infested with parasites, would help decrease the parasite load in the animals. Condensed tannins and other secondary metabolites in plant sources have recently been investigated for their potential use as natural anthelmintics to agricultural animals (Athanasiadou et al., 2001; Aas, 2003; Min and Hart, 2003; Nguyen et al., 2005; Kerboeuf et al., 2008). Small ruminants, such as sheep and goats, are having issues with 53

71 GIP becoming more resistant to current commercial anthelmintics being administered (Fleming et al., 2006; Hoste et al., 2006). In some regions, parasites in these animals are becoming multidrug resistant (Aas, 2003; Min and Hart, 2003; Fleming et al., 2006). There is now a growing interest, especially among organic farmers, to use natural means to help with the parasitism, including utilizing integrated pest management practices (Aas, 2003; Min and Hart, 2003).Wine GP was used in the current study due to it being readily available in Ohio and it being a cheap source of CT that may have efficacy as a natural anthelmintic. Grape pomace is a by-product of the juicing and wine industry that consists of mainly seeds, skin, and pulp (~18 to 20 kg/100 kg of grapes). Traditionally, uses of GP have been largely restricted to land applications, such as an organic way to add nutrients back into the soil or as a heavy metal absorbent (Arvanitoyannis et al., 2006; Spanghero et al., 2009). Grape pomace has not had a lot of consideration as an animal feed for ruminants due to some varieties having low nutritional value. There have been several studies, however, that has looked at GP as a feed additive and has found benefits to adding the pomace to growing lamb diets, such as helping increase weight gain and feed efficiency without negative effects (Famuyiwa and Ough, 1982; Bahrami et al., 2010). There has been little to no research done that examines GP as a natural anthelmintic for small ruminants, even though it could potentially be a renewable, cheap source of CT. The objective of this trial was to determine the effects of GP to reduce the parasite load of grazing lambs in vivo and to determine if the efficacy of GP is comparable to commercial anthelmintics. For Trial 1, we hypothesized that GP would greatly reduce the 54

72 viability of intestinal parasites against a control (no treatment; negative control) and would have similar effectiveness as a commercial anthelmintic (positive control). For Trial 2, we hypothesized that as CT concentration increased in the diet, parasitism in the lambs would decrease by decreased FEC, increased PCV, FAMACHA, and weight gains. Material and Methods Animal Diet The GP used in this study had an average of 4.1% CT on a DM basis and was a combination of 1/3 Shiraz and 2/3 Cabernet Sauvignon varieties of grapes. A specialized feed was made from the GP mix and pelleted as 2/3 GP variety mix and 1/3 of an alfalfa/corn mixture. The ingredients were mixed according to the specifications in Table 6 and fed to animals as a pelleted feed. The lambs in the GP treatment group were fed for a 5-d dose that varied in CT concentration depending on the trial. After the GP dosing, the lambs were fed an alfalfa trainer pellet for the remaining 16 d. All other treatment groups were fed the alfalfa pellet for the entire 21 d trial period. The animals were fed approximately 4% BW once daily at 0800 h every morning, except for sampling days in which lambs were fed at 0900 h. Refusals were collected, weighed, and recorded every morning prior to feeding. Samples of the diets were sent to Cumberland Valley Analytical Services (14515 Industry Drive, Hagerstown, MD 21742) for nutritional analysis and the procedure references used are provided in Appendix A. 55

73 Animal Trials Trial 1. Fifty-four Dorset cross lambs with known natural GIP infestation were randomly allotted to treatment groups and blocked by FEC using a randomized block design. The treatment groups included a negative control with untreated animals, a positive control in which animals were treated with 1 ml/5 kg BW of Cydectin (Boehringer Ingelhiem Vetmedica, Inc., St. Joseph, MO) and an experimental treatment in which the animals were given 0.9 kg of the specialized GP diet containing 28 g CT/kg DM. The FEC blocking was broken into low (< 2100 eggs/g, EPG), medium (2100 to 4200 EPG), or high (> 4200 EPG). Lambs were housed three per pen, with 18 total pens and six pens per treatment. Lambs were housed in pens with slotted floors to prevent build up of urine or fecal matter in pens. The study was conducted over 21 d and data collected weekly for FEC, PCV, BW gain, and mucous membrane eye scores (FAMACHA ; Kaplan et al., 2004). The FAMACHA eye scoring was done by the same trained individual each week and example of the eye scoring chart is provided in Appendix B. Fecal samples were collected directly from the anal cavity of lambs for FEC. FEC was done using a modified McMaster s technique utilizing Sheather s sugar floatation media (JorVet, Loveland, CO). Blood sampling was done by collecting samples from the jugular vein into evacuated 5 ml blood tubes that contained ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. PCV was done according to Bull et al. (2000) and is reported as the volume percentage of red blood cells in blood. All sampling was done before morning feeding. Lambs were fed either 0.9 kg of alfalfa pellet 56

74 or GP pellet for the first 5 d, and then all lambs were given the alfalfa pellet for the duration of the 21 d study. Trial 2. Fifty-four naturally infested Dorset cross lambs were randomly allotted to treatment groups and also blocked by FEC. This trial was a dose titration study examining the efficacy of the GP feed. Treatment groups consisted of an untreated control group, a low dose group (28 g CT/kg DM), and a high dose group (45 g CT/kg DM). The FEC blocking was allotted as previously stated in low, medium, and high. Lambs were placed in pens as stated previously in Trial 1 of three pen and six pens per treatment. The study was conducted over 21 d and similar data as for Trial 1 were collected weekly for Trial 2. Lambs were fed 1.5 kg of only the alfalfa pellet diet for the untreated group, 0.9 kg of GP pellet plus 0.57 kg alfalfa pellet for low dose group (GPD1), and 1.5 kg GP for high dose group (GPD2) for 5 d and then given just the standard alfalfa pellet for the duration of the 21 d study. Statistical Analysis Data were analyzed as a randomized complete block design, with the use of repeated measures using heterogeneous compound symmetry as the covariate matrix for ADG, FI, FEC, PCV, FAMACHA and BW. Pen was the experimental unit and individual lamb data were averaged per pen. There were three blocks since the pens were blocked by low, medium or high FEC. Pen and treatment were analyzed as fixed effects with block analyzed as a random effect. Covariate analysis was done for FEC in trial 2 using d 0 measurements to account for variability. FEC were analyzed as the log transformation of egg counts to normalize the FEC data. The data were analyzed using 57

75 PROC MIXED in SAS 9.3 (SAS Inst., Inc. Cary, NC). Mean separation test was done using Fisher s LSD by analyzing with LSMEANS using DIFF option in SAS. Data was analyzed for significance of treatment, day, or the interaction of treatment x day. Statistical significance was reported if P<0.05 with trends being reported if 0.05< P Results and Discussion Animal Diet The ingredient and nutritional composition for the feeds used in both trials are provided in Table 9. The composition of the feeds was different and not nutritionally the same, but the GP diet was only used for 5 d to provide the amount of CT desired. It was not intended to nutritionally replace the normal alfalfa ration that the lambs were fed for the remainder of the study. Trial 1 BW increased (P < 0.01) as weeks progressed, which was expected for growing lambs (Table 10). The average weight was 26±0.5 kg among treatments. There was no significant difference (P > 0.10) for treatments or the interaction of treatment x day. There was a significant (P < 0.01) interaction for treatment x day for ADG (Table 10). As the weeks progressed, the treatments varied in ADG with the Cydectin treated lambs having the highest (P < 0.05) ADG by the third week. For the first week, the negative control had the highest (P < 0.05) ADG, but there was no difference (P > 0.10) between the GP and Cydectin treated lambs. In week 2, the GP treated lambs had the highest (P < 0.05) ADG, but there were no differences (P > 0.10) between the negative 58

