domain: sdv shs info

text:
Irene Mueller-Harvey
email: mueller-harvey@reading.ac.uk

Giuseppe Bee

Frigga Dohme-Meier

Herve Hoste

Maarit Karonen

Roland Kölliker

Andreas Lüscher

Vincent Niderkorn

Wilbert F Pellikaan

Juha-Pekka Salminen

Hervé Hoste

Juha-Pekka Salminen

Leif Skøt

Lydia M J Smith

Stig M Thamsborg

Paul Totterdell

Ian Wilkinson

Andrew R Williams

Blasius N Azuhnwi

Nicolas Baert

Anja Grosse Brinkhaus

Giuseppe Copani

Olivier Desrues

Chris Drake

Marica Engström

Christos Fryganas

Marion Girard

Nguyen T Huyen

Katharina Kempf

Carsten Malisch

Marina Mora-Ortiz

Jessica Quijada

Aina Ramsay

Honorata M Ropiak

Garry C Waghorn

A G Brinkhaus

H Hoste

J.-P Salminen

Benefits of condensed tannins in forage legumes fed to ruminants: importance of structure, concentration, and diet composition

Keywords: BSA, bovine serum albumin, CT, condensed tannin, mDP, mean degree of polymerization, MYB, myeloblastosis, NIRS, nearinfrared reflectance spectroscopy, PC, procyanidin, PD, prodelphinidin, PEG, polyethylene glycol, RuBisCo, ribulose-

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

This review focuses on progress achieved during the past decade and considers the following topics:

1. bioactive CTs in plants in general and forage legumes in particular; 2. intra-and interspecies variations in CT contents and composition, which will be termed CT traits from here on; 3. effects of agronomic, harvesting, and processing practices on CT efficacies; 4. effects on animal health, nutrition, product quality, and environmental emissions; and 5. characteristics useful for plant breeders and tools for selecting or engineering forages with novel CT traits. This review also summarizes results from a multidisciplinary research consortium that focused on sainfoin (Fig. 2). In this project, agronomists and plant breeders assembled germplasm collections of sainfoin (Onobrychis Mill. spp.), which is a traditional forage legume in Europe, and identified molecular markers and strategies for weed control. Ruminant nutritionists studied various accessions for their in vitro fermentation characteristics and in vivo feeding trials, nitrogen balances, and the quality of meat and dairy products. Parasitologists explored the antiparasitic properties of a wide range of CT traits, and chemists developed tools for analyzing the CTs in sainfoin pellets, silages, and digesta.

The background to this work was the pioneering research in New Zealand that first studied fresh sainfoin (Onobrychis viciifolia Scop.) and Lotus L. spp. in relation to legume bloat and nutritive value [START_REF] Reid | Plant tannins, bloat and nutritive value[END_REF]. Sainfoin research included measurements of the protein-binding characteristics of CTs (Fig. 1) [START_REF] Jones | Complexes of the condensed tannins of sainfoin (Onobrychis viciifolia Scop.) with Fraction 1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH[END_REF] and aspects of nitrogen digestion in sheep [START_REF] Egan | Quantitative digestion of fresh herbage by sheep. VI. Utilization of nitrogen in five herbages[END_REF], but problems with sainfoin persistence in swards diverted attention to Lotus species. Initial research by Barry and colleagues focused on big trefoil (Lotus pedunculatus Cav.), examining effects of CT concentration [START_REF] Barry | The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 4. Sites of carbohydrate and protein digestion as influenced by dietary reactive tannin concentrations[END_REF], whereas [START_REF] Waghorn | Nutritive value of Lotus corniculatus L. containing low and medium concentrations of condensed tannins for sheep[END_REF] demonstrated the beneficial effects of CTs in birdsfoot trefoil (Lotus corniculatus L. var. corniculatus) on absorption of essential amino acids from the intestine. Other forages were also evaluated in New Zealand, including sulla (Hedysarum coronarium L.; [START_REF] Stienezen | Digestibility and effects of condensed tannins on digestion of sulla (Hedysarum coronarium) when fed to sheep[END_REF] and dock (Rumex obtusifolius L.; [START_REF] Waghorn | Bloat in cattle 46. The potential of dock (Rumex obtusifolius) as an antibloat agent for cattle[END_REF], but the importance of CT composition, in addition to concentration, was demonstrated more recently (Waghorn et al., 1997). Subsequent research included CT effects on livestock parasites and greenhouse gas emissions, but financial support decreased because of a lack of competitiveness of tanniniferous forages, especially in fertile soils. It also became apparent that detailed chemical characterization was required to elucidate mechanisms of action, because the two Lotus species differed in their biological effects and tannin types. The question was: were the CTs responsible for these different biological effects?

