Agronomic Characteristics of Birdsfoot Trefoil (Lotus corniculatus L.) in the Northeast

Tanniferous forages, such as birdsfoot trefoil (Lotus corniculatus L.), are a potential control for the parasitic nematode Haemonchus contortus (barber pole worm). Gastrointestinal parasites cause huge economic loss in small ruminant production. Tanniferous forages have been shown to reduce counts of parasite eggs in feces of small ruminants. Currently the tanniferous forage Lespedza cuneate L. is used in the southern United States in control of gastrointestinal nematodes of small ruminants. It is not a viable option in the Northeast due to insufficient winter hardiness. Birdsfoot trefoil is adapted to the Northeast and may be a viable option as a tanniferous forage for small ruminant production if populations exist that combine anthelmintic efficacy with strong agronomic performance. To address this need, I collected data on the agricultural performance of 51 Plant Introductory (PI) accessions and six commercial cultivars. In a three-year field study (2013, 2014, and 2015) I evaluated growth habit, winter hardiness, pest resistance, uniformity and vigor for 57 populations. In 2015, I tested 19 PI accessions and six commercial cultivars for grazing tolerance, regrowth, and uniformity and vigor in a rotationally grazed pasture trial. Birdsfoot trefoil accessions were chosen for the studies based on condensed tannin contents reported in the literature. Tannin content and in vitro anthelmintic efficacy were compared to agronomic characteristics. Growth habit was assessed using height/width ratios measured once each year. Winter hardiness was assessed as survival rate counted each spring. Birdsfoot trefoil is susceptible to potato leafhopper; damage by pests were visually evaluated. Quality was visually evaluated based on vigor and uniformity within each population. Vigor and uniformity was assessed yearly in the nursery plantings and immediately before each grazing event in the grazing trial. The eight-week grazing trial allowed for two grazing events for each of the nine replications. Grazing tolerance and regrowth were measured weekly for each replication throughout the eight weeks of grazing. Accessions and cultivars showed a significant difference in 2013 and 2015 for potato leaf hopper damage (P <0.0001). There were also significant differences between accessions and cultivars for population uniformity and vigor scores and winter survival (P <0.0001). Winter survival ranged from 0 to 100% while uniformity scores of surviving plots ranged from 1 to 5. A positive correlation was seen between plant survival and uniformity and vigor scores both in 2014 and 2015. PI306182, S3149, PI262530, PI262529, and PI255305 were high in vigor and uniformity scores averaged across 2014 and 2015; 2013 scores were not significantly different. ‘Pardee’, ‘Leo’, ‘Empire’ and ‘Bull’ were highest in uniformity and vigor scores for commercial cultivars averaged across 2014 and 2015. Growth habit was measured as height/width ratio and significant differences were seen in 2013 and 2015 but not in 2014. Cluster analysis divided the 51 accessions and six commercial cultivars into 6 clusters. Clusters did not correlate with tannin levels or efficacy but cluster means allowed for evaluation of agronomic characteristics of similar populations. All six commercial cultivars were clustered together in cluster 1 (N= 18) with favorable agronomic characteristic results. All the commercial cultivars also showed high anthelmintic efficacy, with the exception of ‘Bruce’. PI193725, PI162425, PI180171, PI228286 and PI325379 are promising accessions that were included in cluster 1, ranking high across agronomic characteristics and in in vitro anthelmintic efficacy. In the rotational grazing trial regrowth and combined uniformity and vigor differed significantly between populations for both grazing events. Plant survival varied widely with a low of 56.92% for PI232098 and a high of 95.06% for ‘Empire’. After agronomic characteristics were assessed, anthelmintic efficacy and condensed tannin content were compared. In vitro efficacy and condensed tannin content showed no correlation. High efficacy was positively correlated with plant survival rates and uniformity and vigor. Regrowth was independent of anthelmintic efficacy. This research identifies commercial cultivars and PI accessions with potential for anthelmintic efficacy against H. contortus and successful agronomic performance as a legume forage in the northeastern U.S. PI193725, PI180171, PI162425, PI228286, PI325379, ‘Empire’,’Norcen’,’Bull’, ‘Pardee’ and ‘Leo’ performed well agronomically and were efficacious in in vitro tests against H. contortus.

PI325379 are promising accessions that were included in cluster 1, ranking high across agronomic characteristics and in in vitro anthelmintic efficacy.
In the rotational grazing trial regrowth and combined uniformity and vigor differed significantly between populations for both grazing events. Plant survival varied widely with a low of 56.92% for PI232098 and a high of 95.06% for 'Empire'.
After agronomic characteristics were assessed, anthelmintic efficacy and condensed tannin content were compared. In vitro efficacy and condensed tannin content showed no correlation. High efficacy was positively correlated with plant survival rates and uniformity and vigor. Regrowth was independent of anthelmintic efficacy. Thank you to Tim Sherman and the agronomy farm crew for many hours of work on the field plots and to Nick Miniter and the Peckham Farm crew for care of the sheep that were used. Thank you to the students that set up the project before me.
I would like thank my husband, Joseph Fox for support throughout graduate school, it was not always easy! I would also like to thank Lorraine Perricone-Dazzo who inspired me to go to graduate school.
