Effect of Birdsfoot Trefoil on Exsheathment of Haemonchus contortus in Rumen Fistulated Sheep

This study has been developed to address the issue of small ruminant parasite resistance to commercial anthelmintics and to examine the possibility of controlling these parasites using feeds with condensed tannin containing plants. The goal of the research was to determine whether birdsfoot trefoil hay prevents the exsheathment of Haemonchus contortus and whether efficacy differs among birdsfoot trefoil cultivars. During the first phase of research, a method for testing the exsheathment of H. contortus in vivo was developed. Various larvae containment capsules were tested to see whether the larvae could escape from the capsules. The most successful capsules were then tested in the rumens of fistulated ewes. Larvae were placed in capsules and suspended in the rumens by cords of various lengths for several different amounts of time. Using the methods developed, it was found that after eight hours in the rumen 82 ± 1% of the larvae were exsheathed. For the second phase of the research, four rumen fistulated ewes were fed diets of birdsfoot trefoil or a control. Three cultivars of birdsfoot trefoil were fed: Pardee, Empire, and Bruce. These diets were fed to the each of the ewes for 28 days in a Latin 4x4 design. During exsheathment tests, capsules containing third-stage H. contortus larvae were placed in the ewes' rumens for 8 hours. They were then examined under a microscope for any changes in exsheathment or motility. It was found that for all three cultivars, feeding birdsfoot trefoil hay did not affect exsheathment percentages. These results indicate that while further studies should be conducted to confirm these results, research on effectively incorporating condensed tannin containing plants should focus on other life stages of the H. contortus parasite.


ABSTRACT:
This study has been developed to address the issue of small ruminant parasite resistance to commercial anthelmintics and to examine the possibility of controlling these parasites using feeds with condensed tannin containing plants. The goal of the research was to determine whether birdsfoot trefoil hay prevents the exsheathment of Haemonchus contortus and whether efficacy differs among birdsfoot trefoil cultivars.
During the first phase of research, a method for testing the exsheathment of H. contortus in vivo was developed. Various larvae containment capsules were tested to see whether the larvae could escape from the capsules. The most successful capsules were then tested in the rumens of fistulated ewes. Larvae were placed in capsules and suspended in the rumens by cords of various lengths for several different amounts of time. Using the methods developed, it was found that after eight hours in the rumen 82 ± 1% of the larvae were exsheathed.
For the second phase of the research, four rumen fistulated ewes were fed diets of birdsfoot trefoil or a control. Three cultivars of birdsfoot trefoil were fed: Pardee, Empire, and Bruce. These diets were fed to the each of the ewes for 28 days in a Latin 4x4 design. During exsheathment tests, capsules containing third-stage H. contortus larvae were placed in the ewes' rumens for 8 hours. They were then examined under a microscope for any changes in exsheathment or motility.
It was found that for all three cultivars, feeding birdsfoot trefoil hay did not affect exsheathment percentages. These results indicate that while further studies should be I would also like to extend a special thanks to my fellow graduate student Carly Barone for the countless hours that you put into training and mentoring me during the past few years. You were always ready to put aside your work to answer my endless barrage of questions, or to simply encourage me when the going got rough.
Thank you also to Sydney Day and Nick Miniter for your support at Peckham Farm. iv

PREFACE:
This thesis has been prepared using the Manuscript Format. Chapter I contains a literature review, while chapters II and III each contain a manuscript that will be submitted for publication. Chapter IV covers a summary of future directions that this research should take. v

Economic Impact
Gastrointestinal nematodes are a major economic concern for small ruminant producers across the globe (Nieuwhof & Bishop, 2005;Sackett et al., 2006;Qamar et al., 2011). In the United States, the estimated death loss of sheep due to parasites in 2009 was valued at $2.8 million US dollars (National Agriculture Statistics Service,

2010). A report published by the Meat and Livestock Australia Limited in 2006
estimated that Australia's annual sheep loss due to internal parasites is $283 million US dollars (Sackett et al., 2006). In Great Britain, it is estimated that there is an annual loss of $104 million US dollars due to internal parasites in sheep, $79 million of which is due to reduced growth, and $25 million due to treatment costs (Nieuwhof & Bishop, 2005). As of 2011, the small ruminant herds in Pakistan consisted of about 24.6 million sheep and 52.6 million goats (Qamar et al., 2011). It is estimated that in Pakistan parasite infections in sheep and goats cause a total annual loss of over $2.6 billion US dollars, $1364 million of which is due to parasite associated animal mortality, $1179 million due to reduced milk production, $84 million due to abomasa condemned at slaughter, $0.38 million due to weight loss, and $0.24 million spent on parasite treatments (Qamar et al., 2011).

Production loss
A review by Charlier et al. (2014) of studies looking at production losses due to internal parasites found that infection could reduce weight gains by 10%-47% and wool production by 0%-21%. They also found that treating parasites could increase milk yield from 9%-40% (Charlier et al., 2014). Parasite infection also causes reduced feed intake and reduced feed efficiency (Coop & Holmes, 1996). An experimental infection of 3000 Haemonchus contortus larvae was found to reduce milk production of ewes by 32.6% (P < 0.01) (Cobon & O'Sullivan, 1992). Approximately five weeks after an experimental infection of 2000 H. contortus larvae, infected lambs gained an average of 0 grams/day for the next 52 days while control lambs gained 98 grams/day; wool growth was also significantly reduced in infected lambs (Cobon & O'Sullivan, 1992).

Resistance in the United States
Anthelmintic resistance is prevalent in the United States (Terrill et al., 2001;Howell et al., 2008;Crook et al., 2016). Forty-six small ruminant farms located in the southern United States, including Puerto Rico and St. Croix, were evaluated for parasite resistance (Howell et al., 2008). It was found that H. contortus were resistant to benzimidazole at 98% of the farms, levamisole at 54%, ivermectin at 76%, and moxidectin at 24% (Howell et al., 2008). Thirty-four small ruminant farms from the mid-Atlanic United States were evaluated for anthelmintic resistance (Crook et al., 2016). It was found that H. contortus were resistant to benzimidazole at 100% of the farms, levamisole at 24%, ivermectin at 82%, and moxidectin at 47% (Crook et al., 2016). Two goat farms in Georgia were evaluated for anthelmintic resistance (Terrill et al., 2001). Resistance was found at both farms to ivermectin and levamisole, with one farm additionally having parasitic resistance to benzimidazole (Terrill et al., 2001).

Global Resistance
Parasite resistance to anthelmintics is a problem for producers all over the globe (Ramos et al., 2002;Howell et al., 2008;Manikkavasagan et al., 2013;Chandra et al., 2015). Resistance to benzimidazole in H.
contortus was examined in 20 locations covering the five regions of Uttar Pradesh, India, and was present in all five regions (Chandra et al., 2015). Another study in southern Queensland, Australia, tested 20 farms and found that there was resistance to levamisole at 42% of the farms and moxidectin at 50% ( . Parasitic infections combined with anthelmintic resistance have been blamed for losses of 10%-50% of weaned lambs in southern Queensland during wet seasons . Twenty-seven goat farms in Tamil Nadu (India) were evaluated for parasite resistance to anthelmintics and resistance was found at 81% of the farms to albendazole and 92% for levamisole (Manikkavasagan et al., 2013). An evaluation of the parasite resistance to benzamidizol on eleven farms in Ontario (Canada) found that 91% of the farms had resistant parasites ( Barrere et al., 2013). In Santa Catarina (Brazil), sixty-four flocks of sheep were evaluated for anthelmintic resistance (Ramos et al., 2002). Of these flocks, 67% had resistance to ivermectin, 65% to albendanzole, and 15% to levamisole (Ramos et al., 2002). Thus, parasite resistance was highly prevalent in all the locations tested.