This review seeks to provoke discussion on how to progress this area of research, how to optimize the Fig. 1. Example of a condensed tannin (CT) molecule that consists of four flavan-3-ol subunits (= monomeric building blocks). Procyanidins are composed of catechin or epicatechin and prodelphinidins of gallocatechin or epigallocatechin subunits (for further details, see [START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF]. a few CT-containing feeds with ruminant animals (e.g., sheep, goats, and cattle;[START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF]. The nutritional benefits include improved growth, milk yields, fertility, and tolerance to some intestinal parasites and arise from protection of dietary protein from excessive fermentation in the rumen. Other benefits include bloat prevention, which is associated with tannins reducing the stability of a foam that traps ruminal fermentation gases, and antiparasitic effects against ruminant and nonruminant gastrointestinal parasites [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF][START_REF] Kingston-Smith | Plant-based strategies towards minimising 'livestock's long shadow[END_REF][START_REF] Terrill | Experiences with integrated concepts for the control of Haemonchus contortus in sheep and goats in the United States[END_REF][START_REF] Wang | Forthy bloat in ruminants: Cause, occurrence, and mitigation strategies[END_REF]Hoste et al., 2015Hoste et al., , 2016;;[START_REF] Macadam | Beneficial effects of temperate forage legumes that contain condensed tannins[END_REF].

Although plants synthesize many different tannin types, this review will focus on CTs, as these are of particular interest in forage legumes and several other pasture plants. Hydrolyzable tannins are not considered here, although evidence is emerging that some may exert similarly useful bioactive effects [START_REF] Baert | A study of the structure-activity relationship of oligomeric ellagitannins on ruminal fermentation in vitro[END_REF][START_REF] Engström | Chemical structures of plant hydrolyzable tannins reveal their in vitro activity against egg hatching and motility of Haemonchus contortus nematodes[END_REF][START_REF] Bee | Hydrolysable tannin-based diet rich in gallotannins has a minimal impact on pig performance but significantly reduces salivary and bulbo-urethral gland size[END_REF].

A major impetus for research and utilization of CT forage legumes by livestock producers has been the drive to reduce bloat, to improve farm profitability, to control parasites, and to reduce greenhouse gas and ammonia emissions [START_REF] Kingston-Smith | Plant-based strategies towards minimising 'livestock's long shadow[END_REF][START_REF] Wang | Forthy bloat in ruminants: Cause, occurrence, and mitigation strategies[END_REF][START_REF] Mccaslin | New strategies for forage quality improvement in alfalfa[END_REF][START_REF] Hoste | Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock[END_REF]. Nutritional responses to CTs have been variable, and this has led to contradictory reports about their benefits [START_REF] Min | The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: A review[END_REF][START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF]. This is not surprising given the complexity of plant CTs, their impact when forages are fed as a sole diet or as a dietary component, and their interactions with feed components, host tissues, and the microbiome, plus the effects stemming from the animal's nutrient requirements and parasitism. A concerted multidisciplinary research approach is required to harness the full potential of CTs for livestock production [START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF]. However, obtaining funding for such a wide-ranging set of topics has been challenging and reflects common barriers to interdisciplinary research, such as narrowly focused funding goals and short funding timelines. Progress in the study of complex plant-livestock systems is inherently slow, and CTs also pose interesting analytical and experimental challenges. Taken together, these facts account for the relatively slow progress in identifying the relationships between CT traits and bioactivities.

CHALLENGES AND OPPORTUNITIES POSED BY PLANTS WITH DIFFERENT CONDENSED TANNIN TYPES

Research on plant CTs has come a long way since the early literature described these compounds of mysterious composition and function as "accidents of [plant] metabolism" or metabolic "waste products" that served to bioactivity of CT forages, and how to develop on-farm applications. It will also consider whether farmers might require plants with different CT traits for either antiparasitic or nutritional purposes. The reader is also referred to the accompanying article by [START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF] for a detailed description of CT structures and the review by [START_REF] Tedeschi | Developing a conceptual model of possible benefits of condensed tannins for ruminant production[END_REF] for a mechanistic model describing the overall interactions between CTs and ruminants.

ROLES OF TANNINS IN PLANTS AND CHALLENGES TO HARNESSING THEIR BENEFITS FOR LIVESTOCK PRODUCTION

The reader of the tannin literature is repeatedly reminded that tannins are "secondary" plant metabolites and provide a defense against herbivory [START_REF] Barbehenn | Tannins in plantherbivore interactions[END_REF][START_REF] Agrawal | Insect herbivores drive real-time ecological and evolutionary change in plant populations[END_REF][START_REF] Lattanzio | Plant phenolics: A biochemical and physiological perspective[END_REF]. However, herbivores comprise a range of species, from insects to ruminants, and have distinctly different gut systems. Although tannins can account for antiherbivory effects in insects [START_REF] Salminen | Chemical ecology of tannins and other phenolics: We need a change in approach[END_REF], ruminant behavior suggests that their herbivory effect is marginal because forages containing CTs are consumed, and selection is often in preference to grasses [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF]. Within plants, leaves are selected in preference to stems despite higher CT concentrations, even when CTs accounted for ~20% of the dry matter in erect canary clover [Dorycnium rectum (L.) Ser.] leaf [START_REF] Waghorn | Effect of condensed tannins in Dorycnium rectum on its nutritive value and on the development of sheep parasite larvae[END_REF]. Current thinking suggests that these secondary metabolites provide plants with a plasticity that can support their development and interaction with the environment [START_REF] Bidel | Biological activity of phenolics in plant cells[END_REF][START_REF] Neilson | Plant chemical defense: At what cost?[END_REF][START_REF] Mouradov | Flavonoids: A metabolic network mediating plants adaptation to their real estate[END_REF]. This concept, that CTs may have multiple and interrelated functions, is now gaining traction. For example, [START_REF] Chen | Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year[END_REF] showed that the temperatures experienced by the mother plant elicit CT pathways that can pass information to the next generation. In addition, the flavan-3-ol monomers, which are precursors of CTs, appear to play a vital role in protecting chromosomes during periods of high cell activity, but not during dormancy or drought stress [START_REF] Feucht | Loss of nuclear flavanols during drought periods in Taxus baccata[END_REF]. It is important to appreciate that a multitude of different CT compounds exist [START_REF] Khanbabaee | Tannins: Classification and definition[END_REF][START_REF] Hümmer | Analysis of proanthocyanidins[END_REF][START_REF] Salminen | Chemical ecology of tannins and other phenolics: We need a change in approach[END_REF][START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF], and that their synthesis in the plant kingdom has not converged on a single structure. These findings suggest that the function of CTs deserves a closer look in plant and crop science.