Finally funding for this project was provided by USDA OREI Grant No. 2012viii

INTRODUCTION
Despite an overall decline in sheep and goat farming in the United States the northeast region has seen an increase in production and demand in the past few decades (Jones, 2004). This is due to the increasing demand for fresh sheep and goat products from the local food movement. However, due to sporadic availability, imported meat from Australia and New Zealand supplements the U.S. market (Stanton, 2012). The factors limiting U.S. production are the ability to produce pasture and gastrointestinal nematode parasites. Climate, soil, and landscape are conducive to forage production including hay and pasture. Challenges include over grazing and underutilizing pastures (Jones, 2004).
Heamonchus contortus, a gastrointestinal nematode parasite, is responsible for the majority of economic loss to parasites in small ruminants . The spread of H. contortus takes place on the pasture. Helminth eggs are deposited in the feces of infected animals; the larvae are then ingested by animals while grazing. The susceptible animal becomes anemic and loses weight and death is likely (Santos et al., 2012). Heamonchus contortus larvae can live in a hypobiotic state in an animal throughout the winter and then resume development to the adult stage and lay eggs on pastures in the spring. With female adult nematodes depositing 5,000 to 10,000 eggs per day H. contortus is detrimental to all sheep and goat producers (Leite-Browning, 2006). H. contortus needs to be controlled to reduce economic loss in sheep production.
Currently synthetic anthelmintic treatments are used to control H. contortus . Organic producers cannot market meat as organic if they treat animals with synthetic anthelmintics, but have no other economically viable option . Routine use of synthetic anthelmintics leads to development of drug resistance in the H. contortus population; resistance has been documented to all three available drug classes . Rotational grazing is routinely used to reduce infection, but it is labor intensive and requires large amounts of land. In the Northeast high prices for farm land and the rapid colonization of vacant land by woody species means that sufficient land is often not available (Colvin et al., 2008).
Forages containing condensed tannin (CT) (complexes of proanthocyanidin), a secondary plant metabolite, have the ability to suppress gastrointestinal parasites . Condensed tannins are a heterogeneous series of monomers that vary in structure. The structure of condensed tannins are species specific and change throughout plant development (Haring et al., 2007). Condensed tannin content is highest in the leaves of forages and varying tannin structures allow astringency to vary . Tanniferous forages have shown a direct suppression of gastrointestinal parasites by disrupting the lifecycle of the nematode and preventing larvae from developing to the reproductive stage in in vitro tests .
Birdsfoot trefoil (Lotus corniculatus L.) is a tanniferous forage that can be adapted to the Northeast's harsh climate and grows well on poorly drained and poorly aerated soils. Birdsfoot trefoil (BFT) incorporation in ruminants' diets is beneficial even in the absence of parasites, resulting in increased protein and amino acid intake without bloating, increased wool production and increased growth .
Birdsfoot trefoil production has further environmental benefits including reducing methane and ammonia emissions and converting atmospheric nitrogen to forms useable by plants and animals .
Birdsfoot trefoil has an established economic importance as a forage and for hay production. Breeding programs in the United States and Canada have been developing new varieties adapted to the Midwest with traits that will produce higher yield, but few varieties adapted to New England have been produced since the 1970s, and old varieties are generally unavailable . None of the existing varieties have been developed specifically for use as anthelmintics. The agronomic challenges associated with using BFT include seed harvesting, stand establishment, and susceptability to root and crown rot. Once pasture is established BFT can be productive for many years with minimal input due to self seeding.
This research evaluated agronomic performance of BFT populations selected for potential as an anthelmintic forage in the northeastern U.S. Evaluation of data from field plantings determined agronomic performance based on growth habit, stand establishment, winter hardiness, grazing tolerance, regrowth speed and pest damage.
Fifty-one accessions from the core subset at the National Plant Germplasm System and six commercially available varieties were evaluated. The results were cross referenced with the tannin profile and anthelmintic efficacy of each accession. The research was designed to test the following hypotheses.
There will be significant difference in plant establishment and uniformity and vigor between populations. The different phenotypes in the numerous accessions of BFT will result in a wide range of adaptability to Northeast weather conditions .
There will be significant differences between populations in winter survivability. A wide range of origins and adaptability affects the ability of the populations to survive the frost and harsh winters of the Northeast .
The populations that show pest and disease resistance will have a higher growth rate. Pests and disease can limit the amount of biomass a variety can produce. Birdsfoot trefoil is particularly susceptible to root and crown rot and potato leaf hopper (Seaney and Hensen, 1970).
European type growth habit populations will show a faster regrowth after grazing than Empire type growth habit populations. Empire type BFT is more prostate in growth habit but are slower to establish and regrow after harvest or grazing than European type .

Birdsfoot Trefoil Agronomic Traits
Birdsfoot trefoil (Lotus corniculatus L.) originated in the Mediterranean region and now occurs worldwide . Reports from the late 1800s show birdsfoot trefoil (BFT) being cultivated in cattle pastures across Europe . It is unknown how BFT was introduced to North America . In 1957 it was estimated 770,000 acres in the U.S. were planted to BFT; ten years later acreage had more than doubled to 2 million acres (Saunders et al., 1969). Diaz et al. (2005) estimated that there were 3.43 million acres of the Lotus species sown for agronomic use in North America with 90% being BFT. It is clear that the economic value of BFT as a pasture forage and for hay production is well known.