76 control and Cydectin treatment groups. Overall, the accumulative ADG was not different (P > 0.10) between any of the treatment groups. FI increased (P < 0.05) as the weeks progressed but was not different (P > 0.10) between treatments or for the interaction of treatment x week (Table 10). The increase in FI corresponded with the increase in BW gain weekly and averaged 1.12±0.01 kg/head/d over the entire trial period. There was a positive (P < 0.01) interaction of treatment x day for PCV (Table 10). There was no difference (P > 0.10) observed between treatments until the second week in which PCV was higher (P < 0.05) for the positive control (Cydectin) than for lambs on the negative control and GP diets. The positive control had an average PCV of 33.2±1.9%, whereas the negative control and GP treatments had 30.0±1.8 and 30.1±1.9%, respectively. The Cydectin treatment group continued to have the highest (P < 0.05) PCV for the duration of the trial with the GP treatment group having the lowest (P < 0.05) PCV. The Cydectin treatment group tended to have a higher (P = 0.09) average PCV than GP but neither were different (P > 0.10) from the negative control. The PCV being above 25% for all treatments correlated with FAMACHA eye scores being generally low around 2, with no difference between treatments or the interaction of treatment x day (Table 10). FAMACHA is a very quick and useful way for farmers to use to help in integrated farm management practices and was developed as a way to clinically identify anemia status in sheep and goats (Kaplan et al., 2004). The results observed in this study helped to further validate the usefulness of using this method on farm; however, it is not for use in determining parasite load as it does not 59

77 correspond with the number of FEC but only the anemia status of the animal and how the parasite is affecting the animal s health. FEC was significantly affected (P < 0.01) by the interaction of treatment x time (Table 10). All treatments started at approximately the same FEC but significantly differed (P < 0.01) from each other by the end of the first week. The positive control (Cydectin) continued to have the lowest (P < 0.01) FEC for the duration of the trial with there being no difference (P > 0.10) between the negative control and the GP treatment. The positive control treatment was expected to have a low FEC, but what was unexpected was for there to be no difference between the GP treatment and the negative control. This rejected our hypothesis that the GP treatment would be comparable to the positive control. There were several issues that arose from the results of this trial. First, the Cydectin used for the positive control never decreased the EPG to zero or even below 500 EPG over the 3-wk period. This validated suspicions with on farm management that they were experiencing increased resistance to Cydectin. The second issue relates to the nutritional differences between the GP diet and normal alfalfa ration. The lambs receiving only 0.9-kg of the GP treatment may have been limited in nutrition too much in the first 5 d to observe a desired decreased response to CT in the diet. Most of the feeding trials that fed optimum CT to lambs observed a decrease in parasitism only when the protein and other nutritional components were administered in adequate and equal concentrations in the diet (Bahrami et al., 2010; Min et al., 2012). 60

78 The observation that the current GP dose of 0.9 kg (25 g CT/kg DM) was not comparable to Cydectin and may not be a high enough dose to observe a treatment response against GIP led to the development of the proceeding Trial 2. We decided that CT in the diet should be tested as dose titration response and that lower dose (0.9 kg, 25g CT/kg DM) should be supplemented with the trainer alfalfa pellet so that all lambs received equal amounts of feed (4% BW) but still allowing for the determination of the proper dose for CT addition to the diet. This would hopefully result in a significant effect on decreasing parasitism in the lambs. Trial 2 BW increased (P < 0.01) as the weeks progressed (Table 11), but there was no difference (P > 0.10) between treatments or for the interaction of treatment x day, which was the same as observed in Trial 1. The average BW was 33.0±0.9, 32.5±0.8, and 33.6±0.8 kg for the control, GPD1, and GPD2, respectively. ADG varied significantly (P < 0.05) for the treatments as the weeks progressed (Table 11). The variation was more (P < 0.05) between treatments for week 1 and 2 but the accumulative ADG was not different (P > 0.10) between treatments. In the first week, GPD1 had the highest (P < 0.05) ADG at 202±42 g/d when compared to GPD2 at 79.3±42 g/d but was not different (P > 0.10) from the negative control. By the second week, both GP diets had the lowest (P < 0.05) ADG than the control and the control tended (P = 0.08) to be different than the GP diets at week 3. The biggest differences observed during the first and second week could be expected when the negative control diet was more nutritious when given during the first week and could have given the 61

79 control group lambs more of a growing start than the GP diet groups, but all lambs were given the same diet at the end of week 1 until the end of the trial so it makes sense why the lambs eventually caught up with each other on gains. FI followed a similar pattern as BW and increased (P < 0.01) as the weeks progressed but was not significantly different (P > 0.10) between treatments or the interaction of treatment x day (Table 11). The lambs averaged a FI of 1.4±0.01 kg/head/d among treatment groups. PCV increased (P < 0.01) as the days progressed, but there was no difference (P > 0.10) between treatments or the interaction of treatment x day (Table 11). The average PCV for all treatments was approximately 28±1.3%. In general, there was an increase of PCV in all the treatments with time, but lambs fed GPD2 had the largest increase in PCV from 26.3±1.50% in wk 1 to 32.2±1.56% in wk 3. This was a noticeable difference than from Trial 1, where the PCV appeared to be decreasing as weeks progressed. This decrease was not significant in Trial 1 due to the high variability observed because of the overall variable health of the animals in that trial. The lambs in Trial 1 were younger, smaller in weight, and not as healthy as the lambs in Trial 2, which were older, bigger, and had better weight gain. No lambs were lost or needed to be rescued in Trial 2, unlike in Trial 1 there were at least three lambs that required rescue for respiratory complications not related to the trial and one lamb that needed rescue due to the effects of parasitism. This was accounted for in trial 1 because pen was the experimental unit not individual lambs and lambs were averaged per pen. 62

80 FAMACHA improved (P < 0.01) through the course of the trial period, but there was no differences (P > 0.10) between treatments or the interaction of treatment x day. The average FAMACHA score was low and ranged from 1.4 to 1.7 among the treatments (Table 11). These low eye scores corresponded to the high PCV values seen in the lambs, and the increase in scores over time that was also observed with increase in PCV. The negative control had the lowest (P < 0.01) FEC when compared to both GP treatments. There was no differences (P > 0.10) in day or the interaction of treatment x day. There was also no differences (P > 0.10) observed for FEC between the GP treatments. It was expected that there would be significance observed among GP treatments, but it is unclear as to why there was no differences seen between the GP treatments. There is the possibility that more experimental units were probably needed to observe the expected results or more control of the parasite infestation by experimentally infecting the lambs instead of using naturally infested lambs. Min et al. (2012) fed CT from pine bark in a dose titration study and their highest concentration was at 32 g CT/kg DM, which is a concentration between our GP treatment CT concentration, and their study results showed that final BW, ADG, and FI increased (P < 0.05) linearly with increase of CT in the diet. The scientist also observed a linear reduction (P < 0.01) in FEC with increased CT in the diet. We did not observe this result in our study but they fed their treatment diets for 83 d instead of just 5 d. In a private communication with one of the authors, it was suggested that the length of time for a natural anthelmintic such as CT would take greater than 40 d to see any results pertaining to FEC reductions 63