Overview
The parasite Haemonchus contortus (barber pole worm) is known for being one of the most pathogenic gastrointestinal nematodes (GIN) of small ruminants . H. contortus do not generally cause diarrhea, but since they feed on the blood of the host, they do cause anemia (Roeber et al., 2013). A study of lambs infected with 10,000 stage-three (L3) larvae found that onset of anemia began ten days after the infection (Hunter & Mackenzie, 1982). These parasites use a single lancet that extends from their buccal cavity to slice the lining of the abomasum; blood was visible in the mucosal lining seven days after infection (Hunter & Mackenzie, 1982).

Haemonchus contortus life-cycle
Adults measure approximately 2.5cm and females can lay up to 10,000 eggs per day (Gilleard, 2013. After exiting the host via feces, these eggs remain on the pasture while hatching and developing to the infective stage (Roeber et al., 2013). Larvae are identified by five stages during their development into adults, they are referred to as stage-one larvae (L1) through stage-five larvae (L5) (Silverman & Patterson, 1960 18-21 days (Roeber et al., 2013). Silverman and Patterson (1960) found that the rate of larval maturity varied by the age and susceptibility of the host. In young, susceptible lambs, parasites could reach maturity in as few as 12 days, while in older hosts this may take as long as 24 days (Silverman & Patterson, 1960). In resistant animals, the parasites were inhibited at the L4 or L5 stages (Silverman & Patterson, 1960). During the L4 stage, larvae are capable of entering a hypobiotic period in the abomasum of the host, particularly when environmental conditions are not favorable for egg/larva development on pasture (Gatongi et al., 1998;Roeber et al., 2013). Adult H. contortus have a short lifespan of only a few months (Roeber et al., 2013). Developing H. contortus larvae molt their outer cuticles a total of four Here the eggs hatch and develop from L1 larvae to L3 larvae. When pasture containing L3 larvae is consumed these larvae enter the host's rumen and undergo exsheathment. They then migrate to the abomasum and develop into adults.
times (Sommerville, 1957). The second molt, which occurs during the L3 stage, is generally referred to as exsheathment and is a notable stage because when it occurs the larvae have entered the parasitic portion of their life-cycle (Sommerville, 1957).

Exsheathment
When infective L3 H. contortus larvae are consumed by a small ruminant, they enter the rumen and exsheathment is triggered (Sommerville, 1957). Sommerville (1957) found that in H. contortus, and other species in general, exsheathment was triggered in the gastrointestinal tract just anterior to where that specie's adults reside; these observations were confirmed by Hertzberg (2002) for trichostrongylid species.
The cuticle is a transversely striated (Ozerol & Silverman, 1972) protective covering that can shield the larva from digestion by nonspecific proteases during its free-living stages (Fetterer & Rhoads, 1996). While the process of triggering exsheathment is poorly understood, it is thought that the presence of CO2, which is mediated by carbonic anhydrase, is sensed by chemoreceptors present in the amphids of larvae and triggers the release of noradrenalin which leads to downstream activation of exsheathment (Nikolaou & Gasser, 2006). When exsheathment is triggered, larvae release an exsheathing fluid into the area under the cuticle (Sommerville, 1957;Rogers & Sommerville, 1960). Exsheathing fluid is thought to be released by excretory cells (Wharton, 1991) and is composed of 80% proteins (Ozerol & Silverman, 1969). After the exsheathing fluid is released, a refractile ring forms near the anterior end of the larva, creating a loose cap at the tip of the sheath and allowing the larva to wriggle out (Wharton, 1991).

Lespedeza cuneata
Consumption of several condensed tannin (CT) containing plants has been found to reduce gastrointestinal nematode burdens in small ruminants Shaik et al., 2006). One such plant, Lespedeza cuneata (sericea lespedeza), has been extensively researched and found to have anti-parasitic effects Shaik et al., 2006;Terrill et al., 2007;Gujja et al., 2013). Sericea lespedeza is a legume that is native to east Asia and was introduced to the United States for its potential uses including use as a hay for livestock (Ohlenbush et al., 2007). In general, feeding trials have shown that consumption of sericea lespedeza reduces fecal egg counts by greater than 50%, while reduction in adult worm counts are inconsistent (Table 1). Sericea lespedeza hay was fed to Boer goats with GIN infections for 6 weeks, and by the final week, fecal egg counts dropped by 88% compared to control animals (Shaik et al., 2006). The adult abomasal worm count of H. contortus was also reduced by 62-77% (male-female) (Shaik et al., 2006).  fed sericea lespedeza leaf meal to young male goats for up to 63 days. A 23% non-significant reduction in adult worms was found after 63 days compared to control animals, and while fecal egg counts were significantly reduced to approximately 90% lower than control animals . Ewe lambs were fed sericea lespedeza hay for 49 days and this diet was associated with 67-86% lower fecal egg counts than those of control animals . Worm counts from the treatment groups were lower than the control, but the difference was not statistically significant . Terrill et al. (2007) fed young male goats a diet of pelleted sericea lespedeza for 4 weeks. Compared to control animals a 70% reduction in the fecal egg count for the experimental diet was found, as well as a 75% reduction in adult worm burdens (Terrill et al., 2007). Young male goats were fed a sericea lespedeza leaf meal pellet for 11 weeks as a supplement to grazing, and fecal egg counts as well as combined abomasal and small intestine worm counts were both significantly lower for goats on the experimental diets than those on a control diet (Gujja et al., 2013).
Generally, it was found the birdsfoot trefoil reduced adult abomasal worm counts, but fecal egg count reductions were not consistent (Table 2). Marley et al. (2003) had male lambs grazing on birdsfoot trefoil cultivar (cv.) Leo for 35 days, and although there was no significant difference for the fecal egg counts at the end of the study, there were significantly fewer adult worms found in the abomasa of lambs on the birdsfoot trefoil diet than those on the control diet. Birdsfoot trefoil cv. Odenwälder was grazed by lambs for 17 days and, compared to the control animals, was associated with a 58% lower H. contortus fecal egg counts, but no significant difference was found in worm counts (Heckendorn et al., 2007). For two consecutive years, ewes and their lambs grazed on pastures of perennial ryegrass/white clover or birdsfoot trefoil cv.
Grasslands Goldie for 86 days and 91 days respectively ( Ramirez-Restrepo et al., 2004). Fecal egg counts of the ewes consuming birdsfoot trefoil were significantly lower than those on the control pasture both years ( Ramirez-Restrepo et al., 2004).
Fecal egg counts of the lambs were lower for the birdsfoot trefoil groups for most of the study, however, they increased to approximately equal or exceeded the control groups near weaning ( Ramirez-Restrepo et al., 2004). One-hundred twenty lambs were grazed for 95 days on either birdsfoot trefoil cv. Grasslands Goldie or perennial ryegrass/white clover with each group further split to equal groups of regularly dewormed lambs and "trigger-drenched" lambs; ivermectin was used for deworming (Ramirez-Restrepo et al., 2005a). Trigger-drenched groups were dewormed when mean fecal egg counts reached 1000 eggs/gram for either group ( Ramirez-Restrepo et al., 2005a). For the trigger-drenched groups, the lambs grazing on birdsfoot trefoil actually had significantly higher fecal egg counts on day 49 ( Ramirez-Restrepo et al., 2005a). While trigger-drenched lambs grazing birdsfoot trefoil had significantly lower abomasal worm counts for H. contortus than control lambs, they had higher abomasal worm counts of both Teladosargia circumcincta and Trichostrongylus axei (Ramirez-Restrepo et al., 2005a).