From an animal's perspective, when dietary CT concentrations are too high, or protein concentrations are too low, as in tropical environments where grasses may have little nitrogen and tree leaves may have high CT concentrations, CTs can be antinutritional [START_REF] Cooper | Foliage acceptability to browsing ruminants in relation to seasonal changes in the leaf chemistry of woody plants in a South African savanna[END_REF]. The benefits of CTs have been demonstrated in only support the primary plant metabolism [START_REF] Haslam | Vegetable tannins[END_REF]. It is now well established that CT synthesis is under genetic control [START_REF] Szczyglowski | Lotus genome: Pod of gold for legume research[END_REF][START_REF] Scioneaux | Qualitative variation in proanthocyanidin composition of Populus species and hybrids: Genetics is the key[END_REF][START_REF] Cheynier | Plant phenolics: Recent advances on their biosynthesis, genetics and ecophysiology[END_REF][START_REF] Escaray | Lotus tenuis ´ L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes[END_REF] and that expression depends on the plant species and plant parts [START_REF] Larkin | Modifying condensed tannin content in plants[END_REF][START_REF] Gebrehiwot | Seasonal variations in condensed tannin concentration of three Lotus species[END_REF][START_REF] Abeynayake | Biosynthesis of proanthocyanidins in white clover flowers: Cross talk within the flavonoid pathway[END_REF][START_REF] Ferreyra | Flavonoids: Biosynthesis, biological functions, and biotechnological applications[END_REF][START_REF] Hancock | Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa[END_REF][START_REF] Verdier | MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula[END_REF][START_REF] Cheynier | Plant phenolics: Recent advances on their biosynthesis, genetics and ecophysiology[END_REF][START_REF] Harding | Condensed tannin biosynthesis and polymerization synergistically condition carbon use, defense, sink strength and growth in Populus[END_REF][START_REF] Mouradov | Flavonoids: A metabolic network mediating plants adaptation to their real estate[END_REF][START_REF] Pérez-Díaz | VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L[END_REF][START_REF] Zhou | Production and transcriptional regulation of proanthocyanidin biosynthesis in forage legumes[END_REF][START_REF] Zhu | Ectopic expression of the Coleus R2R3 MYB-type proanthocyanidin regulator gene SsMYB3 alters the flower color in transgenic tobacco[END_REF][START_REF] Chezem | Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs[END_REF].

Chemotaxonomic surveys on the distribution of CTs in plants in general and of forage legumes in particular have found that CT compositions tend to follow distinct biosynthetic patterns in terms of their flavan-3-ol subunit composition and polymer sizes, which are described in terms of mean degree of polymerization (mDP). Plants with procyanidin (PC)-type CTs are much more widespread than plants with prodelphinidin (PD)-type CTs (Fig. 1), but many more plant species contain PC/PD mixtures [START_REF] Porter | Flavans and proanthocyanidins[END_REF][START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Sivakumaran | Variation of proanthocyanidins in Lotus species[END_REF][START_REF] Mechineni | Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections, skin and carcass microbial load, and meat quality of growing goats[END_REF][START_REF] Laaksonen | Proanthocyanidins and their contribution to sensory attributes of black currant juices[END_REF][START_REF] Quijada | Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are influenced by structural features of condensed tannins[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF]Ropiak et al., 2016b). Most plant CTs have cis-flavan-3-ol subunits, especially as extension units (Fig. 1), whereas CTs with predominantly transflavan-3-ol subunits in extension units are relatively rare [START_REF] Porter | Flavans and proanthocyanidins[END_REF][START_REF] Hernes | Tannin signatures of barks, needles, leaves, cones, and wood at the molecular level[END_REF][START_REF] Klongsiriwet | Near infrared spectroscopy (NIRS) for screening condensed tannins in a willow germplasm collection[END_REF]. We have also observed other trends in the composition of CTs in forage legumes, which are illustrated with examples from a few plants that are "specialists" in producing particular CT types:

• CTs that comprise PCs tend to be mixtures of oligomers and smaller polymers (e.g., cocoa [Theobroma cacao L.] bean CTs with mDP values of 2-5).