81 (Solaiman, 2014). This longer feeding period may be used for consideration in a future trial. Also, all of their diets were isocaloric and isonitrogenous so it may be beneficial to have a better GP diet formulation so that the GP diet can be fed for longer with comparable results. Not observing significance among treatments in trial 2 rejected our hypothesis that with increasing CT in the diet, there would be a decrease in parameters/variables associated with assessing parasitism in the lambs. Even though the concentration of CT increased with increasing GP in the diet, but the best application for GP inclusion in the diet may be to keep protein concentrations at or above 0.23 kg/head with optimum levels of CT (32 g CT/ kg DM) to achieve beneficial antiparasitic effects. There may be potential for GP to be incorporated in diets of growing lambs to help with decreasing parasitism, but that was not observed from these trials. There needs to be more research conducted and animal trials performed before actual on farm application can come to fruition. References Aas, E. A practitioner s perspectives: Traditional tannin-treatment against intestinal parasites in sheep and cattle. Ethnobotany Res. and Apps. 2003, 1: Arvanitoyannis, I. S., D. Ladas, and A. Mavromatis. Potential uses and applications of treated wine waste: A review. Int. J. Food Sci. Tech. 2006, 41: Ash of Animal Feed (942.05). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. Athanasiadou, S., I. Kyriazakis, F. Jackson, and R. L. Coop. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies. Vet. Parasitol. 2001, 99: Bahrami, Y., A.-D. Foroozandeh, F. Zamani, M. Modarresi, S. Eghbal-Saeid and S. Chekani-Azar. Effect of diet with varying levels of dried grape pomace on dry matter 64

82 digestibility and growth performance of male lambs. J. Ani. Plant Sci. 2010, 6(1): Bull, B. S., J. A. Koepke, E. Simson, and O. W. van Assendelft. Procedure for determining packed cell volume by microhematocrit method; Approved standard-3 rd ed. Clinical and Laboratory Standards Institute. 2000, 20 (18): AOAC Official methods of analysis of the AOAC. 17th ed., Association of Official Analytical Chemists, Eden Prairie, MN. AOAC Official methods of analysis of the AOAC. 18th ed., Association of Official Analytical Chemists, Eden Prairie, MN. Famuyiwa, O. and C. S. Ough. Grape pomace: possibilities as animal feed. Am. J. Enol. Vitic.1982, 33(1): Fleming, S. A., T. Craig, R. M. Kaplan, J. E. Miller, C. Navarre, and M. Rings. Anthelmintic resistance of gastrointestinal parasites in small ruminants. J. Vet. Intern. Med. 2006, 20: Goering, H.K. and P.J. Van Soest Forage Fiber Analysis. USDA Agricultural Research Service. Handbook number 379. U.S. Dept. of Agriculture. Superintendent of Documents, US Government Printing Office, Washington, D.C Hoste, H., F. Jackson, S. Athanasiadou, S. M. Thamsborg, and S. O. Hoskin. The effects of tannin-rich plants on parasitic nematodes in ruminants. TRENDS in Parasitol 2006, 22(6): Kaplan, R. M., J. M. Burke, T. H. Terrill, J. E. Miller, W. R. Getz, S. Mobini, E. Valencia, M. J. Williams, L. H. Williamson, M. Larsen, and A. F. Vatta. Validation of the FAMACHA eye color chart for detecting clinical anemia in sheep and goats on farms in the southern United States. Vet. Parasitol. 2004, 123: Kerboeuf, D., M. Riou, and F. Guégnard. Flavonoids and related compounds in parasitic disease control. Mini-Reviews in Medicinal Chemistry. 2008, 8: Min, B. R. and S. P. Hart. Tannins for suppression of internal parasites. J. Anim. Sci. 2003, 81 (E. Suppl. 2): E102-E109. Min, B. R., S. Solaiman, N. Gurung, J. Behrends, J.-S. Eun, E. Taha, and J. Rose. Effects of pine bark supplementation on performance, rumen fermentation, and carcass characteristics of Kiko crossbred male goats. J. Anim. Sci. 2012, 90:

83 Nguyen, T. M., D. V. Binh, and E. R. Orskov. Effects of foliages containing condensed tannins and on gastrointestinal parasites. Anim. Feed Sci. Tech. 2005, 121: SAS/STAT Software User s Guide: Statistics, Version 9.3 Edition SAS Inst., Inc. Cary, NC Solaiman, S. Tuskegee University, Tuskegee, AL. Personal communication with S. LeShure, Department of Animal Sciences, The Ohio State University, March 24, Spanghero, M., A. Z. M. Salem, and P. H. Robinson. Chemical composition, including secondary metabolites, and rumen fermentability of seeds and pulp of Californian (USA) and Italian grape pomace. Ani. Feed Sci. Tech. 2009, 152: Van Soest, P. J. van, J. B. Robertson, and B. A. Lewis. Methods for dietary fiber, neutral detergent fiber and nonstructural polysaccharides in relation to animal nutrition. J Dairy Sci 1991, 74(10):

84 Table and Figures Table 9. Composition (DM basis) of diets in Trial 1 and Trial 2 fed to lambs as a control or experimental diet Item Alfalfa pellet GP pellet Ingredient % of DM % of DM Alfalfa Grape pomace Ground Corn Animal Fat Ammonium chloride Trace Mineral Salt Monosodium phosphate Selenium, 201 mg Se/kg Vitamin A, 30,000 IU/g Vitamin D 3, 3,000 IU/g Vitamin E, 44 IU/g Lasalocid Nutritional Composition NDF, % ADF, % CP, % EE 4, % Ash, % Na, % NE 5 m, Mcal/kg NE 6 g, Mcal/kg Grape pomace pellet consisted of two-thirds Cabernet Sauvignon wine GP and one-third Shiraz wine GP; Trial 1 28 g condensed tannin (CT)/kg DM given in a 0.9 kg/head dose, Trial 2 28 g CT/kg DM given in a 0.9 kg/head dose and 45 g CT/kg DM given in a 1.5 kg/head dose 2 Included: 95% NaCl; 0.35% Zn, as ZnO; 0.28% Mn, as MnO 2 ; 0.175% Fe, as FeCO 3 ; 0.040% Cu, as Cu 2 O; 0.007% I, as Ca 5 (IO 6 ) 2 ; and 0.007% Co, as CoCO 3 3 Fed as 25.5 mg lasalocid/kg of diet DM (Bovatec ; Alpharma Animal Health, Bridgewater, NJ) 4 EE = Ether extract 5 NE m (Net energy maintenance) = ((0.029 x TDN) ) x NE g (Net energy gain) = ((0.029 x TDN) ) x