Onobrychis viciifolia
Onobrychis viciifolia (sainfoin) is a legume that is palatable to sheep and has first cut yields that are comparable to alfalfa (Tilleyet al., 2008). In general, fecal egg counts were reduced by consumption of sainfoin, but adult worm counts were not reduced ( Table 3). Lambs grazing sainfoin cv. Visnovsky for 17 days had a 57% difference in fecal egg counts compared to the control group, but no significant change in worm counts (Heckendorn et al., 2007). Sainfoin hay was fed to lambs for 56 days with a trickle infection of Trichostrongylus colubriformis being given after the first two weeks of sainfoin consumption and continuing throughout the study ( Rios-De Alvarez et al., 2008). Lambs consuming sainfoin had lower fecal egg counts than those on the control diet, but no significance was found between the post-trial worm counts ( Rios-De Alvarez et al., 2008). Lactating dairy goats living on pasture were brought indoors and fed sainfoin hay or a control hay for periods of 10 days each month; fecal egg counts were lower for does consuming sainfoin hay (Hoste et al., 2005). Cull goats living on pasture were fed sainfoin hay or ryegrass (control) hay for seven days each month (Paolini et al., 2005a). Fecal egg counts of the sainfoin group were significantly lower after 6 weeks and 8 weeks of the study (P < 0.05 and P < 0.001 respectively); around week 8 two goats from the control group died and five more were dewormed due to low packed cell volumes while no animals from the sainfoin group required treatment (Paolini et al., 2005a). Total worm counts for both groups were not significantly different (Paolini et al., 2005a). Young Alpine goats were fed sainfoin hay for 9 days, and on days 4, 5, and 6 they received trickle infections of H. contortus larvae (Paolini et al., 2005b). The goats were slaughtered and although there were lower total worm counts for the control group, the difference was not statistically significant (Paolini et al., 2005b).

Other Plant Species
Male lambs grazed Cichorium intybus (chicory) for 35 days and worm counts showed that there were fewer adult abomasal worms infecting lambs on the experimental diet than those on the control diet (P < 0.001), but no significant difference was found between the final fecal egg counts (Marley et al., 2003). Chicory cv. Grasslands Puna was also grazed by lambs for 17 days and although no significant difference was found for the worm count, a 69% difference in H. contortus fecal egg count was found (Heckendorn et al., 2007). In another study, heather (61% Calluna vulgaris L.; 25% Erica Umbellata L.; 12% Erica cinerea L.) was offered free choice to goats every three days for five months and these goats had lower fecal egg counts (P < 0.001) and no deaths, while in the control group the two goats with the highest fecal egg counts died during the study (Frutos et al., 2008). Ram lambs grazing on sulla (Hedysarum coronarium) for 28 days had lower (P < 0.05) egg counts than the control group (Niezen et al., 1995).

Structure
The anthelmintic properties of plants are primarily attributed to the plant's condensed tannin content . The term condensed tannin    (proanthocyanidin) is used to refer to polymers composed of flavan- 3-ol sub-units (Reed, 1995). The condensed tannin contents of plants vary in concentration and structure, and it is hypothesized that both of these factors contribute to the level of anthelmintic efficacy (Quijada et al., 2015). The most abundant flavan-3-al sub-units found in condensed tannins are procyanidins and prodelphinidins ( Figure 3) (Reed, 1995). The ratio of procyanidin to prodelphinidin has been proposed as a potential factor related to the efficacy of condensed tannins, with Quijada et al. (2015) finding this ratio to show a non-significant trend in efficacy in an in vitro exsheathment assay. When purified monomers of either procyanidin or prodelphinidin were tested against an in vitro exsheathment inhibition assay, the prodelphinidins were found to be more efficacious (Brunet & Hoste, 2006). For their in vitro tests, the catechin and epicatechin forms of procyanidins did not inhibit exsheathment; the gallocatechin form of prodelphinidin showed total inhibition of exsheathment, but the epigallocatechin form did not inhibit exsheathment (Brunet & Hoste, 2006). Two additional structural features that are being examined is the length of the polymer, which is measured by its molecular weight, as well as the stereochemistry of the flavan-3-ol sub-units ( Figure 1) (Quijada et al., 2015). An in vitro larval migration study by Naumann et al. (2014) looked specifically at average molecular weights of the condensed tannin containing plants and found that it had a slight correlation with efficacy, but probably is not the only factor involved.

Measuring condensed tannin concentration
The concentration of condensed tannins can be determined using 4-(dimethylamino)cinnamaldehyde method (DMAC) (Neilson et al., 2016). Since condensed tannins have binding properties, the concentration measurements can be broken down into several categories: extractable, protein bound, fiber bound, and total condensed tannins (Naumann et at., 2014). Protein bound condensed tannins are thought to be the most biologically relevant to anthelmintic properties (Naumann et al., 2014). These measurements can be determined using the butanol-HCL method, where, following a series of extractions, the concentration is determined by a measure of light absorbance at a wavelength of 550nm (Naumann et al., 2014). Condensed tannin concentration can also be measured using UV spectra (Azuhnwi et al., 2013).
The molecular weight of condensed tannins can be determined using Gel Permeation Chromatography and can be reported as either an average or a range (Huang et al., 2010). Percentages of cis or trans stereochemistry, as well as ratios of procyanidin to prodelphinidin, can also be determined by forms of chromatography (Quijada et al., 2015). Another method that can be used to determine structural characteristics of condensed tannins is matrix assisted laser desorption/ionization and time of flight mass spectral analysis (MALDI-TOF) .

Changes in condensed tannin content over the life-cycle of a plant
Condensed tannin content can vary over the course of the plant's life; a single cultivar of birdsfoot trefoil had higher condensed tannin content in 2-year vs 1-yearold plants (Hedqvist et al., 2000). Sainfoin was also found to vary in condensed tannin concentration, % cis versus % trans ratios, and prodelphinidin to procyanidin ratios over two harvests 42 days apart (Azuhnwi et al., 2013). Birdsfoot trefoil was examined for changes in condensed tannin content and it was found that for both cultivars tested the concentration was significantly lower in the fall than in the spring and summer (Gebrehiwot et al., 2002).

Variations between cultivars
Hedqvist et al. (2000) measured the variation in condensed tannins content of seven cultivars of birdsfoot trefoil and found that they varied extensively. The concentration of the condensed tannins ranged from 0.3%-1.0%, and the ratios of prodelphinidin to procyanidin ranged from 16:84 to 33:67 (Hedqvist et al., 2000). Six cultivars of sainfoin was also found to vary significantly in condensed tannin concentration, cis/trans ratios, prodelphinidin to procyanidin ratios, and molecular weight (Azuhnwi et al., 2013). For birdsfoot trefoil, cv. ARS-2620 was found to have 60%-70% more condensed tannins than cv. Norcen (Gebrehiwot et al., 2002).

Proposed Mechanism of Action
The effects of anthelmintic plants were first attributed to the broad category of plant secondary metabolites (Athanasiadou & Kyriazakis, 2004) and the search for a more specific cause of efficacy has led to the condensed tannins (Quijada et al., 2015).
Although no consensus has been reached as to the mechanism of action for the condensed tannins, there have been several hypotheses suggested Cedillo et al., 2015). The possible mechanisms can be divided into two categories: a direct mode of action, where the condensed tannins act directly on the parasite, and an indirect mode of action, where the condensed tannins increase immune response by the host . This higher immune response may be due to the ability of condensed tannins to increase the amount of protein bypassing the rumen, which allows increased uptake by the host in the small intestine . For example, a feeding trial by Rios-De Alvarez et al. (2008) found that feeding sainfoin to lambs with T. colubriformis infections resulted in increased levels of Pan T cells, eosinophils, and mast cells in the lambs' intestinal tissue. One proposed mechanism of action, where the condensed tannins act directly on the parasite, stems from evidence that the condensed tannins may be binding to the cuticle of the parasites, which may interfere with feeding, or other physiologic processes . Another proposed mechanism is that the condensed tannins may be binding to enzymes secreted by the parasite and preventing their utilization by the parasite . The mechanism of action specifically relating to inhibition of exsheathment also remains unknown ( Alonso-Diaz et al., 2008). Most exsheathment inhibition testing has been performed in vitro, where an indirect mode of action is not feasible, so the inhibition seen in these assays can be attributed to a direct effect of either condensed tannins or other compounds on the larvae. It has been hypothesized that the condensed tannins may act directly on the sheath of larvae (Williams et al., 2014), however, incubating L3 H. contortus in sainfoin extract for three hours prior to electron microscopy was not associated with any visible change to the sheath (Brunet et al., 2011). Incubation in sainfoin was associated with an internal accumulation of vesicles in L3 H. contortus larvae, rupturing of the hypodermis in Trichostrongylus colubriformis larvae, and intracellular disorganization in both (Brunet et al., 2011). It is possible that these inner changes negatively affect the exsheathing mechanism in larvae.