Birdsfoot trefoil is a well-adapted legume that can grow on poorly drained soil often unsuited to other forages . Due to genetic diversity and the easy adaptability of birdsfoot trefoil, many benefits can be expected from cross breeding varieties for specific traits .
The National Plant Germplasm System (NPGS) has 627 accessions of BFT (USDA, 2016). There are 48 accessions of BFT in the core subset collection chosen from 21 different countries of origin including 12 cultivars, 2 germplasms, 27 wild or naturalized old world accessions, and 7 old world accessions with origin unknown.
The NPGS core subset was divided into four genetic geographical diversity pools: Asia Minor (n = 8), European (n=26), Mediterranean (n = 5) and North American (n=9) .  used nine descriptors of each accession to describe each diversity pool including leaf size, pod weight, susceptibility to seed chalcid (Bruchophagus platypterus), herbage tannin content, flowering intensity, early flowering, seed globulin polypeptides, random amplified polymorphic DNA markers and flowering response index. The descriptors were analyzed with a cluster analysis and divided into interpretive groups for comparison.
Birdsfoot trefoil is currently a minor crop because of its low seedling vigor, slow establishment and poor persistence, particularly with the commonly grown North American varieties, such as 'Norcen' (Beuselinck, 1995. On well drained fertile land, BFT does not yield as well as alfalfa .
Birdsfoot trefoil plants only live for two to three years but reseed themselves, creating a population matrix with different developmental stages within a stand . A matrix stand increases tolerance of stresses such as drought that only affect seedlings, not established plants . Plantings managed to allow for reseeding require minimal maintenance for stand persistence. Birdsfoot trefoil requires a sunlight day length of 16 hours for optimal seed production; the Northeast's long summer days are ideal for a reseeding pasture . In years when BFT is allowed to reseed it can be hayed after seeds are dispersed . Birdsfoot trefoil shows two distinct growth habits. The European type is more erect and adapted to haying, while the Empire type is more prostrate and adapted to grazing . Although the European type is more vigorous after being harvested and regrows quickly the Empire type has thin stems and indeterminate growth making a better quality forage .
A study using 12 different cultivars of BFT and 12 different cultivars of Lotus peduniculatus compared both species to monocultures and mixed plantings with non tanniferous grass . Plots were harvested seven times over three years to measure dry matter and weed competition and to quantify tannin content in plant tissue . It was found that the dry matter yield was greater from mixed plantings with Festuca pratensis than from monoculture for both Lotus species . It was also noted that weed composition was less in mixed swards compared to monocultures of BFT . In the second year of the study both forages produced more stem increasing dry matter production but decreasing the quality of the forage .
The Northeast can have harsh winters that a perennial forage must be able to survive. In this region winters can be highly variable and legumes such as BFT must be able to survive repeated freeze-thaw cycles without frost heaving. Grazing between September 1 st and the first killing frost will decrease winter survival of BFT . Birdsfoot trefoil is considered to be winter hardy, but hardiness differs among populations . Because varieties are from more than 20 different countries, winter hardiness is not always a strong point for each variety .   American varieties were also highly successful for herbage yield .
Winter tolerance, herbage yield and vigor were all positively correlated. There were also two accessions from Germany and Turkey that did well in winter tolerance, herbage yield and vigor .  and Steiner (2001)  Pests and disease can limit the amount of biomass a cultivar can produce.
Birdsfoot trefoil is particularly susceptible to root and crown rot and potato leaf hopper (Seaney & Hensen, 1970). 'Norcen', 'Empire' and 'Leo' have ranked high in many categories making them candidates for the Northeast but have low condensed tannin content and are still susceptible to root and crown rot (Carlson, 1983). It has also been noted that wilt has previously reduced stand persistance in the Northeast . Breeding programs of the 1990s in New York, Minnesota,  .

Medicinal Forages for Parasite Control
Interest in tannin-containing forages is growing and is driving a renewal of interest in birdsfoot trefoil. Tannins are produced in plants as a secondary metabolite and are classified as hydrolysable tannins or condensed tannins .
Condensed tannins (CT) are more common and are complexes of oligomers and polymers of flavonoid units with a carboncarbon bond that build up in cell vacuoles in many tissues of plants . Condensed tannins are found at varying levels in many forages and can have positive and negative effects on animal health and production (Frutos et al., 2004). Tanniferous forages are particularly popular for use in small ruminants (sheep and goats). Tanniferous forages are low cost, highly nutritious and prevent bloat (Salem, 2010). Current tanniferous forages used for small ruminants include Lespedza cuneata in the southern U.S., sanfoin (Onobrychis viciifolia L.) in the western U.S., sulla (Hedysarum coronarium) in the Mediterranean basin and birdsfoot trefoil (Lotus corniculatus L.) in the northern US (MacAdam et al., 2003).
Gastrointestinal nematode parasites have caused widespread production losses in small ruminant production . Moderate levels of condensed tanning containing forages have shown direct suppression of gastrointestinal parasites in in vitro tests by disrupting the lifecycle of the nematodes and preventing larvae from developing to the reproductive stage .