85 Table 10. Body weight (BW), average daily gain (ADG), packed cell volume (PCV), FAMACHA, and fecal egg counts (FEC) by week and overall for lambs treated with a negative control, Cydectin or Grape pomace pelleted feed in Trial 1 Treatment CON CYD GP SEM BW (kg) D a 25.9 a 25.3 a 0.4 D a 25.3 a 24.8 a 0.3 D b 27.0 b 27.3 b 0.3 D c 28.6 c 27.9 c 0.3 Average 26.4 a 26.7 a 26.3 a 0.5 ADG (g/d) Wk a b b 25.1 Wk a 242 a b 24.5 Wk a,b 226 a 86.5 b 28.3 Accumulative 154 a 129 a 126 a 12.0 FI (kg/head/d) Wk a 0.91 a 0.89 a 0.01 Wk b 1.13 b 1.12 b 0.01 Wk c 1.32 c 1.32 c 0.02 Average 1.12 a 1.12 a 1.11 a 0.01 PCV D a 32.1 a 32.6 a 1.9 D a 34.3 a 31.8 a 1.8 D a 33.2 b 30.1 a 1.8 D a,b 32.5 a 28.1 b 1.8 Average 31.5 a 33.0 a 30.6 a 1.9 FAMACHA D a 2.17 a 1.98 a 0.13 D a 2.02 a 2.27 a 0.22 D a 1.97 a 2.28 a 0.14 D a 1.68 a 2.00 a 0.10 Average 1.91 a 1.96 a 2.13 a 0.21 FEC D a 3.46 a 3.46 a 0.18 D a 2.78 b 3.63 c 0.20 D a 2.78 b 3.70 a 0.19 D a 2.68 b 3.70 a 0.19 Average 3.55 a 2.92 b 3.62 a CYD = Cydectin (Boehringer Ingerlheim Vetmedica, Inc., St. Joseph, MO), positive control; administered at 1mL/5 kg BW, (n=17 lambs) 2 CON = Negative control, no treatment given (n=16 lambs) 3 GP = Grape pomace pellet (25 g CT/kg DM); administered for 5 d (n=17 lambs) 4 SEM = standard error of the mean; reported as weighted average for the day or week 5 FAMACHA is the anemia status of an animal done by checking mucous membrane of the eye 6 FEC reported in log transformation of counted eggs per gram abcde Means with unlike superscripts are different (P < 0.05) 68

86 Table 11. Body weight (BW), average daily gain (ADG), packed cell volume (PCV), FAMACHA, and fecal egg counts (FEC) by week and overall for lambs treated with a negative control, 0.9 kg grape pomace pellet (GPD1), or 1.5 kg grape pomace pellet (GPD2) diet in Trial 2 Treatment CON GPD1 GPD2 SEM BW (kg) D a 30.7 a 32.2 a 0.5 D b 31.8 b 32.7 b 0.4 D c 32.3 c 33.3 c 0.4 D d 35.2 d 36.1 d 0.5 Average 33.0 a 32.5 a 33.6 a 0.8 ADG (g/d) Wk a,b 202 a,b 79.3 a 25.4 Wk a 73.9 b 75.7 b 17.8 Wk a 420 a 405 a 16.3 Accumulative 217 a 232 a 187 a 19.0 FI (kg/head/d) Wk a 1.33 a 1.34 a 0.01 Wk b 1.40 b 1.42 b 0.01 Wk c 1.54 c 1.53 c 0.02 Average 1.46 a 1.42 a 1.44 a 0.01 PCV D a 26.1 a 26.3 a 1.3 D b 28.7 b 27.1 b 1.2 D b,c 30.3 b,c 29.6 b,c 1.2 D c 30.2 c 32.2 c 1.3 Average 28.4 a 28.8 a 28.8 a 1.3 FAMACHA D a 1.75 a 1.93 a 0.13 D b 1.50 b 1.73 b 0.12 D b,c 1.30 b,c 1.50 b,c 0.09 D c 1.12 c 1.47 c 0.10 Average 1.56 a 1.42 a 1.66 a 0.15 FEC D a 3.39 b 3.62 b 0.08 D a 3.67 b 3.69 b 0.03 D a 3.75 b 3.69 b 0.05 Average 2.73 a 3.60 b 3.67 b CON = Negative control, no treatment given (n=18 lambs) 2 GPD1 = Grape pomace diet given at 0.9 kg (25 g CT/kg DM) plus 0.6 kg alfalfa pellet; administered for 5 d (n=18 lambs) 3 GPD2 = Grape pomace pellet given at 1.5 kg (45 g CT/kg DM); administered for 5 d (n=18 lambs) 4 SEM = standard error of the mean; reported as weighted average for the day or week 5 FAMACHA is the anemia status of an animal done by checking mucous membrane of the eye 6 FEC reported in log transformation of counted eggs per gram abcde Means with unlike superscripts are different (P < 0.05) 69

87 Chapter 5: Effects of wine grape pomace on egg hatchability and larval development of helminth parasites: Organic versus conventional farming methods Abstract Gastrointestinal helminth parasite resistance in agricultural ruminants is becoming an increasing problem worldwide, especially for small ruminant species. There has been a need for farmers in both organic and conventional farming practices to find a way to control gastrointestinal parasites (GIP) through other alternatives to chemical anthelmintics. This research examined the use of wine grape pomace (GP) extract to decrease egg hatchability of these parasites into infective larvae. A fecal culture was done in the presence and absence of the extract on feces from sheep collected on both conventional and organic farms (n=5, 3 conventional and 2 organic farms). Approximately 300 g of feces was randomly collected off the ground from fresh excrement from each farm. The GP extract resulted in 100 % inhibition of egg hatching into third stage larvae (P < 0.01). These results indicate that wine GP has efficacy in decreasing hatchability of helminth eggs and could potentially be a natural way to decrease GIP in small ruminants, regardless of farming method. 70

88 Introduction Small ruminants, such as sheep and goats, are having issues globally with GIP becoming more resistant to current commercial anthelmintics being administered with some parasites becoming multidrug resistant (Min and Hart, 2003; Min and Hart, 2003; Fleming et al., 2006). The most relevant GIP to small ruminants are from the order Strongylida, superfamily Trichostrongyloidea (Balic et al., 2000; Zajac, 2006). The transmission of Trichostrongylide sp. parasites occurs through adult females in the abomasum or small intestine producing eggs that are passed out in the feces. Under proper conditions, the eggs hatch and go through several developmental stages until they reach a free-living third larval stage. The third stage larva (L 3 ) retains its outer coat as a protective cuticle and will migrate out of the fecal matter onto grass leaves. The larvae can move horizontally and vertically up blades of grass by way of moisture accumulation on the grass leaves. Once the larvae get mid-way or to the top of forages, they can be ingested by grazing ruminants (Zajac, 2006) and can either develop into fourth larval (L 4 ) and adult stages (L 5 ) once it reaches the abomasum or small intestines where the cycle starts again (Balic et al., 2000; Zajac, 2006). Resistance occurs when anthelmintics are administered and a small population survives; those are the parasites that have resistant genes. There is a vast amount of variability in the genome of helminth parasites. With such genetic diversity of nematode populations, random mutations can arise. Parasites that have high reproduction rates can cause resistance to occur rapidly within the next generation produced (Fleming et al., 2006; Papadopoulos, 2008). Multidrug resistance has been attributed to multiple gene 71