Other benefits of birdsfoot trefoil consumption
Other benefits of small ruminants consuming birdsfoot trefoil have been found, and these benefits may be due to condensed tannins. Lambs grazing birdsfoot trefoil cv. Grasslands Goldie were found to have higher levels of carcass weight gain per day than control groups ( Ramirez-Restrepo et al., 2004;Ramirez-Restrepo et al., 2005a).
Lambs also had higher clean-fleece weights and longer staple lengths than those on the control pastures ( Ramirez-Restrepo et al., 2004). Ramirez-Restrepo et al. (2005b) also found that there was an increase in reproductive efficiency for ewes grazing birdsfoot trefoil during the breeding season, which may be due to the condensed tannins increasing protein availability.

In vitro assays
There are several in vitro assays that are used to screen for potential anthelmintic plants. These assays include the larval exsheathment inhibition assay, larval migration inhibition assay, egg hatching assay, larval development assay, and adult motility inhibition assay (Bachaya et al., 2009;Moreno-Gonzalo et al., 2013). These methods can be used to screen large amounts of potentially anthelmintic plants to determine which plants might warrant further examination (Mengistu et al., 2016). In vitro assays are also useful for testing differing isolated types of condensed tannins (Brunet & Hoste, 2006).

In vitro exsheathment assays
The exsheathment inhibition assay has been widely used for testing the in vitro anthelmintic effects of plants as shown in Table 5 (Bahuaud et al., 2006;Alonso-Diaz et al., 2008;Azando et al., 2011;Oliveira et al., 2011a;Oliveira et al., 2011b; von Son-de Fornex et al., 2012; Moreno-Gonzalo et al., 2013;Mengistu et al., 2016). For the exsheathment inhibition assay, the larvae are placed in the chosen concentration of leaf/plant extract for a period of three hours prior to being artificially exsheathed using sodium hypochlorite and sodium chloride (Mengistu et al., 2016).
Another less harsh method of artificially inducing exsheathment, developed by Conder and Johnson (1996), bubbles CO2 gas into larvae in an Earl's Balanced Salts solution. This method has less of a negative impact on the viability and infectivity of the larvae (Conder & Johnson, 1996).

In vivo exsheathment assays
Exsheathment of H. contortus larvae in vivo has only been attempted a few times and is done by placing L3 larvae into a porous container and placing it into the rumen of a fistulated sheep (Sommerville, 1957;Hertzberg et al., 2002; ( Table 6). Sommerville (1957) used a "Cellophane dialysis sac" (p. 19) to contain the larvae and defined exsheathed larvae as those that had a refractile ring. H. contortus were found to exsheath in the rumen, and exsheathment was examined at several time points up to 5.3 hours, at which point 85% had exsheathed (Sommerville, 1957). Sommerville (1957) also reported that some lower levels of exsheathment were observed and not included in the data. Hertzberg et al. (2002) placed larvae in 5 µm mesh bags each closed with a cord and suspended them approximately 25 cm deep in the rumen. They found that larvae were 90% exsheathed after 1 hour (Hertzberg et al., 2002).  fed sainfoin or a control to fistulated sheep and compared exsheathment rates between the different diets. Larvae were placed in a microtube capped with a Nunc TM Cell Culture Insert which was then placed in a 50 µm mesh bag and suspended 20 cm deep in the rumen . After 2.7 hours the control larvae averaged about 78% exsheathed while the larvae from the sheep fed sainfoin averaged just over 30% exsheathed .

Development of a procedure for in vivo ruminal exsheathment of Haemonchus contortus L3 larvae
To be submitted as a short communication to Veterinary Parasitology Highlights: • A reproducible method for in vivo rumen exsheathment of Haemonchus contortus was developed for use in fistulated sheep.
• Over 190 capsules were tested with minimal infection of the fistulated sheep.

Abstract:
The Parasitic infections of livestock are a significant concern due to their global economic impact caused by heavy production loss and death of the hosts (Roeber et al., 2013). Haemonchus contortus, a blood feeding parasite that can cause severe anemia in the host, is the most pathogenic parasite of small ruminants (Qamar et al., 2011;. Haemonchus contortus parasites have a life-cycle that requires several conditions to be met. A single adult female parasite residing in the abomasum can lay up to ten thousand eggs in a single day, which must then exit the host via feces and remain undisturbed on the pasture until they have developed to third-stage infective larvae (L3) . In order to successfully infect a host, the L3 larvae must be ingested and undergo a critical exsheathment stage in the rumen (Sommerville, 1957;Roeber et al., 2013).
The potential for preventing H. contortus infections through inhibition of exsheathment has been widely explored through in vitro testing Azando et al., 2011;Alonzo-Diaz et al., 2011;von Son-de Fornex et al., 2012). Very few studies have reported in vivo exsheathment testing of H.
contortus (Sommerville, 1957;Hertzberg et al., 2002;, and only one has examined potential exsheathment inhibition . This may, in part, be due to the lack of an established validated procedure for conducting in vivo testing of H. contortus exsheathment in rumen fistulated sheep. Reported studies have varied in their time to exsheathment (Sommerville, 1957;Hertzberg et al., 2002; as well as recovery of L3 from exsheathment containers ).
An effort was made to reproduce published methods for in vivo exsheathment (Sommerville, 1957;Hertzberg et al., 2002;, and we were unable to replicate their results. Difficulties in reproducing the in vivo protocols utilized in previous studies (Sommerville, 1957;Hertzberg et al., 2002; included an inability to procure the supplies used, finding that a

Rumen Cannula Placement:
In the spring of 2015, rumen cannula (8C, Bar Diamond TM , Inc., Parma, ID) were placed into a fistula that was created by surgically opening the rumen wall in each of four ewes (Tufts Ambulatory Service, Woodstock, CT). Surgery was done using a paravertebral block, and needle pricks were used to ensure sufficient anesthetic was used. A portion of the ewe's skin was removed and the abdominal muscles were incised to allow access to the rumen. The rumen wall was then also incised and sewn to the cut edge of the skin. The incision area was cleaned, and the cannula was inserted. Post-surgery pain medications were administered for a minimum of five days. The surgical area was cleaned daily for the first week and as needed thereafter using a modification of a previously established procedure (Penn State, 2011).

Larvae
Haemonchus contortus L3 larvae used in these experiments were either obtained directly from Dr. Anne Zajac (Virginia Maryland College of Veterinary Medicine, Blacksburg, Virginia), or cultured from the manure of donor lambs that had been infected with larvae obtained from Dr. Zajac. Larvae were isolated from manure using the Baermann Technique (Todd et al., 1970). Each batch of larvae was under four months at the time of usage, with day zero defined as the day of Baermann collection.
During initial testing, some batches of larvae were found to not exsheath well using the in vivo methods described. These batches were eliminated from further study.
Batches that were found to exsheath well (≥ 80%) were used in future experiments and were called 'pre-tested' batches. For exsheathment tests, approximately 2000 ensheathed L3 larvae were pipetted into each containment capsule.

Metal Capsules:
Another containment capsule that was used for exsheathment testing was made out of a brass metal hose union (HU22-12MHX P, Brass Craft ® , Novi, MI) capped at each end by a female hose swivel barbed adaptor (HU126- Brass Craft ® ,Novi,MI) with the barbed swivel removed and replaced with a 5 µm CellMicroSieves TM piece of membrane (N5R, BioDesign Inc., Carmel, NY) ( Figure 1B). Post-exsheathment larvae were placed in 15 mL falcon tubes (Globe Scientific Inc., Paramus, NJ) and centrifuged at 1000 RPM for three minutes, and the top supernatant was pipetted off so that the larvae was suspended in less than 2 mL of liquid for easier microscopic examination.