The mechanism of the anthelmintic effect in vitro is still not clearly understood . Tannins also decrease the effects of gastrointestinal parasites indirectly by increasing available protein and amino acids, improving animals' ability to tolerate nematode infection . The CT can bind to protein and prevent degradation in the rumen, then dissociate in the abomasum when the pH falls below 3.5 allowing more amino acids to be absorbed in the small intestine Frutos, 2004). The identity of the anthelmintic tannin(s) is unknown, so previous studies have correlated efficacy to total condensed tannin levels . However, the specific compounds within the condensed tannin pool vary by species, so the relationship between total CT and efficacy is specific to the species or cultivar.  found that fecal egg counts in goats were reduced 50% when fed L. cuneata with condensed tannin levels ranging from 45 to 55 g of CT/kg of dry matter when compared to non-CT containing forages. When CT concentrations exceeded 55g of CT/kg of dry matter or were below 45g of CT/ kg of dry matter results varied. Forages high in CTs can have negative effects such as limiting growth and wool production, and lower palatability for grazing ruminants.
High concentrations of CT in forage can result in free CT in the rumen that will react with enzymes secreted by bacteria and prevent rumen carbohydrate fermentation .
It has been shown that lambs with high parasite burden will choose forages and plants high in condensed tannin when grazing freely while sheep with low parasite burden prefer plants low in condensed tannins. In the study by Lisonbee (2008) groups of 8 lambs with varying fecal egg counts were given different options of CT containing and non-CT containing forages. Lambs with high parasite burden were able to display self-medicating behavior by choosing the CT containing forage (Lisonbee, 2008).
Currently in the southern U.S. Lespedza cuneata is used as a medicinal forage to control H. contortus in small ruminants . L. cuneata is a moderate CT containing forage that is well adapted to the southern US. Studies have found that goats grazing on L. cuneata have lower fecal egg counts than goats only grazing on blue grass (Poa pratensis) . Supplementing L.
cuneata pellets in a grazing sheep's diet decreases worm burden and dependence on the use of synthetic anthelmintics (Gujja, et al., 2013). Lespedeza cuneata is not a viable forage for the Northeast because it cannot tolerate frost, however, birdsfoot trefoil is an adapted forage in the Northeast with varying levels of CTs. Birdsfoot trefoil has been in the forefront of tanniferous forage research in the Northeast because it has anthelmintic effects similar to Lespdeza and will reliably overwinter in the Northeast.
The accessions in the NPGS core subset of BFT have a large range of total condensed tannins ranging from 2.0 m eq. g -1 to 98.0 m eq. g -1 .  found that the North American and Asia Minor geographic diversity pools had the lowest herbage tannin content (2.0 to 9.0 m eq. g -1 , mean = 4.0 m eq. g -1 ) while the Mediterranean diversity pool had the highest tannin content (53.0 to 98.0 m eq. g -1 , mean = 71.3 m eq. g -1 ). Grabber et al. (2014)  'Norcen' to 31.6 g/kg -1 in 'Rodeo' .  concluded that environment and cultivar equally influenced CT concentrations.
Condensed tannin concentrations were found to vary depending on maturity of the plant and the environment but relative rankings among cultivars remained consistent; 'Norcen' always remained lowest in CT levels .  studied tannin content in 97 accessions of BFT from the USDA Northeast Regional Plant Introduction Station in Geneva, New York. The study was conducted at two locations in Missouri with three harvest dates for each location over one growing season (May-August). Accessions were found to have a wide range of condensed tannin concentrations based on harvest date and location. Tannin content fluctuated more in high-tannin accessions than low-tannin accessions .
A United Kingdom study found that lambs grazing on BFT, specifically the cultivar 'Leo', had lower fecal egg counts after 7 days than those grazing on chicory or white clover, neither of which contain condensed tannins (Marley, 2003). At the end of the study slaughtered lambs that had grazed BFT had fewer adult helminth parasites in the abomasum than did lambs that had grazed on the other two forages (Marley, 2003). The decreases in fecal egg counts and helminth parasites are correlated to an increase in wool production, daily weight gains and increase reproductive ability (Athanasiadou, 2001).

Grazing Trials
Grazing trials are needed in agronomic performance studies of forages to identify accessions that will be able to tolerate trampling and defoliation and to determine the amount of rest time needed in between grazing events . Grazing trial results may be very region specific depending on characteristics being tested . Time and space limitations make grazing trials challenging. However, short term, small scale studies can predict long term outcomes .
Birdsfoot trefoil, once established, is persistent under grazing when grown in mixtures with grass . Pastures must be maintained as overgrazing will decrease regrowth. Heavy grazing is not recommended because BFT keeps energy reserves in the top growth as opposed to the roots . The height of 7cm will allow for regrowth to occur without damaging the BFT.
Overgrazing of BFT will prevent regrowth and promote winter injury ).