• CTs that comprise PDs are usually mixtures of larger polymers (e.g., sericea lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don] CTs with mDP values of 10-30).

However, exceptions exist, as lime tree flowers (Tilia L. spp.) and some varieties of cider apples (Malus domestica Borkh.) have PCs with higher mDP values of 8 and ~100, respectively (Guyot et al., 2001a;[START_REF] Ropiak | Identification of structural features of condensed tannins that affect protein aggregation[END_REF].

Other sources of special CT types include:

• Leaves from several willow (Salix L. spp.) accessions and black currant (Ribes nigrum L.) have high proportions of PCs and PDs with trans-flavan-3-ol subunits, respectively [START_REF] Porter | Flavans and proanthocyanidins[END_REF].

• Tea [Camellia sinensis (L.) Kuntze] leaves and shea (Vitellaria paradoxa C. F. Gaertn.) nuts are unusual in having high proportions of galloylated flavan-3-ol monomers and galloylated low molecular weight PDs [START_REF] Henning | Catechin content of 18 teas and a green tea extract supplement correlates with the antioxidant capacity[END_REF][START_REF] Ramsay | Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum[END_REF].

• Water dock (Rumex hydrolapathum Huds.) roots and persimmon (Diospyros kaki Thunb.) fruits contain highly galloylated smaller PCs (mDP = 6; galloylation = 52%; Ropiak et al., 2016b) and larger PDs (mDP = 26; galloylation = 72%; [START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF], respectively.

• An entire series of oligomeric and polymeric PC xylosides are present in birch (Betula pendula Roth) bark [START_REF] Liimatainen | Procyanidin xylosides from the bark of Betula pendula[END_REF].

Not surprisingly, these biosynthetic patterns can generate contradictory and confounding effects when attempting to align CT structure with bioactivity [START_REF] Laaksonen | Proanthocyanidins and their contribution to sensory attributes of black currant juices[END_REF][START_REF] Hixson | Survey of the variation in grape marc condensed tannin composition and concentration and analysis of key compositional factors[END_REF]. However, the particular CTs in these 'specialist' plants can provide unique opportunities for research, because it would be very difficult to separate sufficient quantities of a particular CT type from the complex CT mixtures, which are typical of most plants, for laboratory or in vitro studies. This problem is illustrated by the CT mixtures in different sainfoin, sericea lespedeza and Lotus accessions (Table 1), in which the PC/PD ratios ranged from 84:16 to 3:97, cis-/trans-flavan-3-ol ratios from 90:10 to 66:34, and mDP values from of 12 to 84 [START_REF] Meagher | Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry[END_REF]Stringano et al., 2012;Azuhnwi et al., 2013a;[START_REF] Mechineni | Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections, skin and carcass microbial load, and meat quality of growing goats[END_REF].

An alternative approach is to use CTs from "CT specialist plants." The already "pure" groups of either PCs or PDs, having either cis-or trans-flavan-3-ol stereochemistry, can be isolated from these specialist plants, separated in the laboratory into mDP variants, and used to explore the bioactivities of different PC/PD ratios, cis-/trans-flavan-3-ol ratios, and polymer sizes [START_REF] Brown | Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition[END_REF]. Currently, this is the most straightforward approach to structure-activity studies, because chemical synthesis of CTs is even more challenging.

Another aspect of CT composition that is poorly researched concerns the galloylated CTs. This is a group of particularly potent antioxidants (Fig. 1; [START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF] that appear to possess strong antiparasitic, nematocidal, and antimicrobial activities but have received little attention for their nutritional or health effects [START_REF] Brunet | Monomers of condensed tannins affect the larval exsheathment of parasitic nematodes of ruminants[END_REF]Ropiak et al., 2016a). Acacia nilotica (L.) Delile leaves, carob (Ceratonia siliqua L.) pods, grape (Vitis vinifera L.) seeds, persimmon fruits, lentisk (Pistacia lentiscus L.) leaves, shea nuts, tea leaves, and Rumex L. spp. are good sources of galloylated CTs [START_REF] Self | Fast atom bombardment mass spectrometry of polyphenols (syn. vegetable tannins)[END_REF][START_REF] Henning | Catechin content of 18 teas and a green tea extract supplement correlates with the antioxidant capacity[END_REF][START_REF] Papagiannopoulos | Identification and quantification of polyphenols in carob fruits (Ceratonia siliqua L.) and derived products by HPLC-UV-ESI/MSn[END_REF][START_REF] Spencer | Isolation and characterisation of procyanidins from Rumex obtusifolius[END_REF][START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF][START_REF] Rodríguez-Pérez | A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves[END_REF][START_REF] Derksen | 3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus[END_REF] It is also essential to use CT mixtures that are specific to the plant species being investigated, because CT composition affects ultraviolet-visible absorption maxima and reaction yields that result from the hydrochloric acid (HCl)-butanol-acetone and thiolysis assays [START_REF] Grabber | Acetone enhances the direct analysis of procyanidin-and prodelphinidin-based condensed tannins in Lotus species by the butanol-HCl-iron assay[END_REF][START_REF] Krueger | Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health[END_REF][START_REF] Engström | Rapid qualitative and quantitative analyses of proanthocyanidin oligomers and polymers by UPLC-MS/MS[END_REF][START_REF] Hixson | Evaluation of direct phloroglucinolysis and colorimetric depolymerization assays and their applicability for determining condensed tannins in grape marc[END_REF]Ropiak et al., 2016b;[START_REF] Wang | Influence of degree-of-polymerization and linkage on the quantification of proanthocyanidins using 4-dimethylaminocinnamaldehyde (DMAC) assay[END_REF]. We emphasize the unsuitability of commercially available CTs from quebracho [Schinopsis quebracho-colorado (Schltdl.) F. A. Barkley & T. Mey.] as a standard because these have 5-deoxy-flavan-3-ol subunits, which give particularly low reaction yields that lead to overestimation of CT concentrations [START_REF] Schofield | Analysis of condensed tannins: A review[END_REF][START_REF] Rautio | Bitter problems in ecological feeding experiments: Commercial tannin preparations and common methods for tannin quantifications[END_REF], and tannic acid is even less appropriate, as it contains none of the CT flavan-3-ol subunits.