89 mutations, such as specific mutations in a P-glycoprotein (Kerboeuf et al., 2008) or in generalized mutations that affect the receptor binding site(s) where the drugs work, or in differences in enzymes, transport mechanisms, or metabolism of anthelmintics (Papadopoulos, 2008). This resistance to chemically synthesized anthelmintics has lead researchers to look for alternatives to controlling GIP. There also is now a growing interest, especially among organic farmers, to use natural means to help with the parasitism, including utilizing integrated pest management practices. Breaking the transmission cycle is a way to help with not only decreasing infestation in the animal herd, but also a way to combat resistance by not allowing young larval stages that may be carrying the resistant genes to develop into adults that will harm the host animal and produce more resistant larvae. GP has shown efficacy in decreasing larval parasites in vitro in the previous experiments in chapter 3, but GP was only tested on the free living L 3 larval stage and adults. GP has not been investigated to test efficacy against other developmental stages such as eggs or younger larval stages of development. The compounds found in crude GP extract, such as flavonoids, flavon-3-ols and condensed tannins (CT), have been shown to deactivate eggs or adhere to outer membranes and prevent egg hatching, as well as prevent early larval stages from shedding their outer cuticle which leads to starvation and death of the parasite (Athanasiadou et al., 2001; Min and Hart, 2003; Hoste et al., 2006; Kerboeuf et al., 2008). Even though there was no efficacy seen in the previous trial in chapter 4 on decreasing FEC in growing lambs, I thought that GP may have decreased the viability of those eggs to hatch on pasture. There has not been any research conducted that has 72

90 examined GP or its extracts as a means to decrease transmission of helminth parasites by stopping the development cycle or examined the effects of GP on genetically resistant helminth parasites. This study addresses the gap in knowledge by investigating the effects of GP on the egg development/hatchability to free living L 3 larval stages of mixed helminth parasites from both conventional and organic farming practices. The purpose of examining both farming practices is that the conventional farms selected will be those farms in which resistance to current commercial anthelmintics has been observed, whereas the organic farms should have parasites that have not been introduced to any chemical treatment therefore being more susceptible to treatment. Our objectives were to examine the egg development/hatchability of mixed helminth parasites to determine if the extracts will decrease the number of eggs that will hatch and develop into infective larval stages when compared to a negative control. The study was done with parasite egg collection from feces of sheep from organic and conventional farms to determine if there is a difference in efficacy on these parasites exposed to differing environments. We hypothesized that GP would reduce the viability of the helminth parasites against a control (no treatment). This reduced viability will be observed by a decreased number of eggs in the feces that will hatch to free living larval stages when compared to the control fecal cultures. It was also hypothesized that GP would reduce the viability of the helminth parasites in feces from organic farming practices more than conventional farming practices, due to increase susceptibility of the parasites on organic farms, especially if resistance to anthelmintics has been observed on the conventional farms. 73

91 Materials and Methods Extraction Dried GP (1.85 kg, > 95% DM) was finely ground (1-mm sieve) and extracted with 70% aqueous ethanol for 24 h. The extract was then strained to remove all liquid from solid material and the residue was discarded. The ethanol was evaporated for extract (< 5% remaining) using vacuum rotary evaporation and the resulting residue was dissolved in distilled water to give a final concentration of 37.5 mg/ml crude extract. This concentration was comparable to the gram amount of CT assumed to be left in fecal matter of lambs (assuming an estimated digestibility of GP at approximately 17%) from a subsequent study after being administered an experimental GP diet containing 45 g CT/kg DM. Fecal Culture Feces were collected from sheep with known parasite infestation on both commercial and organic farms. Approximately 300 g of feces was randomly collected off the ground from fresh excrement from each farm (n=5, 3 conventional vs. 2 organic) and kept at ambient temperature in insulated containers so that samples could be cultured fresh within 3 h of collection. The samples from each farm were pooled by individual farm to give a well mixed sample to represent each farming practice. Six subsamples were taken from each pooled farm fecal sample for replicates in fecal culture. The feces were then subjected to a control treatment group with no pomace extract added and to a concentrated pomace extract dose. The experimental procedure for fecal culture was done using a procedure from Hall (1987). A fecal egg count (FEC) was done at the initiation of 74

92 the experiment to determine the number of eggs per gram (EPG) of feces going into culture. This assisted in determining the hatchability of the eggs that develop into larvae. Thirty grams of fecal matter were placed into a 250-mL disposable polystyrene toxicology jar along with 20 ml of water. The feces and water were mixed well to get a slurry that were lightly mixed with 5 g of vermiculite, making sure not to pack the mixture. The lid was loosely placed on the jars and incubated at ambient temperature for 14 d in a dark area. After incubation, the cultures were exposed to light for 1 h, then the culture jars were filled with warm water (30 C) and inverted into a Petri dish. The moat that was formed was filled with water and the fecal culture was left to stand for 3 to 8 h until larvae collected in the moat. The liquid plus larvae was then pipetted off and placed in a conical centrifuge tube. The larvae were then counted by serial dilution to determine the number of larvae that hatched for each treatment. One to two drops of Lugol s iodine was added to each slide before counting larvae microscopically to insure an accurate count. Statistical Analysis Results were analyzed statistically using the PROC MIXED procedure in SAS 9.3 (SAS Inst., Cary, NC, 2011) to determine difference for treatments, farm, farming practices and potential interactions. Farming practice and treatment were analyzed as fixed effects and farm was analyzed as a random effect. The interaction of farm x farming practice and treatment x farm x farming practice were also analyzed as random effects. Treatment differences were compared using Fisher s LSD by using LSMEANS with the 75

93 DIFF option in SAS. Statistical significance was reported if P 0.05, with trends being reported if 0.05 P Results and Discussion In all culture jars for the GP extract, there was 100% inhibition of egg hatch into larvae (P < 0.01) (Data not shown). On the contrary, in all of the control jars that did not receive treatment, there was an abundance of larvae present. There were more larvae counted than the number of eggs estimated at the beginning of the experiment. This result is to be expected because there is no way of counting all eggs present in fecal samples, no matter how well mixed the sample can be. This is why most FEC are estimates of the average number of EPG of feces and is usually why more than one count is done. There was no difference (P > 0.10) in farm or farming practice, meaning that neither farming practice nor farm had any influence on the treatment being administered or alternatively the treatment was effective in prohibiting larval development, regardless of farming practices on a particular farm. There was also no significant interaction observed between farming practice and the treatment. This was a different result than what was hypothesized in the beginning of the experiment because it was thought that due to the resistance being observed to chemical anthelmintic therapies on conventional farms; it was assumed there would be some resistance observed from these farms against the GP extracts, but this was not the case. Regardless of resistance observed on conventional farms, the extract still inhibited parasite egg hatch and larval development. This result could be attributed all the bioactive compounds present in the GP, such as CT and flavonoids, which are considered to be the most abundant polyphenol in the GP 76

94 extracts (Lu and Yeap Foo, 1999). Molan et al. (2003) observed 100% inhibition of egg hatchability at concentrations of 1 mg/ml against T. colubriformis parasite eggs and effectively inhibited L 3 larval migration at concentrations of 500 µg/ml. The extracts showing the most efficacies against these parasites were the flavan-3-ol galloyl derivatives, with epigallocatchin (a CT) being the most active in both egg hatch and larval development inhibition assays (Molan et al., 2003). The combination of bioactive compounds in the crude extract of GP seems to make it very effective in controlling GIP transmission through contaminated feces. This surprising result of the GP extract showing efficacy in both farming methods allows for implications that the treatment could be used in feed to help with integrated pest management of decreasing parasite burden in farm animals. There is still further research that needs to be done to validate this implication before it can be fully put into practice, but it provides promise for natural means to help reduce GIP in agricultural animals on both organic and conventional farms. References Aas, E. A practitioner s perspectives: Traditional tannin-treatment against intestinal parasites in sheep and cattle. Ethnobotany Res. and Apps. 2003, 1: Athanasiadou, S., I. Kyriazakis, F. Jackson, and R. L. Coop. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies. Vet. Parasitol. 2001, 99: Balic, A., V. M. Bowles, and E. N. T. Meeusen. The immunobiology of gastrointestinal nematodes infestations in ruminants. Advances in Parasitology 2000, 45: Gil, M. I., F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft, and A. A. Kader. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agric. Food Chem. 2000, 48:

95 Hall, C. A. Diagnosis of anthelmintic resistance in sheep and goats. Austrilian Standard Diagnostic Techniques for Animal Diseases. 1987, 46:1-17. Kerboeuf, D., M. Riou, and F. Guégnard. Flavonoids and related compounds in parasitic disease control. Mini-Reviews in Medicinal Chemistry. 2008, 8: Min, B. R. and S. P. Hart. Tannins for suppression of internal parasites. J. Anim. Sci. 2003, 81 (E. Suppl. 2): E102-E109. Nguyen, T. M., D. V. Binh, and E. R. Orskov. Effects of foliages containing condensed tannins and on gastrointestinal parasites. Anim. Feed Sci. Tech. 2005, 121: Lloyd, J. U. Punica granatum. The Western Druggist. 1897, 3-9. Lu, Y., and L. Yeap Foo. The polyphenol constituents of grape pomace. Food Chem. 1999, 65: 1-8. Li, Y., C. Guo, J. Yang, J. Wei, J. Xu, and S. Cheng. Evaluation of antioxidant properties of pomegranate husk extract in comparison with pomegranate pulp extract. Food Chem. 2006, 96: Papadopoulos, E. Anthelmintic resistance in sheep nematodes. Small Rum. Res. 2008, 76: SAS/STAT Software User s Guide: Statistics, Version 9.3 Edition SAS Inst., Inc. Cary, NC Zajac, A. M. Gastrointestinal nematodes of small ruminants: Life cycle, anthelmintics, and diagnosis. Vet. Clin. Food Anim. 2006, 22:

96 General Conclusions The dry matter degradability study was conducted to examine if adding PH or GP would detrimentally change the degradation kinetics. In hindsight, it would have been more beneficial if the study had looked at concentrations similar to what was expected to be fed in the diet instead of the ratios chosen. Also, if the study were to be repeated, then it would be better to get nutritional analysis of all treatments beforehand, combine with a feedstuff at ratios comparable to what would be fed in an animal diet, and do in situ fermentation to get a better model of kinetics and degradability. Conducting a protein binding capacity assay would also be beneficial because CT are known to bind to protein and assessing the binding capacity of the tannins in both GP and PH would help better understand if they are affecting digestibility of protein in the feed. The in vitro parasite study examining effects of the PH and GP extracts provides promise but needs to be expanded. The extracts should be fractionated and tested for efficacy to determine what compound(s) is responsible for observed efficacy in decreasing viability of the parasites or if efficacy is best observed from crude extraction. Extracts should be tested to determine if the bioactive compounds in the extracts are killing the parasites or just paralyzing them. Studies should be designed to observe the 79

97 same parasites when the extract is present and then once the extract is removed. This will determine if the extract is cidial or static, which either way could be beneficial to host assisted parasite removal. It would also be beneficial to examine effects of extracts on other economically important parasites, such as Haemonchus contortus. The lamb trials were helpful but would be considered preliminary. In hindsight, both trials should have been conducted over a longer time period administering the GP diet for longer than 5 d, but this would also require better GP diet formulation to meet NRC nutritional requirements for sheep. There would have been added benefit to use experimentally infect the animals instead of using naturally infested animals. This would have controlled the variability and high parasite infestation so that affects of treatment would have been more noticeable. At the moment, it is clear that GP is not comparable to commercial anthelmintics but may have an effect in assisting the animal to be more resilient to the parasite infestation until host immunity can occur. This would be especially useful for those farms that do not use commercial anthelmintics. With the lambs being so heavily parasitized, it should be a considerate to increase protein and vitamin A, E and selenium in the diets. These components are known to help with hostparasite immunity and fight off infestation of the gut (Balic et al., 2000) and by adding them in additional amounts there is the possibility that there could be positive associative effects. The purpose of the fecal culture was to examine the effects of decreasing transmission of the parasite by inhibiting the life cycle at initial and developmental stages. It was a beneficial result to observe 100% inhibition of egg hatchability and larval 80

98 development with the GP treatment. This leads to implications of how undigested GP in the diet would potentially assist with decreasing transmission of the parasites to other animals in the herd, but the same research still needs to be conducted as discussed with the in vitro parasite trial. There should be studies done to determine whether the GP is actually inactivating eggs and killing the parasite larvae or if it is just inhibiting growth. In conclusion, this research has shown potential of PH and GP in becoming a natural way to control GIP in ruminants, but there still needs to be lot of work done before either of these by-product could be incorporated in integrated pest management systems. This research has illustrated that there is efficacy of decreasing parasite viability in the by-products from pomegranate and grape fruit industries but only within in vitro settings. Determining the exact mechanism of the efficacy and what exact compounds are responsible are still aspects that need to be researched. The results from the studies in the previous chapters, such as the efficacy against helminth larvae observed from GP and PH extracts and the GP extract resulting in 100% inhibition of egg hatchability and larval development, show that there is promise in these by-products as natural anthelmintics. Even though no results were observed in the lamb trial on decreasing parasite load, utilizing GP as a feed additive to decrease transmission of parasites on farm by inhibiting the growth cycle of certain Stronglylide sp. parasites maybe a useful benefit for the by-product. 81

99 References Aas, E. A practitioner s perspectives: Traditional tannin-treatment against intestinal parasites in sheep and cattle. Ethnobotany Res. and Apps. 2003, 1: Abarghuei, M. J., Y. Rouzbehan, and D. Alipour. The influence of the grape pomace on the ruminal parameters of sheep. Livestock Science 2010, 132: Amin, M. R., M. Mostofa, M. E. Hoque, and M. A. Sayed. In vitro anthelmintic efficacy of some indigenous medicinal plants against gastrointestinal nematodes of cattle. J. Bangladesh Agril. Univ. 2009, 7, 1: AOAC Official methods of analysis of the AOAC. 13 th ed., Association of Official Analytical Chemists, Arlington, Va. AOAC Official methods of analysis of the AOAC. 17th ed., Association of Official Analytical Chemists, Eden Prairie, MN. AOAC Official methods of analysis of the AOAC. 18th ed., Association of Official Analytical Chemists, Eden Prairie, MN. Arvanitoyannis, I. S., D. Ladas, and A. Mavromatis. Potential uses and applications of treated wine waste: A review. Int. J. Food Sci. Tech. 2006, 41: Athanasiadou, S., I. Kyriazakis, F. Jackson, and R. L. Coop. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: In vitro and in vivo studies. Vet. Parasitol. 2001, 99: Bahrami, Y., A.-D. Foroozandeh, F. Zamani, M. Modarresi, S. Eghbal-Saeid, and S. Chekani-Azar. Effect of diet with varying levels of dried grape pomace on dry matter digestibility and growth performance of male lambs. J. Ani. Plant Sci. 2010, 6(1):