An eight-year study to evaluate birdsfoot trefoil pasture took place in Iowa from 1956 -1963. The study compared BFT to white clover and Kentucky bluegrass (Poa pretensis L.) for long term persistency, yield, and suitability for grazing. Wedin (1967) set up three treatment pastures, replicated three times, that were each grazed continuously by three cows with calves for each grazing season. Tester cows were weighed for each treatment. Cages placed in each pasture allowed for dry matter weight and botanical composition estimations. Forage yields were found to be higher for BFT pasture (Wedin, 1967). Weed composition was also higher in the BFT pasture (Wedin, 1967). Over the eight year period BFT pasture was also most productive in beef production (Wedin, 1967). Davis and Bell (1957) researched a BFT and Kentucky bluegrass (Poa pretensis L.) mixture for pasture in comparison with a Ladino clover (Trifolium repens L.) and Kentucky bluegrass mixture over a three year grazing study. They measured lamb weight gains, total digestible nutrients and carrying capacity. There were sixteen paddocks of pasture divided between the BFT bluegrass mixture and the Ladino clover blugrass mixture, 4 being continuously grazed and the remaining being rotationally grazed. Rotationally grazed paddocks were grazed for 10 to 16 days and rested for 24 to 30 days. In the establishment year the lambs on the Ladino clover bluegrass mixture gained more weight, the forage contained more total digestible nutrients and the pasture had a higher carrying capacity than the BFT bluegrass mixture. However, for the following two years the BFT pasture excelled in all three criteria (Davis and Bell, 1957). The Ladino clover bluegrass mixture stand was dramatically reduced in the last two years due to drought, BFT pasture under continuous grazing was also reduced due to overuse but the rotationally grazed BFT stands persisted the three years (Davis and Bell, 1957).
Many birdsfoot trefoil grazing trials in the literature focus on the animal outcome. Often the study will only use one or two BFT cultivars and may not even specify which cultivar is being used. Studies have shown a positive outcome of more wool production, higher weight gains, and lower fecal egg counts in animals fed BFT.
Studies that look at BFT tannin content and the medicinal advantages of grazing on BFT do not include agronomic performance of the BFT accessions used. Regional variety trials compare performance of different BFT cultivars, but these trails do not subject the cultivars to grazing. As a result, little is known about the suitability of BFT cultivars and accessions for rotational grazing.
Grazing variety trials have been done on other species that can be used as examples for birdsfoot trefoil. One hundred and forty-two red clover accessions were evaluated for forage persistence during a rotational grazing trial using cattle that lasted 3.5 years in New Zealand (Ford, 2011). The study measured persistence and regrowth based on ratings of all 142 accessions 15 times over the course of the study (Ford, 2011). Each population was given an establishment score; a rating of 1 indicated lowest establishment and 10 was highest establishment. Growth rate ranges increased over the four-year study to show significant differences in species' persistence under cattle grazing. Upon termination of the study plant survival ranged from 4% to 60% showing significant differences in grazing tolerance (Ford, 2011).
A study in Wisconsin of different cool season perennial grasses under an intensive grazing system collected data on ninety-one different varieties on three different dairy farms. The study specifically looked at available bulk density, winter hardiness and ground cover of perennial grasses (Casler, 2013). Each paddock was rotationally grazed five to six times over a two year period with measurements taken before and after each grazing session (Casler, 2013). The measurements of each characteristic presented significance among species and within species (Casler, 2013  . Resistance of parasites to currently used commercial anthelmintics is wide spread . Tanniferous forages containing moderate amounts of condensed tannins (CTs) have been shown to suppress the symptoms of H. contortus infection in small ruminants .
Condensed tannin containing forages have been shown to suppress gastrointestinal parasites by disrupting the lifecycle of the nematode and preventing larvae from developing to the reproductive stage in in vitro tests . Tannins are produced in plants as a secondary metabolite and are classified as hydrolysable tannins or condensed tannins . Condensed tannins (CT) are more common and are complexes of oligomers and polymers of flavonoid units with a carboncarbon bond that build up in cell vacuoles in many tissues of plants . Varying amounts of condensed tannins in different varieties of forages can have differing results; the identity of the anthelmintic tannins are unknown, so previous studies have correlated efficacy to total condensed tannin levels . However, forages high in condensed tannins (above 55g CT/kg of dry matter) are less palatable and can have negative effects on grazing ruminants such as limiting growth and wool production .
Birdsfoot trefoil (Lotus corniculatus L.) is a tanniferous legume forage that is adapted to the Northeast and has been shown to decrease nematode burden as measured by fecal egg counts in small ruminants . Birdsfoot trefoil (BFT) populations vary in condensed tannin content. North American varieties are generally low in CTs compared to Mediterranean and European varieties . Birdsfoot trefoil has potential as a small ruminant forage in the northeastern U.S. because of the improved production seen in small ruminants grazing on BFT. Birdsfoot trefoil is drought tolerant and can flourish on poorly drained acidic soils; it also grows well on well drained soils although when compared to alfalfa (Medicago sativa L.), alfalfa had a higher yield . The challenges that limit use of BFT are poor stand establishment and low seedling vigor (Seaney and Hensen, 1970). Pests and disease can limit the amount of biomass a variety can produce. Birdsfoot trefoil is particularly susceptible to root and crown rot and potato leafhopper (Empoasca fabae) (Seaney and Hensen, 1970). Once established, BFT is a low maintenance forage due to self-seeding with individual plants living for two to three years .