Nunc TM Capsules:
Finally, a capsule using the same Nunc TM Cell Culture Inserts as used by Brunet et al.   Figure 1A). Metal capsules were made with a metal hose union (Brass Craft ® ) capped at each end by a female hose swivel barbed adaptor (Brass Craft ® ) ( Figure 1B). Nunc TM capsules were assembled by capping each end of Tygon ® tubing (ID 3/8 in, OD 9/16 in) with an Nunc TM top (white arrow) ( Figure 1C). A cannula stopper with a U-bolt fixed to it was used for exsheathment testing (Bar Diamond TM , Inc.) ( Figure 1D). Two methods of suspending capsules in the rumen were used ( Figure 1E; Figure 1F).

Larval Escape Tests:
Potential capsules were tested for the ability of larvae to escape by placing a capsule containing larvae into small (various sizes based on the capsule of interest) containers filled with tap water. These were then placed in a Daisy incubator (ANKOM Technology, Macedon, NY) set at 37 ºC with the rotation function on (1.1 RPM). This not only simulated the temperature of the rumen, but also the movement.
The capsules were left overnight and the liquid in the container but exterior to the containment capsules, was examined microscopically to determine if any larvae had escaped from the capsule, and the number of larvae that escaped was quantified.

Suspension of exsheathment capsules in Rumen:
For secure attachment of capsules, a cannula stopper with a U-bolt permanently fixed to it was used during exsheathment testing (Bar Diamond TM , Inc., Parma, ID) ( Figure 1D). Two methods were used for attachment of larvae capsules to the U-bolt.
For the first method, capsules were placed inside a short piece of capped PVC pipe (polyvinyl chloride) that contained numerous holes to allow ruminal fluid flow into and out of the PVC ( Figure 1E). These were modeled after PVC containers used for holding digestion bags in rumen fistulated animals (#3T, Bar Diamond TM , Inc., Parma, ID). During exsheathments, this container was placed inside the rumen and attached to the U-bolt securely by a cord. Metal capsules were primarily tested using this method.
A second method of attachment was also used ( Figure 1F). Each capsule was contained within a 5x10 cm ANKOM heat-sealed 50 µm concentrate bag (R510, ANKOM Technology, Macedon, NY) to prevent clogging of the capsule membranes with large particles . In order to suspend the larvae capsules beneath the fiber mat of the rumen, cords with loops at the bottom were fixed to the Ubolt so that the distance from the U-bolt to the bottom of the loop in the cord was 20 cm, which is similar to distances used by Hertzberg et al. (2002, 25 cm) and Brunet et al. (2007, 20 cm). Each capsule was then attached to its own cord (Hertzberg et al., 2002). An easy and secure attachment was achieved by using two small Zip ties and wrapping them around the capsule and through the loop in the cord. During testing of Nunc TM capsules this method was generally used.

Timing of Capsule Placement:
It has been observed that the time since a host feeds can affect the ability of rumen fluid to cause the in vitro exsheathment of H. contortus (Whitlock et al., 1959). Thus, it is important to establish a consistent exsheathment testing protocol relative to feeding time. Hertzberg et al. (2002) report feeding approximately 1 hour after insertion of larvae, while  report feeding 1 hour prior to insertion of larvae. Although initially exsheathment was tested at multiple timepoints relative to feeding, for most experiments conducted by this lab, sheep were fed just after larvae capsule insertion. This made placement of the capsules in the rumen easier as the rumens were not as full.

Length of Larval Exposure to Rumen:
Capsules with larvae were left in the rumens of the fistulated sheep for between 1.5 hours and 12 hours. As the goal was to determine the length of time that was required for larvae to consistently exsheath in high numbers, differing lengths of time were tested more or less extensively depending on results obtained. The timepoints tested included 3, 6, 8, 9, and 12 hrs.

Exsheathment and Motility Determination:
After removal from the rumen, the larvae were moved to non-membranous containers and examined under a microscope for exsheathment and motility. A larva was considered motile if movement occurred within five seconds of viewing it (Skantar et al., 2005) and exsheathed only if it had completely exited the cuticle.

Nalgene TM Capsules:
When tested for larval escapes, Nalgene TM capsules showed inconsistent results.
The results ranged from zero larvae escaping to numbers of larvae escaping that were too numerous to count. Because of the potential for high numbers of larvae escaping, these capsules were not tested in vivo.

Metal Capsules:
The metal capsules averaged 5 ± 2 larvae (0.3%) escaping per test, which was considered acceptable, and they were then used for in vivo testing. Metal capsules were tested for exsheathment percentages after 3, 6, 9, and 12 hours of rumen exposure ( Figure 2). The percent exsheathment (mean ± SEM) for these timepoints were 66 ± 2%, 81 ± 2%, 80 ± 5%, and 88 ± 2%, respectively. Tarnishing of the brass was observed, and since the brass capsule was not inert, its use was discontinued.

Nunc TM Capsules:
The Nunc TM topped capsules averaged 3 ± 2 larvae (0.2%) escaping per test. After the transition to Nunc TM capsules was made from metal capsules, exsheathment was most extensively examined after 6 and 8 hours of rumen exposure ( Figure 2). The mean percent exsheathment found at these two timepoints were 73 ± 4% and 77 ± 1%, respectively. The most variable factor observed was the larvae themselves. Some batches of larvae had much better exsheathment than others. By pre-testing batches of larvae to determine which had the highest exsheathment percentages, and using only those batches that exsheathed well, the variation in the percent exsheathment was greatly reduced. Twelve tests were run with two capsules per ewe using only pretested larvae with a total of 96 capsules being tested ( Figure 2). The larvae from these tests had average exsheathments of 82 ± 1%. Exsheathment percentages were examined at four timepoints. Nunc TM capsules: Results from exsheathment percentages examined at two timepoints. The eight hour timepoint is further split into 8a and 8b. While 8a represents all of the applicable exsheathments measured at eight hours, 8b shows only the exsheathments that were completed using the final methods including pre-testing the larvae.

Fecal Egg Counts:
During the period of Nunc TM top capsule testing, fecal samples were taken weekly, and fecal egg counts were performed. The average fecal egg count for the four ewes during the testing was 25 ± 4 eggs per gram. The average for the eight weeks prior to the testing had been 25 ± 7 eggs per gram.

Discussion:
Testing of three containment capsules for the in vivo exsheathment of H. contortus showed that the Nunc TM capsules were most suited to these tests. This was determined through the measurement of larval escapes and exsheathment percentages attained in fistulated animals.
The Nalgene TM capsules did not fulfill the requirement for sufficiently containing the larvae and thus were not tested in vivo to see if larvae exsheathment was attained.
While the metal capsules described in this paper fulfilled both of these requirements, due to the observed tarnishing of the brass these capsules were discontinued. It was also hypothesized that the size and weight of the metal capsules combined with the PVC suspension chamber (500g total) would allow only minimal movement of the capsules within the rumen and therefore might not accurately represent in vivo larval experience. Thus, neither of these capsules are recommended for in vivo exsheathment testing.
While the containment bags used for in vivo exsheathments by Sommerville It was found that results with only pre-tested batches of larvae had more consistently successful exsheathments. The finding that exsheathment was not consistent between different larvae batches may explain the comments of Sommerville (1957) who reported finding that in regards to in vivo exsheathment (referred to here as ecdysis) "Occasionally slower rates of ecdysis than those recorded here were observed, particularly with H. contortus" (p. 21). Identifying the variable that causes some batches of larvae to not exsheath well could prove useful not only to researchers by making their in vivo results more consistent, but could itself be considered as a potential control for parasite infection.

Conclusion:
This procedure opens the way for increased in vivo testing of H. contortus exsheathment, and using these methods, potential exsheathment inhibitors that have high efficacy in vitro can more readily be evaluated in vivo.

Effect of birdsfoot trefoil hay on in vivo exsheathment of Haemonchus contortus
Highlights: • Birdsfoot trefoil hay did not inhibit exsheathment of Haemonchus contortus.
• Contrary to previous in vitro studies, condensed tannin plants may not inhibit exsheathment of Haemonchus contortus in vivo.