Additional challenges include variation in extractability of CTs. Some can be extracted with water or aqueous methanol, and others require aqueous acetone, but many CTs are tightly bound to the plant matrix and cannot be extracted with these solvents. By using techniques that only measure the easily extractable CTs, researchers may risk missing a large fraction (Table 2) that al., 2014; [START_REF] Ramsay | Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum[END_REF]Ropiak et al., 2016b;our unpublished data).

PROGRESS IN TANNIN ANALYSIS

Several new techniques have been developed recently for analyzing CT mixtures. As [START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF] has addressed this topic in detail, only a few additional techniques are described below. An important constraint in the quest for valid tannin data is the requirement for high purity standards for quantitation, which means that the CT concentration and purity of the standards needs to be assessed by CT-specific methods such as thiolysis [START_REF] Gea | In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening[END_REF][START_REF] Grabber | Acetone enhances the direct analysis of procyanidin-and prodelphinidin-based condensed tannins in Lotus species by the butanol-HCl-iron assay[END_REF]Williams et al., 2014a) or nuclear magnetic resonance spectroscopy (Zeller et al., 2015a). We emphasize that the widely used elution of plant extracts with 70% aqueous acetone from Sephadex LH-20 columns for tannin "purification" can lead to CT concentrations as low as ~13 g CTs 100 g -1 "purified sample" (Williams et al., 2014b). Their use would overestimate CT concentrations, so additional steps are required to increase their purity [START_REF] Stringano | Analysis of sainfoin (Onobrychis viciifolia) proanthocyanidins by complementary and newly developed techniques[END_REF][START_REF] Fryganas | Investigations into the chemical analysis and bioactivity of plant proanthocyanidins to support sustainable livestock farming[END_REF][START_REF] Brown | Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition[END_REF][START_REF] Ropiak | Identification of structural features of condensed tannins that affect protein aggregation[END_REF]. may have potentially important bioactivities [START_REF] Gea | In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening[END_REF][START_REF] Pérez-Jiménez | Analysis of nonextractable phenolic compounds in foods: The current state of the art[END_REF][START_REF] Cheynier | Polyphenols: From plants to a variety of food and nonfood uses[END_REF][START_REF] Hixson | Survey of the variation in grape marc condensed tannin composition and concentration and analysis of key compositional factors[END_REF]our unpublished data). The impact of extractable vs. unextractable CTs on ruminant nutrition and health has not yet been determined. Thiolysis or phloroglucinolysis (analytical degradation of CTs with thiols or phloroglucinol) can be used to determine the composition of CTs by depolymerization, enabling characterization of the flavan-3-ol subunits [START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF]. The use of thiolysis to analyze CTs in whole plant material (removing the need for extraction) was first reported by Guyot et al. (2001b) for apple residues and then adapted to sainfoin and food samples [START_REF] Hellström | HPLC determination of extractable and unextractable proanthocyanidins in plant materials[END_REF][START_REF] Gea | In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening[END_REF]. However, quantification remains problematic, and higher CT yields have been reported in some samples with the HCl-butanol-acetone assay than with the thiolysis or phloroglucinolysis assays. Low values have been linked to oxidative processes affecting reaction yields [START_REF] Hixson | Evaluation of direct phloroglucinolysis and colorimetric depolymerization assays and their applicability for determining condensed tannins in grape marc[END_REF][START_REF] Klongsiriwet | Phytochemical analysis of a willow germplasm collection and its pharmacological activities[END_REF][START_REF] Brillouet | Isolation of native proanthocyanidins from grapevine (Vitis vinifera) and other fruits in aqueous buffer[END_REF][START_REF] Desrues | Condensed tannins in the gastrointestinal tract of cattle after sainfoin (Onobrychis viciifolia) intake and their possible relationship with anthelmintic effects[END_REF].