Birdsfoot trefoil originated in the Mediterranean and has since been adapted to many climates worldwide. As varieties are from more than 20 different countries, winter hardiness can be variable . In the northeast region winters can be highly variable and legumes such as BFT must be able to survive repeated freeze-thaw cycles without frost heaving.
A previous study by   The main objective of this research was to evaluate BFT populations selected for condensed tannin content for strong agronomic performance as a forage in the northeastern U.S. Evaluation of data from field plantings determined the agronomic performance based on growth habit, uniformity and vigor, winter hardiness, and pest tolerance.

Material and Methods
Site Description: The Cornell University, Ithaca, NY) from varying origins. These accessions were chosen based on the condensed tannin content. Condensed tannin content of the accessions in the study ranged from 2 to 105 g*kg 1 *DM  GRIN database).

Plant Production:
The  A score of 1 with at least 50% of leaves suffering from A score of 6 with moderate visible damage not reaching A score of 9 with no visible signs of leafhopper damage.

Statistical Analysis:
The nursery was laid out in a randomized block design with three replications of 51 PI accessions and six commercial cultivars. Statistical analysis was done using cluster analysis in JMP statistical discovery software (Statistical Analysis System Inst., Cary, NC); a resulting dendrogram was used to identify like performing populations. Analysis of variance tests were done to test for significance between populations and between years. A P-value (P) of 0.05 or below was considered significant. Tukey's HSD test was used for means comparison when differences were significant to identify top performing accessions. Regression tests were used to determine correlations between agronomic characteristics. In vitro anthelmintic efficacy and condensed tannin concentrations were compared agronomic data to determine agronomic characteristic independence from tannin concentrations and in vitro efficacy.

Results and Discussion:
There were significant differences between populations ( foliage growth (Zandt, 2007). Birdsfoot trefoil keeps energy reserves in its top growth as opposed to its' roots making foliage growth imperative to winter survival . These results are consistent with   Birdsfoot trefoil has two different growth habits, European, which is erect and optimal for haying and Empire which is prostrate and ideal for grazing . The European type plants are vigorous and regrow quickly after being harvested. The Empire type plants have thin stems and indeterminate growth which produce a better-quality forage . Birdsfoot trefoil with grass mixtures is persistent under grazing once established but heavy grazing will decrease regrowth . Regrowth time should be allowed prior to winter to promote winter survival .
Height/width ratio is a measure of growth habit. Height/width ratio data were  above 4.5. These four populations are all cultivar type with tannin content ranging from 11 to 29 g*kg -1 *DM GRIN database). Cultivars are produced for agronomical use with desired characteristics selectively bred into the population which could explain why these populations had more vigor and uniformity than other populations. The least uniform and vigorous populations were PI249753, PI246730, PI273938 and PI235525 with uniformity and vigor scores at 1.0. These populations were all wild type with a higher range of tannin content from 53 to 105 g*kg -1 *DM GRIN database). This could be explained by the tradeoff of plant growth and producing secondary metabolites causing uniformity and vigor to be lower (Zdant, 2007).  Accessions were visually scored from 1 to 5. A score of 1 was assigned to populations with weak and variable looking plants and a score of 5 was assigned to populations with vigorous and similar looking plants. 1 Uniformity and vigor scores are between 1 and 5. A score of 1 was given for weak variable looking plots and a score of 5 was giving to similar PI237278 was largely damaged with leafhopper burn both years. Leafhopper damage did not seem to be related to condensed tannin content. Leafhopper is slower to develop on BFT than on the currently used forage, alfalfa . Both years there were populations that showed no leafhopper damage with a few populations that consistently showed little or no leafhopper damage showing ability of having a leafhopper resistant variety. given to populations that showed damage on more than 50% of the plants. A Score of 9 was given to populations that showed no leaf hopper damage.
Anthelmintic efficacy was measured in vitro using an aqueous extract of lyophilized BFT leaf tissue (Barone,unpublished) and was based on percent inhibition of hatching in H. contortus eggs, and mortality of first instar larvae. Acessions were divided into two groups using anthelmintic efficacy data, greater than 89% inhibition and 89% and lower inhibition at 3mg/ml. There were populations with undetermined efficacy due to lack of tissue samples from poor germination and survival in the field.

Introduction:
Gastrointestinal parasites cause economic losses in sheep production worldwide, with the most pathogenic parasite being Haemonchus controtus . Currently commercial anthelmintic treatments are used to control H.
contortus . There are three different classes of drugs available on the U.S. market including benzimbadazole, nicotinic agonist and macrocylic lactone . Organic producers cannot market product as organic if they treat animals with synthetic anthelmintics, but have no other economically viable option . Routine use of synthetic anthelmintics leads to development of drug resistance in the H. contortus population; parasite resistance has been documented to all three drug types currently available . Studies have shown that tanniferous forages suppress H. contortus in sheep . The mechanism of the direct anthelmintic effect is still not clearly understood . Moderate amounts of tannins suppress worm burden symptoms indirectly by allowing an increase in available protein and amino acids, building an increased tolerance to the nematode . The condensed tannins can bind to protein and prevent degradation in the rumen allowing more amino acids to be absorbed in the small intestine . As the structural profile of the anthelmintic tannins is unknown, previous studies have correlated efficacy to total condensed tannin levels . However, forages high in condensed tannins (over 55g CT/kg of dry matter) are less palatable for grazing ruminants and can have negative effects on sheep such as limiting growth and wool production . In the southern US Lespedza cuneate L. is used as a medicinal forage to control H. contortus in small ruminants . Lespedza cuneata is not an option for the northeastern US because it is a perennial and does not overwinter where temperatures are below -18°C.