Abstract:
Although extensive research has been done on the inhibition of exsheathment of Keywords: exsheathment, ecdysis, Haemonchus contortus, barber pole worm

Introduction:
Internal parasites are a detriment to the health of small ruminants and place a major economic burden on small ruminant producers worldwide (Nieuwhof & Bishop, 2005;Sackett et al., 2006;Qamar et al., 2011). The main parasites include Teladorsagia circumcincta, several Trichostrongylus species, and Haemonchus contortus (Roeber et al., 2013). Haemonchus contortus is found globally and is the most pathogenic gastrointestinal nematode (GIN) of small ruminants (Qamar et al., 2011;Roeber et al., 2013;. This parasite feeds on the blood of its host, and infections can cause anemia, reduced production of wool, milk, and meat, and in severe cases may lead to the death of the host (Roeber et al., 2013;Preston et al., 2014). While commercial anthelmintics are commonly used for the control of internal parasites in small ruminants, H. contortus have become increasingly resistant to all of the commercially available anthelmintics (Howell et al., 2008;Gilleard, 2013). Parasite resistance to anthelmintics in small ruminants has impacted Australia since the 1980s, and more recently it has become a global problem (Waller et al., 1995;Manikkavasagan et al., 2013;Chandra et al., 2015).
A variety of condensed tannin containing plants have been tested for potential antiparasitic properties with several being found to affect fecal egg counts or worm burden counts . Sericea lespedeza (Lespedeza cuneata) has been well documented as a plant with anthelmintic properties (Shaik et al., 2006;Terrill et al., 2007;Gujja et al, 2013). Feeding sericea lespedeza hay to buck kids was associated with 88% lower fecal egg counts and 61% lower abomasal worm burdens than animals receiving a control diet (Shaik et al., 2006). Unfortunately, sericea lespedeza's area of adaptation does not include the northeastern United States (Ohlenbush et al., 2007). Unlike sericea lespedeza, birdsfoot trefoil (Lotus corniculatus) has a large area of adaption that includes the northeastern United States (Steiner, 1999). Feeding a fresh fodder of birdsfoot trefoil (BFT) cultivar (cv.) Odenwalder to lambs with established worm burdens for 17 days was found to reduce fecal egg counts by 63% compared to control animals (Heckendorn et al., 2007). The fodder contained approximately 68% BFT and had a condensed tannin content of 15g/kg (Heckendorn et al., 2007). Lambs grazing birdsfoot trefoil cv. Leo for 35 days were also found to have less total adult helminths in their abomasum and intestines than lambs on a control diet (Marley et al., 2003).
Although the mechanism of action that causes some condensed tannin containing plants to have anthelmintic properties has not yet been determined (Cedillo et al., 2015), the level of efficacy that these plants exhibit is thought to be related to the structure of the condensed tannins present (Quijada et al., 2015). One such structural component is the ratio of prodelphinidins to procyanidins (Brunet & Hoste, 2006;Quijada et al., 2015). An in vitro exsheathment inhibition test showed that purified prodelphinidins were more effective at preventing larval exsheathment than procyanidins (Brunet & Hoste, 2006). Other structural features that are being examined as potential indicators of efficacy include the stereochemistry of the condensed tannin sub-units and the molecular weight of the condensed tannins (Quijada et al., 2015). Different cultivars of condensed tannin plants, including birdsfoot trefoil, often have varying condensed tannin contents (Hedqvist et al., 2000;Azuhnwi et al., 2013).
During H. contortus infection, third-stage (L3) larvae are consumed by the host, and while the larvae are in the rumen, exsheathment is triggered (Sommerville, 1957).
Exsheathment is the process by which larvae shed their outer protective cuticle. In vitro testing of certain condensed tannin containing forages have found that these plants are capable of reducing the percentage of H. contortus larvae successfully completing the exsheathment stage, and this assay is used to screen for potential anthelmintic plants Alonzo-Diaz et al., 2011; von Son-de Fornex et al., 2012;Moreno-Gonzalo et al., 2013;Mengistu et al., 2016).
Only one previous study has attempted to show a similar result in vivo . That experiment was done using fistulated sheep and found that feeding increasing amounts of the condensed tannin containing plant sainfoin (Onobrychis viciifolia) slowed the exsheathment process . The study fed sainfoin as a fresh chopped forage , The purpose of this study is to evaluate the in vivo ability of birdsfoot trefoil hay to prevent exsheathment of H. contortus, as well as determine the difference in efficacy between three cultivars of birdsfoot trefoil. In an in vitro test, freeze-dried birdsfoot trefoil was found to reduce the exsheathment of the cattle parasites Cooperia oncophora and Ostertagia ostertagi (Novobilsky et al., 2011). In our laboratory, Barone et al. (2016) found that, during in vitro tests, aqueous extracts of varying cultivars of freeze-dried birdsfoot trefoil reduced the percent exsheathment of H.
contortus larvae. Based on these results, three commercially available cultivars representing a broad range of efficacies were chosen for in vivo testing.

Experimental Design:
Four ruminally fistulated [3][4] year old Dorset cross ewes were fed three cultivars of birdsfoot trefoil hay (Pardee, Empire, and Bruce) and a control hay of alfalfa/grass in a Latin 4x4 design ( The timeline of feed transitions and testing periods.

Ewes:
The This was done as a standing surgery using a paravertebral block. A circular incision equivalent to the inner diameter of the cannula was made through the skin and the skin was removed. The abdominal muscles were incised and part of the rumen was drawn out and an incision was made. The cut wall of the rumen was sewn to the cut edge of the skin, and the cannula was inserted. Post-operative care included daily cleaning of the surgical area for the first week, and cleaning as needed thereafter using a modification of the previously established procedure by Penn State (2011). During the study, the ewes were housed individually in indoor 8'x8' pens at Peckham Farm (University of Rhode Island).

Diet:
Throughout the study, ewes were provided with free choice water and minerals.
The control diet contained a mix of alfalfa and grass hay and was purchased from an outside source. When transitioning between the control hay and birdsfoot trefoil hay, on day 1 of a new diet, ewes were fed 25% of the new diet and 75% of the previous diet. The percentage of the new diet was increased by 25% each day until 100% of the feed was the new diet. Diets were formulated to meet or exceed dietary requirements (National Research Council, 2007). Nutrient analysis of all feedstuffs was conducted by Dairy One (Ithaca, NY). For each cycle, a 16% protein sheep pellet (Central Connecticut Co-op, Manchester, CT; Blue Seal, Muscatine, IA) was fed equally to the ewes on each of the four diets. This pelleted grain was fed at a rate of 68 g/day during cycles one and two, but was gradually increased to 454 g/day for cycles three and four.

Larvae:
Haemonchus contortus larvae used in the exsheathment trials were provided by Dr.  (Todd et. al, 1970) was used to recover the larvae from the fecal samples incubated at room temperature for eight days. After incubation, the manure was placed in cheese cloth and suspended in a funnel with a short piece of tubing attached to the stem. A clamp was affixed to the end of the tubing. The funnel was filled with water covering the fecal matter. The larvae migrated out of the manure and were collected at the bottom of the clamped tube. The tube was then clamped above the larvae, and the lower clamp was removed, allowing the larvae suspended in only a small amount of water to be collected. Larvae were considered to be age zero on the day of collection and were under three months of age at the time of use. After collection from the cultures, larvae were stored at 4°C and adjusted to room temperature for 20-24 hours prior to placement in the rumen. In order to maximize exsheathment rates, batches of larvae were selected by testing their exsheathment rates in the control animal prior to the study's exsheathment tests.