However, thiolysis can also generate up to threefold higher yields than the HCl-butanol-acetone assay (Drake and Mueller-Harvey, unpublished data, 2016); these particular CTs had high degrees of galloylation (Fig. 1), and this demonstrates the variation in reactivity of CTs and yield of assayable end products. These inconsistencies illustrate the challenge of determining the amount and type of CTs in forage material and reinforce the case for using more than one method for analyzing CTs, and for using plant-specific CT standards (see above). Recent observations also suggest that the optimum time for the in situ thiolysis is affected by the plant species. Some samples yielded more consistent CT parameters if thiolysis was performed for 2 to 3 h rather than 1 h, as suggested by [START_REF] Gea | In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening[END_REF]. Clearly, there is no one silver bullet for CT analysis, and methods need to be evaluated and adapted for a particular plant species and research objective.

Infrared spectroscopy techniques are of particular interest to plant breeders due to their speed and suitability for screening large numbers of samples; nearinfrared reflectance spectroscopy (NIRS) can be used in the laboratory, whereas visible-near-infrared and shortwave-infrared spectroscopy have been used for field screening [START_REF] Lehmann | Field spectroscopy in the VNIR-SWIR region to discriminate between Mediterranean native plants and exotic-invasive shrubs based on leaf tannin content[END_REF]. Near-infrared reflectance spectroscopy has potential for measuring not only CT concentrations but also PC/PD and cis-/transflavan-3-ol ratios (Fig. 3) [START_REF] Petersen | Screening sericea lespedeza germplasm for herbage quality[END_REF][START_REF] Larkin | Modifying condensed tannin content in plants[END_REF][START_REF] Dykes | Prediction of total phenols, condensed tannins, and 3-deoxyanthocyanidins in sorghum grain using near-infrared (NIR) spectroscopy[END_REF][START_REF] Grabber | Yield, morphological characteristics, and chemical composition of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental United States[END_REF][START_REF] Klongsiriwet | Phytochemical analysis of a willow germplasm collection and its pharmacological activities[END_REF]Mueller-Harvey et al., unpublished data, 2010). Sample analysis by NIRS is rapid but requires robust calibrations that are based on laboratory analyses. Once calibrated, a single NIRS scan can also generate a large amount of information on other nutritional parameters, such as fiber, protein, soluble carbohydrate, lignin, dry matter, and ash contents, as well as predicted digestibility and gross energy [START_REF] Givens | Current procedures, future requirements and the need for standardisation[END_REF].

TANNIN VARIATION IN GERMPLASM COLLECTIONS AND POTENTIAL FOR TRAIT-DIRECTED PLANT BREEDING

Alignment of CT composition with function offers opportunities for exploiting their bioactivities, and germplasm collections offer a rich source of CT variation (Hayot Carbonero et al., 2011;[START_REF] Klongsiriwet | Phytochemical analysis of a willow germplasm collection and its pharmacological activities[END_REF]. Concentrations of CTs vary greatly not only between plant species but also between accessions [START_REF] Larkin | Modifying condensed tannin content in plants[END_REF][START_REF] Mosjidis | Forage legume breeding and evaluation at Auburn University in the last 16 years[END_REF][START_REF] Sivakumaran | Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria[END_REF][START_REF] Gruber | Variation in morphology, plant habit, proanthocyanidins, and flavonoids within a Lotus germplasm collection[END_REF][START_REF] Häring | Tanniferous forage plants: Agronomic performance, palatability and efficacy against parasitic nematodes in sheep[END_REF][START_REF] Lorenz | Effect of wilting, silage additive, PEG treatment and tannin content on the distribution of N fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties[END_REF][START_REF] Grabber | Protein and drymatter degradability of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental USA[END_REF][START_REF] Hixson | Survey of the variation in grape marc condensed tannin composition and concentration and analysis of key compositional factors[END_REF]. Table 1 lists the variation in forage plants: birdsfoot trefoil tends to have the lowest (<5 g 100 g -1 dry matter) and sericea lespedeza and erect canary clover the highest CT concentrations (6-20 g 100 g -1 dry matter).

The CT traits can also differ markedly between accessions, and between parts of the same plant [START_REF] Springer | Variation of condensed tannins in roundhead lespedeza germplasm[END_REF]. Examples are PC/PD ratios in sainfoin stem CTs of ~50:50 and in sainfoin leaf CTs of ~10:90 [START_REF] Malisch | Large variability of proanthocyanidin content and composition in sainfoin (Onobrychis viciifolia)[END_REF]. Although alfalfa (Medicago sativa L.) seed coats have CTs with mDP values of 4 to 7 and a PC/PD ratio of 93:7 [START_REF] Koupai-Abyazani | Purification and characterization of a proanthocyanidin polymer from seed of alfalfa (Medicago sativa Cv. Beaver)[END_REF], only the smaller PC dimers and trimers have been detected in engineered alfalfa leaves [START_REF] Hancock | Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa[END_REF]. This means that plant breeding can target CT composition and concentration, which is important because these traits have been linked to different bioactivities (see below).