Birdsfoot trefoil (Lotus corniculatus L.) is a tanniferous forage legume that is adapted to the Northeast and has been shown to decrease fecal egg counts in small ruminants . Additional benefits of birdsfoot trefoil (BFT) incorporated in diets include increased protein and amino acid intake without bloating, increased wool production and increased weight gain . Birdsfoot trefoil also has environmental benefits including reducing methane and ammonia emissions from ruminants and converting atmospheric nitrogen to forms useable by plants and animals .
Breeding programs in the United States and Canada have been developing high yielding new varieties adapted to the Midwest, but few varieties adapted to New England have been produced since the 1970s, and old varieties are generally unavailable. On well drained fertile land, BFT does not yield as well as alfalfa . The challenges associated with growing BFT are seed harvesting, stand establishment, and susceptability to root and crown rot (Seaney and Hensen, 1970). Birdsfoot trefoil plants only live for two to three years but reseed themselves, creating a population matrix with different developmental stages within a stand. Once pasture is established BFT can produce yields for many years with minimal input due to self seeding .
Birdsfoot trefoil shows two distinct growth habits: European type which is more erect and adapted to haying and Empire type which is more prostrate and adapted to grazing . Although the European type plants are more vigorous and regrow quickly after being harvested the Empire type plants produce a better quality of forage due to thin stems and indeterminate growth .
New cultivars of BFT are routinely tested in mowed forage trials, but are rarely tested under grazing conditions MacAdam and Griggs, 2013).
Birdsfoot trefoil, once established, is persistent under grazing when grown in mixtures with grass . Heavy grazing is not recommended as BFT keeps energy reserves in the top growth as opposed to the roots . A minimum height of 7 cm will allow for regrowth to occur without damaging the BFT. Overgrazing of BFT will prevent regrowth and promote winter injury . Birdsfoot trefoil should not be grazed within 5 to 6 weeks of the first frost to allow for regrowth and to prevent winter injury .  recommends that BFT not be grazed after mid-August in the North Central US region to allow for recovery to increase stand persistence, in the Northeast BFT can be grazed into September.
Grazing trials are needed in agronomic performance studies of forages to identify accessions that will be able to tolerate trampling and defoliation and to determine the amount of rest time needed in between grazing events .  tested a semi erect germplasm and an erect variety of BFT for grazing tolerance in a four-year trial in Italy. The semi erect germplasm was specifically selected for grazing tolerance. The erect variety persisted as well as or better than the selected semi erect germplasm.
Many birdsfoot trefoil grazing trials in the literature focus on the animal outcome, meaning the cultivars used may not even be identified. Studies have shown a positive outcome of more wool production, higher weight gains, and lower fecal egg counts in animals grazing on BFT. Data have been collected on specific varieties for specific regions but variety trials have not included testing under rotational grazing Haring et al., 2007). Most studies of BFT as a medicinal forage have not attempted to relate agronomic performance and efficacy as a medicine. The objective of this study was to evaluate 19 PI accessions and six commercial cultivars of birdsfoot trefoil for grazing tolerance, uniformity and vigor and regrowth in the northeastern U.S. using a rotational grazing trial, and to relate these characteristics to anthelmintic efficacy against H. contortus.

Materials and Methods:
Site Description: The    The study area was fertilized each year in early May. In 2014 the field was fertilized with 8-1-9 organic fertilizer (Nature's Turf, Bradford, VT) at 489.5 kg/ha. In 2015 the field was fertilized with 33kg/ha nitrogen, 49.5kg/ha phosphorous, and 99kg/ha potassium (Vtown Ag Supply, Voluntown, CT).
Grazing Trial: Grazing and evaluation took place from July 2015 to September 2015. Fifteen sheep and one donkey grazed each replication for a 24-hour period to a target height of 7.5 cm. The donkey was kept with the sheep as a guard against coyotes and feral dogs.
Each replication was grazed twice with 28 days rest in between (Table 2). There were nine grazing sections, each containing one replication. The sections were separated using three strings of electric fence to block off access to additional grazing sections.
The sheep and donkey had access to water and a shade structure at all times. The stocking rate was determined experimentally. Thirty sheep and one donkey took roughly 12 hours to graze the orchard grass in a test strip of the same size as a replication to the optimum height of 7.5 cm and by 24 hours the test strip was completely overgrazed to stubble. By cutting the number of sheep in half overgrazing was prevented and the sheep and donkey only had to be moved once every 24 hours.
Each replication was mowed to 30.5 cm one to two days before the first round of grazing with a tractor-mounted rotary mower (

Results and Discussion:
The first replication was removed from statistical analysis because it differed significantly from the other replications. The birdsfoot trefoil and orchard grass were grazed significantly lower in the first replication. This difference could be because sheep that forage new pastures have an increased intake that levels out as the animals spend longer on a pasture .