Exsheathment:
The exsheathment method used for this study was developed in this laboratory suspended in the rumen of the ewe by a 20 cm cord ( Figure 2B). The capsules were placed in the rumen of each ewe immediately prior to the morning feeding and removed after eight hours. After removal from the rumen, the larvae in each capsule were transferred to a 2 mL capsule and a minimum of 150 larvae were examined for motility and exsheathment. Only motile larvae were included in exsheathment calculations. Motility was defined by movement within 5 seconds of viewing (Skantar et al., 2005). Larvae were defined as exsheathed if they were entirely free of their cuticle. Percentages of exsheathment were adjusted based on pre-experiment larval exsheathment. This was accomplished by using the following formulas.  (Figure 2A). Capsules were each placed in a 50 µm heat-sealed concentrate bag and suspended on a 20 cm cord attached to a U bolt on the inner edge of the cannula plug ( Figure 2B).

Rumen pH
The rumen pH of each ewe was taken immediately prior to every exsheathment test. A sample of rumen fluid was taken from deep in the rumen, and pH was measured within 15 minutes using an accumet TM portable pH meter and automatic temperature compensation (ATC) electrode (AP115, Fisher Scientific, Hampton, NH). The meter was calibrated per manufacturer instructions with buffers of pH 4 and pH 7 prior to each use.

Condensed Tannin analysis:
The condensed tannin concentration was determined using the 4-  (Feliciano et al., 2012;Krueger et al., 2016). Analyses were performed on each cultivar of birdsfoot trefoil using pure freeze-dried samples. Results were adjusted to reflect the % BFT biomass of each cultivar's hay.

Statistics:
Data was analyzed with R (R Core Team, 2016, Vienna, Austria) using an additive effects model appropriate for Latin 4x4 designs (Montgomery, 2013). The experimental model used was γijk = µ + Τi + αj + βk + εijk where µ is the overall mean % exsheathment, Τi is the treatment effect from feeding birdsfoot trefoil (i = 1, 2, 3, 4), αj is the effect of the ewes (j = 1, 2, 3, 4), βk is the effect of Latin square rows (k = 1, 2, 3, 4), and εijk is the error. Differences were considered biologically relevant at P < 0.05. Analysis of food intake, pH, and bodyweight data were completed using the same model as exsheathment data, but the response variables were daily feed intake, pH, and percent change in weight from the pre-study baseline, respectively. Although the feed intake data was not normally distributed, the analysis was justified due to the large sample size (Ghasemi & Zahediasl, 2012).

Diet and Ewes:
The results of the forage analysis are reported in Table 2. The control diet was altered to a higher ratio of grass hay to alfalfa hay after the first cycle to better match the protein levels of the BFT diets. The percentage of dry matter consumed for each diet is reported in Table 3. After the first two cycles, the ewes were allowed free choice access to treatment and control hay, and their grain intake was increased (68 to 454 g/day) in order to maintain body condition, as there was a decrease in percent bodyweight during the cycles 1 and 2 ( Figure 3). Table 5 shows the comparison of average daily nutrient intake to National Research Council (NRC) requirements. The ewes' average change in percent bodyweight by cycle was negative for cycles 1 and 2, and positive for cycles 3 and 4 (Table 4). Fecal egg counts were low throughout the study with the highest egg count at only 150 eggs per gram.

Table 2
Comparison of the nutritional content of the forages and grain fed during study.

Exsheathment:
There was no difference in percent exsheathment between the three cultivars of birdsfoot trefoil and the control diet (P = 0.29), and no difference in percent exsheathment between the cultivars (Figure 4). On average, the motility was similar pre and post exsheathment. Pre-experiment motility was 94 ± 1%.

Rumen pH:
Rumen pH is reported by diet and cycle (Table 6). Rumen pH was higher when BFT Empire was consumed compared to the control (6.60 ± 0.04 vs 6.37 ± 0.10).
Rumen pH did not differ between the control diet and BFT Bruce or BFT Pardee.
There was also no difference in rumen pH between the cultivars of birdsfoot trefoil.

Table 6
Comparison of pH for each diet and cycle.

Condensed Tannin analysis:
The adjusted condensed tannin content of each cultivar of birdsfoot trefoil was 5.3 mg/g, 2.6 mg/g, and 8.4 mg/g for Pardee, Empire, and Bruce, respectively.

Discussions:
This study found that in vivo exsheathment of H. contortus was not affected by consumption of birdsfoot trefoil hay, and there were no differences in exsheathment between the cultivars fed. An additional finding was that the rumen pH of the ewes consuming Empire was higher than those consuming the control diet of alfalfa/grass.
Although a difference was found between the ruminal pH of ewes consuming birdsfoot trefoil cv. Empire and the control diet, these pH values were still within the normal range for sheep and are likely not biologically relevant (Dehority & Tirabasso, 2001). No significant difference in exsheathment was found between the control diet and the three cultivars of birdsfoot trefoil. Thus, feeding birdsfoot trefoil hay to ewes did not inhibit the exsheathment of L3 H. contortus placed in their rumens, despite Barone et al. (2016) finding that the cultivars Empire and Pardee greatly inhibited exsheathment in vitro. The study by Barone et al. (2016) used a 25 mg/mL aqueous extract of freeze dried birdsfoot trefoil and exsheathment was inhibited by over 75% for the cultivars Pardee and Empire, but was not inhibited for Bruce. Exsheathment of H. contortus has been extensively researched in vitro, and is used as a method for screening potential anthelmintic plants von Son-de Fornex et al., 2012; Moreno-Gonzalo et al., 2013;Mengistu et al., 2016).
Prevention of in vivo exsheathment by the feeding of condensed tannin containing plants was examined in a study by , and it was found that in animals consuming 100% sainfoin diets, H. contortus larvae exposed to ruminal fluids for 2.7 hours had 59% lower exsheathment than animals on control diets. Other than the difference in the condensed tannin containing plants that were fed (sainfoin vs birdsfoot trefoil), there are several other variations between the studies that could explain the contrasting results.  fed fresh sainfoin while the birdsfoot trefoil was fed as hay. Condensed tannin content of the birdsfoot trefoil may have been altered by the hay drying process or the extended storage if low levels of fermentation occurred. A previous study found that the condensed tannin containing forage sericea lespedeza had a lower condensed tannin content as hay (15.3%) than as a fresh forage (19.9%) (Puchala et al., 2012). Terrill et al. (1990) found that freeze drying was the preservation method that best maintained the condensed tannins found in fresh plants. Since the condensed tannin content of the hay fed during this study was calculated based on freeze-dried samples, the actual concentration may have been lower. The condensed tannin content of the birdsfoot trefoil fed during this study was calculated to be between 0.3% and 0.8%, while the tannin content of the sainfoin fed by  was 3.9%.
Additionally, while the forage fed by  was reported to be 100% sainfoin, the birdsfoot trefoil hay used in this study was found to be between 63-70% birdsfoot trefoil due to the organic management of the hay fields (Ferguson, unpublished). Thus, there were lower concentrations of condensed tannins in the birdsfoot trefoil hay than in the sainfoin. However, the longest exposure to rumen fluid that the larvae experienced in the tests by   Condensed tannin concentration is likely not the only factor involved in anthelmintic efficacy. Condensed tannins are polymers of flavan-3-ols and can exist in many forms (Reed, 1995). Some of the structural differences of condensed tannins that have been proposed as potentially related to anthelmintic efficacy include the ratio of procyanidins to prodelphinidins, molecular weight, and stereochemistry (Molan et al., 2003;Brunet & Hoste, 2006;Naumann et al., 2014). Brunet & Hoste (2006) found that monomers of prodelphinidins were more effective at inhibiting in vitro larval exsheathment than procyanidins. An in vitro larval migration inhibition experiment found that the molecular weight of condensed tannins has a slight correlation (R 2 = 0.34, P = 0.05) to efficacy (Naumann et al., 2014). The effect of stereochemistry was investigated by Molan et al. (2003) by comparing monomers of condensed tannins through an in vitro egg hatch assay. Significance due to stereochemistry was only found when testing flavon-3-ol gallate derivatives (Molan et al., 2003). It has also been suggested that other secondary compounds besides condensed tannins may be involved in inhibiting exsheathment (Mengistu et al., 2016). An in vitro exsheathment study of ten east African plant species found that Maerua angolensis, which does not contain condensed tannins, inhibited H. contortus exsheathment. Thus, without further research to determine the effects of structural differences on anthelmintic efficacy, lower vs. higher condensed tannin content alone does not provide sufficient information to draw a conclusion as to why there was a difference in results between this in vivo exsheathment study and the one conducted by .
Although in vivo exsheathment trials have been limited, if exsheathment is inhibited by anthelmintic plants, then it would be expected that in feeding trials where infections are given to animals already consuming an anthelmintic plant, experimental animals would have significantly lower established worm burdens than control animals. However, this is often not the case. Feeding sainfoin (Onobrychis viciifolia) to small ruminants has been associated with a decrease in fecal egg counts (Hoste et al., 2005;Paolini et al., 2005a;Heckendorn et al., 2007;Rios-De Alvarez et al., 2008).
However, Paolini et al. (2005b) found by worm burden counts that feeding sainfoin to young goats did not prevent L3 H. contortus from developing and establishing themselves in the abomasa of the goats. This indicates that exsheathment was not inhibited. Small ruminant feeding trials with sericea lespedeza (Lespedeza cuneata) have found this plant to be associated with anti-parasitic effects Shaik et al., 2006;Terrill et al., 2007;Gujja et al., 2013). However, a study by  found that when sericea lespedeza hay was fed during a trickle infection of H. contortus, while the fecal egg counts of the ewe lambs consuming the experimental diet were approximately 67%-82% lower than the control, the adult abomasal worm count was not significantly different between diets.
This is again indicating that exsheathment was not inhibited and implies that the anthelmintic effects must be occurring during a different stage of the H. contortus lifecycle.
In order to determine whether the discrepancy in results between this study and the study by  was due to a difference in anthelmintic properties between the two experimental diets, or due to the shorter ruminal incubation period used by Brunet et al., further in vivo exsheathment studies should be conducted.