Both concentration and composition can change with season [START_REF] Theodoridou | Effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (Onobrychis viciifolia) varieties and lucerne[END_REF][START_REF] Grabber | Protein and drymatter degradability of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental USA[END_REF][START_REF] Muir | Season progression, ontogenesis and environment affect Lespedeza cuneata herbage condensed tannin, fiber and crude protein content[END_REF], but accession differences tend to be much larger [START_REF] Springer | Variation of condensed tannins in roundhead lespedeza germplasm[END_REF][START_REF] Stringano | Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (Onobrychis viciifolia) germplasm collection[END_REF]. Importantly, environment did not affect the ranking of the CT traits of a few sainfoin accessions (i.e., there was no genotype ´ environment interaction) (Azuhnwi et al., 2013a ;[START_REF] Malisch | Drought effects on proanthocyanidins in sainfoin (Onobrychis viciifolia Scop.) are dependent on the plant's ontogenetic stage[END_REF]; this demonstrates that there are opportunities for trait-directed breeding of new cultivars.

All enzymes involved in the biosynthesis of the CT building blocks (i.e., flavan-3-ols) have been identified, apart from the elusive final condensing enzyme(s) [START_REF] Harding | Condensed tannin biosynthesis and polymerization synergistically condition carbon use, defense, sink strength and growth in Populus[END_REF]. Two genes and several myeloblastosis (MYB) transcription factors (i.e., proteins with myeloblastosis DNAbinding domains that regulate CT synthesis) are responsible for the production of two of the flavan-3-ols (i.e., catechin and epicatechin; [START_REF] Ferreyra | Flavonoids: Biosynthesis, biological functions, and biotechnological applications[END_REF][START_REF] Cheynier | Plant phenolics: Recent advances on their biosynthesis, genetics and ecophysiology[END_REF][START_REF] Zhu | Ectopic expression of the Coleus R2R3 MYB-type proanthocyanidin regulator gene SsMYB3 alters the flower color in transgenic tobacco[END_REF][START_REF] Chezem | Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs[END_REF], but the genes and transcription factors for the other flavan-3-ols await identification. The MYB transcription factors from barrelclover (Medicago truncatula Gaertn.) and rabbitfoot clover (Trifolium arvense L.) have been expressed in alfalfa and white clover (Trifolium repens L.), leading to stable and heritable CT production in their shoots and leaves [START_REF] Hancock | Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa[END_REF][START_REF] Hancock | Progress towards developing bloat-safe legumes for the farming industry[END_REF][START_REF] Verdier | MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula[END_REF][START_REF] Albert | Subspecialization of R2R3-MYB repressors for anthocyanin and proanthocyanidin regulation in forage legumes[END_REF]. These developments provide a pathway for introducing CTs into the leaves of alfalfa and white clover. It would be interesting to explore whether the CTs that are already expressed in white clover flowers can be expressed in the leaves.

It should also be possible to alter the CT composition through conventional crossing experiments. [START_REF] Scioneaux | Qualitative variation in proanthocyanidin composition of Populus species and hybrids: Genetics is the key[END_REF] showed that CT composition, especially the average polymer size (the mDP value), in Populus L. was controlled by genetics and that environment (location) and season (months) had only a small effect. In addition, interspecies hybridization can generate plants with novel CT traits, as demonstrated with narrowleaf trefoil (Lotus tenuis Waldst. & Kit. ex Willd.) ´ birdsfoot trefoil hybrids [START_REF] Escaray | Lotus tenuis ´ L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes[END_REF].

Despite considerable advances in plant science, genomic resources for forage legumes are still scarce, especially for CT-containing forages [START_REF] Szczyglowski | Lotus genome: Pod of gold for legume research[END_REF][START_REF] Hayot Carbonero | Sainfoin (Onobrychis viciifolia), a forage legume with great potential for sustainable agriculture, an insight on its morphological, agronomical, cytological and genetic characterisation[END_REF][START_REF] Zarrabian | Genetic diversity in a worldwide collection of sainfoin using morphological, anatomical, and molecular markers[END_REF][START_REF] Vega | Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement[END_REF][START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF]. These are necessary to harness the potential benefits of CTs, and for breeding of new varieties with improved agronomic, nutritional, and antiparasitic traits. The European and Asian sainfoin germplasm is very diverse in terms of morphology, anatomy, drought resistance, CT traits, and genetic polymorphism [START_REF] Hayot Carbonero | Sainfoin (Onobrychis viciifolia), a forage legume with great potential for sustainable agriculture, an insight on its morphological, agronomical, cytological and genetic characterisation[END_REF][START_REF] Zarrabian | Genetic diversity in a worldwide collection of sainfoin using morphological, anatomical, and molecular markers[END_REF][START_REF] Malisch | Large variability of proanthocyanidin content and composition in sainfoin (Onobrychis viciifolia)[END_REF][START_REF] Malisch | Drought effects on proanthocyanidins in sainfoin (Onobrychis viciifolia Scop.) are dependent on the plant's ontogenetic stage[END_REF][START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF][START_REF] Kempf | Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm[END_REF][START_REF] Kempf | Marker-trait association analysis for agronomic and compositional traits in sainfoin (Onobrychis viciifolia)[END_REF]Mora-Ortiz et al., 2016;[START_REF] Kölliker | Promising options for improving performance and proanthocyanidins of the forage legume sainfoin (Onobrychis viciifolia Scop.)[END_REF]. For example, anatomical trait analysis (xylem/phloem ratio, vessel and sieve tube diameters, and phloem width) suggested that large differences exist in water and solute transport mechanisms between accessions, which are important for drought resistance [START_REF] Zarrabian | Genetic diversity in a worldwide collection of sainfoin using morphological, anatomical, and molecular markers[END_REF].