Regrowth differed ( Grazing tolerance was clearly weaker in PI 246730, with the largest decreased growth mean of 4.5 cm. This could indicate that the BFT did not have a long enough rest period in between grazing events. There were significant differences (P = 0.0001) between post grazing measurements among accessions (Figure 3).  Combined vigor and uniformity was recorded for each plot immediately before each grazing event (Figure 4). There was a significant difference (P< 0.0001) between accessions and cultivars immediately before grazing event 2 (HSD: 3.42) but not before grazing event 1(P = 0.06). There was a significant difference in vigor and uniformity scores (P = 0.0001) between PI accessions (mean = 5.02 ± 0.18) and commercial cultivars (mean = 6.32 ± 0.26) ( Figure 5). This was to be expected as cultivars have been bred for uniformity and vigor. The mean uniformity and vigor score for the entire trial across both grazing periods was 5.44 ± 0.69 with the range being 1 to 10 for all entries. There was a decline in uniformity and vigor across all populations between grazing events but it was not significant. This could indicate a slight decline in stand persistence or it could be a response to weather, temperature, moisture or light exposure. Despite the overall decline in uniformity and vigor between grazing events there were several accessions that retained uniformity and vigor including 'Empire', 'Leo', PI306182 and PI304523.   Plant survival was determined at the termination of data collection (week 8) and varied widely with a low of 57% for PI232098 and a high of 95% for 'Empire'.
Post grazing measurements for both grazing events were positively correlated with survival rates (Post 1; R 2 = 0.26 df =21, Post 2; R 2 = 0.79 df =21) ( Figure 6). The lower an accession was grazed the lower the survival rate. 'B0730' had one of the lowest survival rates (70%) and one of the highest rates for plants grazed 7 cm or below (28%). This could indicate that some areas were overgrazed or that that sheep prefer these populations. There was a significant correlation between uniformity and vigor scores and plant survival rate (Uniformity and vigor score 1 and plants survival R 2 = 0.55, df=21; Uniformity and vigor score 2 and plant survival R 2 = 0.60, df =21).
Accessions that showed weak plants prior to grazing often had a decline in plant survival. There was evidence of weak roots allowing the sheep to pull up the plant while grazing. This could also indicate the plants had a lack of vigor and resources so were not available to regrow after grazing. There was also a significant regression (P = 0.0032) between plants that were replaced with transplants and plant survival.
Transplanted plants may have not had enough time to establish a strong root system before grazing. European type plants are more vigorous and regrow quickly after being harvested while the Empire type plants have thin stems and indeterminate growth, which produce a better quality of forage . Height/width ratios is a measurement of growth habit. Accession height/width ratio data from field plantings (Ferguson, unpublished) were correlated with the regrowth rate after both grazing events. European type plants showed more regrowth than empire type plants consistent with . There was a significant regression (P = 0.02) between height/width ratio averaged over three years and the regrowth measurements after the second grazing event but there was a weak correlation (R 2 =0.24, DF = 21).
There was no significant relationship between the regrowth after the first grazing event and the height/width ratio.
Anthelmintic efficacy was measured in vitro using an aqueous extract of lyophilized BFT leaf tissue (Barone,unpublished) and was based on percent inhibition of hatching in H. contortus eggs, and mortality of first instar larvae. Anthelmintic efficacy was divided into two groups, greater than 89% inhibition and 89% and below inhibition at 3mg/ml. Accession 655626 had undetermined in vitro efficacy due to lack of tissue samples from poor germination and survival in the field.. Egg hatch inhibition and mortality of hatched eggs included 14 populations in the low group and 8 populations in the high group. All the commercial cultivars, with the exception of 'Bruce', were highly effective at suppressing egg hatch and killing first instar H.
contortus larvae. Birdsfoot trefoil survival rate did differ significantly between the efficacy groups (P = 0.02), with the accessions with the highest efficacy also having the best survival (Figure 7). Birdsfoot trefoil uniformity and vigor scores also differed significantly between anthelmintic efficacy groups (P = 0.017) (Figure 8). Efficacy was independent from regrowth rate. This study was done on a short term, two rotations of grazing but long term grazing performance needs to be assessed.
Accessions should be tolerant for more than one grazing period and overwinter.
Reseeding should also be observed so that a low maintenance accession could be suggested. PI accessions were also identified with strong agronomic characteristics and efficacy; further research should be done to identify seed production possibilities and consistency of agronomic performance under long term rotational grazing.  This research supported  in that significant differences were seen between populations for winter hardiness and uniformity and vigor. Seaney and Hensen (1970) found that pest and disease would limit biomass of birdsfoot trefoil, resistant plants would not be affected. In 2013 and 2015 leafhopper damage did not correlate with uniformity and vigor or plant survival.  found that Empire type BFT had faster regrowth than European type birdsfoot trefoil.
In this study, no correlation was seen between growth habit and regrowth.
Future studies should focus on long term rotational grazing results, continuous grazing results and seed production ability of promising accessions. Currently available cultivars that performed well agronomically and had high anthelmintic efficacy includes, 'Empire', 'Leo', 'Bull', 'Pardee' and 'Norcen'.