FUTURE DIRECTIONS
Introduction: The goal of this research was to determine whether birdsfoot trefoil inhibits the in vivo exsheathment of Haemonchus contortus L3 larvae and if some cultivars of birdsfoot trefoil have higher efficacy than others. During the first phase of this research, a containment capsule for the in vivo exsheathment of H. contortus was designed and tested, and a method for testing larval exsheathment rates in rumen fistulated sheep was established. One difficulty that presented itself was frequent batches of larvae that did not exsheath well. For the purpose of these experiments, this difficulty was overcome by discarding these batches of larvae. Although preliminary testing was done to try to determine a potential difference between batches of larvae that exsheathed well and those that didn't, no clear answer was found. In the future, testing should be done to determine if there is an environmental factor involved in the growth and storage of these larvae that is contributing to this issue. For the second phase of the research, a feeding trial was completed using four rumenally fistulated ewes fed three cultivars of birdsfoot trefoil hay and a control hay in a Latin 4x4 design. There was no difference observed between the exsheathments of H. contortus

Weed Control:
The birdsfoot trefoil hay that was fed during this exsheathment study was between [63][64][65][66][67][68][69][70]unpublished), while the rest was weeds. This high number of weeds present was due to the goal of organically managing the birdsfoot trefoil hay plots. However, in future exsheathment testing if weed control was used on the plants during their growth, the higher concentration of the anthelmintic plants may show some level of exsheathment inhibition.

Grazing:
The birdsfoot trefoil that was fed during the feeding trial was hay that had been stored for approximately one year. Another variable that could be tested for is the potential for changes in the condensed tannins during the haying process and storage.
In order to remove the hay drying factor, a feeding trial can be done with the animals grazing birdsfoot trefoil prior to in vivo exsheathment tests. Alternatively, plants could be clipped and fed to the animals daily as a fresh fodder.

Other Cultivars or Species:
While three cultivars of birdsfoot trefoil hay were not found to inhibit in vivo Exsheathed non-motile was same reported in the same column as ensheathed non-motile 2 Larvae not set out night before 3 PVC not used (heat sealed bag) 4 Killed with Lugol's iodine 5 Read next day 6 Started using spacers in PCV 7 Less than 2000 larvae used 8 Pre-motility may have been lower (some dried non-motile ones not counted) 9 More than 2000 larvae used 10 Less than 100 larvae examined larvae (determined previous day) into softened tube.

Carefully insert second Nunc TM top into open end of tube.
11. Place in 37ºC water after seal is made and push Nunc TM top into tube so that 3/4ths is covered by the tubing.
12. Using the syringe and needle, insert the needle into the capsule near the middle of the capsule, but at a nearly parallel angle so that the needle enters the inner part of the tube near one of the Nunc TM tops.
13. Submerge at least one end of the capsule underwater in the 37ºC water and draw back on the syringe to remove the air pocket. The goal is to make the air pocket as small as possible without removing any liquid containing the larvae.
14. Leave the completed capsule in the water and repeat steps 7-13 until all eight capsules are completed.
15. Fill another large bucket half way with 37ºC water.
16. Remove a capsule from the water and dry the outsides using paper towels.
17. Place in one heat seal-able bag and seal end using impulse sealer.
18. Using two zip ties, attach the capsule to one end of a cannula plug string.
• Wrap one zip tie around the tube and bag and through the loop on the cannula plug string; tighten the zip tie to a snug position.
• Repeat with the second zip tie.
19. Repeat steps 16-18 until  • Using arm with shoulder length glove, cup capsules in hand and insert as deep as possible into rumen.
• Orient strings to be at the bottom of the U-bolt they are tied to.
• Insert cannula plug and orient so that the outer U-bolt is parallel to the ground (this makes the inner U-bolt perpendicular to the ground).
23. Repeat for the rest of the fistulated sheep in the order of increasing ear-tag numbers (ex: 1206, 1301, 1308, 1314).
25. Release the ewes and give them their morning feeding. 26. Rinse regular cannula plugs and place somewhere where their smell won't bother others.
27. Read remaining larvae used for set-up to determine pre-experiment motility and exsheathment percentages.
• Look at a minimum of 150 motile larvae.
• Be sure to record the age of the larvae and other flask information.
28. Clean-up from set-up. 29. Get together afternoon supplies • Fill empty bucket with: more shoulder length gloves, extra regular gloves, 4 halters, small scissors, and labeled cups.
• label the eight 2mL capsules with ear tag numbers (two for each sheep).
30. Wait determined amount of time (8 hours) and remove capsules (3-4pm depending on start time).
31. Approximately 30 minutes before removing capsules: • Turn on the Thermo water heater to 37ºC (confirm temp with thermometer).
• Fill bucket with 37ºC tap water.
32. Bring supplies in bucket from step 29 and the bucket with water to sheep.
33. Dump half the 37ºC water in with the cannula plugs that were removed that morning to soften the plugs.
34. Dump half the remaining water into the labeled cups.
35. Catch the sheep with the movable panel and tie them using the halters.
36. Remove capsules starting with sheep with lowest ear-tag number.
• When removing capsules avoid pulling out by the strings. Instead reach into rumen with gloved hand, cup capsules, and remove gently.
37. Replace cannula plug with plain plugs from the morning.
38. Cut and discard both zip ties and cut heat sealed bag off of larvae capsule.
39. Rinse capsule in remaining water and place in appropriately labeled cup.
40. Repeat steps [36][37][38][39][40]  with an additional buffer of Bruce at the end. The field was managed organically and the hay was harvested in 2015. Prior to harvesting, the cultivar rows were measured and marked, and the dividing strips between the cultivars were mowed to provide separation between the cultivars. Three random samples were taken of each cultivar.
These samples were cut approximately 4 inches above the soil to correspond to the hay harvesting height. The birdsfoot trefoil was separated from the other plants and both were dried. The dried samples were weighed and the % biomass was determined for each cultivar. For hay production, the birdsfoot trefoil was cut, allowed to air dry, sprayed with PRESERVOR TM hay and crop treatment (IBA Inc., Millbury, MA), and baled into large round bales. Each bale was labeled with the cultivar name.