100 Bahuaud, D., C. Martinez-Ortiz De Montellano, S. Chauveau, F. Prevot, F. Torres- Acosta, I. Fouraste, and H. Hoste. Effects of four tanniferous plant extracts on the in vitro exsheathment of third-stage larvae of parasitic nematodes. Parasitology 2006, 132(4): Balic, A., V. M. Bowles, and E. N. T. Meeusen. The immunobiology of gastrointestinal nematodes infestations in ruminants. Advances in Parasitology 2000, 45: Baumgärtel, T., H. Kluth, K. Epperlein, and M. Reodehutscord. A note on digestibility and energy value for sheep of different grape pomace. Small Rum. Res. 2007, 67: Besharati, M., and A. Taghizadeh. Evaluation of dried grape by-product as a tanniniferous tropical feedstuff. Anim. Feed Sci. Tech. 2009, 152: British Pharmacopoeia Codex. Granati cortex: pomegranate bark, pomegranate rind, pelletierine, pelletierinetannate. Brit.Pharm.Codex. [Online] (accessed May 22, 2005) Bull, B. S., J. A. Koepke, E. Simson, and O. W. van Assendelft. Procedure for determining packed cell volume by microhematocrit method; Approved standard-3 rd ed. Clinical and Laboratory Standards Institute. 2000, 20 (18): Camberis, M., G. LeGros, and J. Urban, Jr. Animal model of Nippostrongylus barasiliensis and Heligomosomoides polygyrus. Current Protocols in Immunology. 2003, Famuyiwa, O. and C. S. Ough. Grape pomace: possibilities as animal feed. Am. J. Enol. Vitic. 1982, 33(1): Famuyiwa, O. and C. S. Ough. Effect of structural constituents of cell wall on the digestibility of grape pomace. J. Agric. Food Chem. 1990, 38: Fleming, S. A., T. Craig, R. M. Kaplan, J. E. Miller, C. Navarre, and M. Rings. Anthelmintic resistance of gastrointestinal parasites in small ruminants. J. Vet. Intern. Med. 2006, 20: Gil, M. I., F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft, and A. A. Kader. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agric. Food Chem. 2000, 48:

101 Githiori, J. B., S. Athanasiadou, S. M. Thamsborg. Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants. Vet. Parasitol. 2006, 139: Goering, H.K. and P.J. Van Soest Forage Fiber Analysis. USDA Agricultural Research Service. Handbook number 379. U.S. Dept. of Agriculture. Superintendent of Documents, US Government Printing Office, Washington, D.C Hagerman, A., I. Harvey-Mueller, and H. P. S. Makkar. Quantification of tannins in tree foliage Lab manual. FAO/IAEA. 2000, 2-4. Hall, C. A. Diagnosis of anthelmintic resistance in sheep and goats. Austrilian Standard Diagnostic Techniques for Animal Diseases. 1987, 46:1-17. Hoste, H., F. Jackson, S. Athanasiadou, S. M. Thamsborg, and S. O. Hoskin. The effects of tannin-rich plants on parasitic nematodes in ruminants. TRENDS in Parasitol. 2006, 22(6): Hoste, H., J. F. Torres-Acosta, V. Paolini, A. Aguilar-Cabellero, E. Etter, Y. Lefrileux, C. Chartier, C. Broqua. Interactions between nutrition and gastrointestinal infestations with parasitic nematodes in goats. Small Rum. Res. 2005, 60: Hur, S. N., A. L. Molan, and J. O. Cha. Effects of feeding condensed tannin-containing plants on natural coccidian infestation in goats. Asian-Australasian J. Anim. Sci. 2005, 18, no. 19: Hussein, A. Assuit University, Assuit, Egypt. Personal communication with A. Ludwick, Department of Chemistry, Tuskegee University, Aug. 15, Jackson, F. S., W. McNabb, T. N. Barry, Y. L. Foo, and J. S. Peters. The condensed tannin content of a range of subtropical and temperate forages and the reactivity of condensed tannin with ribulose-1, 5-bis-phosphate carboxylase (Rubisco) protein. J. Sci. Food Agric : Jami, E., A. Shabtay, M. Nikbachat, E. Yosef, J. Miron, and I. Mizrahi. Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population. J. Dairy Sci. 2012, 95 (10): Kaplan, R. M., J. M. Burke, T. H. Terrill, J. E. Miller, W. R. Getz, S. Mobini, E. Valencia, M. J. Williams, L. H. Williamson, M. Larsen, and A. F. Vatta. Validation of the FAMACHA eye color chart for detecting clinical anemia in sheep and goats on farms in the southern United States. Vet. Parasitol. 2004, 123:

102 Kerboeuf, D., M. Riou, and F. Guégnard. Flavonoids and related compounds in parasitic disease control. Mini-Reviews in Medicinal Chemistry. 2008, 8: Khanbabaee, K. and T. van Ree. Tannins: Classification and Definition. Nat. Prod. Rep. 2001, 18: Klimpel, S., F. Abdel-Ghaffar, K. A. S. Al-Rasheid, G. Aksu, K. Fischer, B. Strassen, and H. Mehlhorn. The effects of different plant extracts on nematodes. Parsitol. Res. 2011, 108: LeShure, S. Characterizing components of the pomegranate as a natural anthelmintic source for small ruminant animals. Masters in Chemistry Thesis, Tuskegee University, Department of Chemistry, Tuskegee, Alabama. May 9, Li, Y., C. Guo, J. Yang, J. Wei, J. Xu, and S. Cheng. Evaluation of antioxidant properties of pomegranate husk extract in comparison with pomegranate pulp extract. Food Chem. 2006, 96: Lloyd, J. U. Punica granatum. The Western Druggist. 1897, 3-9. Lu, Y. and L. Yeap Foo. The polyphenol constituents of grape pomace. Food Chem. 1999, 65: 1-8. Mangan, J. L. Nutritional effects of tannins in animal feeds. Nutr. Res. Rev. 1988, 1: McKay, D. M. and J. Bienenstock. The interaction between mast cells and nerves in the gastrointestinal tract. Immunology Today 1994, 15: Min, B. R. and S. P. Hart. Tannins for suppression of internal parasites. J. Anim. Sci. 2003, 81 (E. Suppl. 2): E102-E109. Min, B. R., S. LeShure, and S. Solaiman. Personal communication/collaboration, Department of Animal Science, Tuskegee University, Tuskegee, Alabama, Dec. 7, Min, B. R., S. Solaiman, N. Gurung, J. Behrends, J.-S. Eun, E. Taha, and J. Rose. Effects of pine bark supplementation on performance, rumen fermentation, and carcass characteristics of Kiko crossbred male goats. J. Anim. Sci. 2012, 90: Mirzaei-Aghsaghali, A., N. Maheri-Sis, H. Mansouri, M. E. Razeghi, A. Mirza- Aghazadeh, H. Cheraghi and A. Aghajanzadeh-Golshani. Evaluating potential nutritive value of pomegranate processing by-products for ruminants using in vitro gas production technique. J Agric Biol Sci. 2011, 6(6):

103 Molan, A. L., L. P. Meagher, P. A. Spencer, and S. Sivakumaran. Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis. Int. J. Parasitol. 2003, 33: Mueller-Harvey, I. Analysis of hydrolysable tannins. Anim. Feed Sci. Tech. 2001, 91: Mueller-Harvey, I and A. B. McAllen. Tannins: their biochemistry and nutritional properties. Adv. Plant Cell Biochem. 1992, 1: Murthy, K. N. C., G. K. Jayaprakasha, and R. P. Singh. Studies on antioxidant activity of pomegranate (Punica granatum) husk extract using in vivo models. J. Agric. Food Chem. 2002, 50: Natural products as medicinally useful agents. (accessed March 5, 2009) Nguyen, T. M., D. V. Binh, and E. R. Orskov. Effects of foliages containing condensed tannins and on gastrointestinal parasites. Anim. Feed Sci. Tech. 2005, 121: Novobilský, A., I. Mueller-Harvey, S. M. Thamsborg. Condensed tannins act against cattle nematodes. Vet. Parasitol. 2011, 182: Nunez-Hernandez, G., J. D. Wallace, J. L. Holechek, M. L. Galycan, and M. Cardenas. Condensed tannins and nutrient utilization by lambs and goats fed low-quality diets. J. Anim. Sci. 1991, 69: Ohio grape facts. (accessed October 3, 2010) Papadopoulos, E. Anthelmintic resistance in sheep nematodes. Small Rum. Res. 2008, 76: Piwonka, E. J. and J. L. Firkins. Effect of glucose on fiber digestion and particleassociated carboxymethyl cellulose activity in vitro. J. Dairy Sci. 1993, 76:129. Pomegranate profiles. (accessed October 3, 2010) Romani, A., F. Ieri, B. Turchetti, N. Mulinacci, F. F. Vincieri, and P. Buzzini. Analysis of condensed and hydrolysable tannins from commercial plant extracts. J. Pharm. Biomed. Anal. 2006, 41:

104 SAS/STAT Software User s Guide: Statistics, Version 9.3 Edition SAS Inst., Inc. Cary, NC Schofield, P., D. M. Mbugua, and A. N. Pell. Analysis of condensed tannins: a review. Anim. Feed Sci. Tech. 2001, 91: Seibold, L. Contribution to the pharmacy of the pomegranate. Amer. J. Pharm. [Online] 1884, pomegranate.html. (accessed May 20, 2005) Shabtay, A., H. Eitam, Y. Tadmor, A. Orlov, A. Meir, P. Weinber, Z. G. Weinburg, Y. Chen, A. Brosh, I. Izhaki, and Z. Kerem. Nutritive and antioxidant potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed. J. Agric. Food Chem. 2008, 56: Singh, R. P., K. N. C. Murthy, and G. K. Jayaprakasha. Studies on the antioxidant activity of pomegranate (Punica granatum) husk and seed extracts using in vitro models. J. Agric. Food Chem. 2002, 50: Smith, A. H., E. Zoetendal and R. L. Mackie. Bacterial mechanisms to overcome inhibitory effects of dietary tannins. Microb Ecol 2005, 50: Solaiman, S. Tuskegee University, Tuskegee, AL. Personal communication with S. LeShure, Department of Animal Sciences, The Ohio State University, March 24, Spanghero, M., A. Z. M. Salem, and P. H. Robinson. Chemical composition, including secondary metabolites, and rumen fermentability of seeds and pulp of Californian (USA) and Italian grape pomace. Anim. Feed Sci. Tech. 2009, 152: Terrill, T. H., A. M. Rowan, G. B. Douglas, and T. N. Barry. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 1992, 58: Thompson, D. P. and T. G. Geary. The structure and function of helminth surfaces. In Biochemistry and Molecular Biology of Parasites. Marr, J. J. and Muller, M. eds. 1995, Academic Press, New York, NY, p Van Soest, P. J. van, J. B. Robertson, and B. A. Lewis. Methods for dietary fiber, neutral detergent fiber and nonstructural polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74(10):

105 Villalba, J. J., F. D. Provenza, and R. Shaw. Initial conditions and temporal delays influence preference for foods high in tannins and for foraging locations with and without foods high in tannins by sheep. App. Ani. Behav. Sci. 2006, 97: von Samson-Himmelstjerna, G., S. Rickling, and T. Schnieder. Improved in vitro cultivation of larvae of bovine lungworm Dictycaulus viviparous. Parasitol. Res. 1998, 84: Wagland, B. M., W. O. Jones, L. Hribar, T. Bendixsen, and D. L. Emery. A new simplified assay for larval migration inhibition. Int. J. Parasitol. 1992, 22(8): Wibaut, J. P and U. Holstein. Investigation of the alkaloids of Punica granatum L. Arch. Biochem. Biophys. 1957, 69: Yamasaki, M., T. Kitagawa, N. Koyanagi, H. Imamura, H. Tachibana, and K. Yamada. Dietary effects of pomegranate seed oil on immune function and lipid metabolism in mice. Nutrition. 2006, 22: Zajac, A. M. Gastrointestinal nematodes of small ruminants: Life cycle, anthelmintics, and diagnosis. Vet. Clin. Food Anim. 2006, 22:

106 Appendix A: Cumberland Valley Analytical Services, Inc. Procedure References 89

107 ADF Fiber (Acid Detergent) and Lignin in Animal Feed (973.18). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. (Modifications: Whatman 934-AH glass micro-fiber filters with 1.5 um particle retention used in place of fritted glass crucible; ADF method only). Ash Ash of Animal Feed (942.05). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. (Modifications: 1.5 g sample weight, 4 h ash time, hot weigh). Chloride Sample is extracted with 0.5% nitric acid and analyzed by potentiometric titration with silver nitrate using Brinkman Metrohm 848 Titrino Plus. Brinkmann Instruments Inc., One Cantiague Road, P.O. Box 1019, Westbury, NY Dry Matter Grains, mixed feeds, concentrates and by-products: Moisture in Animal Feed, Drying at 135 C (930.15). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. Fat Crude Fat in Feeds, Cereal Grains, and Forages ( ). Official Methods of Analysis, 18th edition Association of Official Analytical Chemists. Tecator Soxtec System HT 1043 Extraction unit. Tecator, Foss NA 7682 Executive Drive, Eden Prairie, MN

108 Lignin Goering, H.K. and P.J. Van Soest Forage Fiber Analysis. USDA Agricultural Research Service. Handbook number 379. U.S. Dept. of Agriculture. Superintendent of Documents, US Government Printing Office, Washington, D.C Modifications: Fiber residue from the ADF step is recovered on a 1.5 um particle retention 7 cm Whatman Glass Fiber Filter in a California Buchner Funnel instead of using a Gooch crucible. Greater surface area allows for better filtration. Fiber residue and filter is transferred to a capped tube and approx. 45 ml of 72% sulfuric acid is added. Tubes are gently agitated for 2 h to insure that all fiber material is continually washed with acid. The contents of the tube after incubation in acid is filtered onto a second filter (same type as above) which is then rinsed, dried, and weighed. The glass fiber filters and lignin residue are than ashed for 2 h in a furnace to remove lignin organic matter. Finally, the filter and ash residue is weighed back and subtracted from the original weight to determine grams of lignin. Metals / Minerals Metals and Other Elements in Plants (985.01). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. Perkin Elmer 3300 XL and 5300 DV ICP. Perkin Elmer, 710 Bridgeport Avenue, Shelton, CT (Modifications include: Ash 0.35 g sample for 1 h at 535 C. Digest in open crucibles for 20 min in 15% nitric acid on hotplate. Samples diluted to 50 ml and analyzed on ICP). 91

109 NDF Van Soest, P.J., J.B. Robertson, and B.A. Lewis Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy Sci. 74: (Modification: Whatman 934-AH glass micro-fiber filters with 1.5 um particle retention) Nitrogen Protein (Crude) in Animal Feed (990.03). Official Methods of Analysis, 17th edition Association of Official Analytical Chemists. Leco FP-528 Nitrogen Combustion Analyzer. Leco, 3000 Lakeview Avenue, St. Joseph, MI

110 Appendix B: FAMACHA Eye Score Chart (Reference: 93

111 94