Molecular markers are helpful in the analysis of genetic diversity, mapping and quantitative trait loci analysis, and genomics-assisted breeding. Next-generation sequencing technology is facilitating the identification and use of molecular markers in plant genetics and breeding. RNAsequencing technology is an efficient way of obtaining sequence information of all the genes that are expressed in a given plant tissue, and it can also be mined for molecular marker polymorphisms. The first such library of expressed genes in sainfoin was obtained from five accessions using this technology (Mora-Ortiz et al., 2016). Annotation of the expressed genes in the library allowed identification of 59 genes involved in the CT biosynthesis pathway (Mora-Ortiz et al., 2016). It also provided the platform for identifying >3800 simple sequence repeat (SSR) markers and 77,000 single-nucleotide polymorphism markers (Mora-Ortiz et al., 2016). Phylogenetic analysis revealed that sainfoin is closely related to red clover (Trifolium pratense L.) and barrelclover. Some of the SSR markers were used to assess the genetic diversity of European sainfoin accessions representing cultivars and noncultivars (ecotypes, landraces) [START_REF] Kempf | Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm[END_REF]. These molecular markers are now available as tools for further genetic and genomic research. They were used to study inbreeding and self-fertilization in sainfoin. In a pilot study, one marker locus was identified that could explain up to 12% of the variation in CT composition (i.e., PC/PD ratio; [START_REF] Kempf | Marker-trait association analysis for agronomic and compositional traits in sainfoin (Onobrychis viciifolia)[END_REF]. We anticipate that the sequence and molecular marker information now available [START_REF] Kempf | Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm[END_REF]Mora-Ortiz et al., 2016) can be used to assist breeding of novel sainfoin cultivars with CT traits that are optimized for animal nutrition and health.

AGRONOMY, WEED CONTROL, HARVESTING, AND PROCESSING OF TANNINIFEROUS FORAGE LEGUMES

There is currently considerable global interest in harnessing the benefits of CTs in forage legumes to support the sustainability agenda of agriculture; birdsfoot trefoil, sericea lespedeza, and prairie clover (Dalea purpurea Vent.) have been evaluated in the United States, Canada, New Zealand, and Switzerland [START_REF] Mosjidis | Forage legume breeding and evaluation at Auburn University in the last 16 years[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF][START_REF] Häring | Tanniferous forage plants: Agronomic performance, palatability and efficacy against parasitic nematodes in sheep[END_REF][START_REF] Berard | Condensed tannin concentrations found in vegetative and mature forage legumes grown in western Canada[END_REF][START_REF] Li | Condensed tannins concentration of selected prairie legume forages as affected by phenological stages during two consecutive growth seasons in western Canada[END_REF][START_REF] Grabber | Protein and drymatter degradability of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental USA[END_REF], sulla in Australia (de [START_REF] De Koning | Solutions for a better environment[END_REF][START_REF] De Koning | Sulla (Hedysarum coronarium) production sown with cover crops[END_REF][START_REF] Heuzé | Sulla (Hedysarum coronarium). Feedipedia[END_REF] and sainfoin in Canada and Europe [START_REF] Häring | Tanniferous forage plants: Agronomic performance, palatability and efficacy against parasitic nematodes in sheep[END_REF][START_REF] Hayot Carbonero | Sainfoin (Onobrychis viciifolia), a forage legume with great potential for sustainable agriculture, an insight on its morphological, agronomical, cytological and genetic characterisation[END_REF][START_REF] Malisch | Large variability of proanthocyanidin content and composition in sainfoin (Onobrychis viciifolia)[END_REF][START_REF] Bhattarai | Sainfoin (Onobrychis viciifolia Scop.): Renewed interest as a forage legume for western Canada[END_REF]. The choice of which forage to grow will depend on the climate, soil, environment, and farming practices. The performance of these forages has been tested in pure stands and in combinations with partner species to increase the overall forage quantity and quality [START_REF] Häring | Tanniferous forage plants: Agronomic performance, palatability and efficacy against parasitic nematodes in sheep[END_REF][START_REF] De Koning | Sulla (Hedysarum coronarium) production sown with cover crops[END_REF]Hayot-Carbonero et al., 2011;[START_REF] Döring | Using legume-based mixtures to enhance the nitrogen use efficiency and economic viability of cropping systems[END_REF][START_REF] Lüscher | Potential of legume-based grasslandlivestock systems in Europe[END_REF][START_REF] Wang | Condensed tannins in sainfoin: Composition, concentration, and effects on nutritive and feeding value of sainfoin forage[END_REF].

Cocultivation of Legumes and Companion Crops

Cocultivation of forage legumes with companion crops can deliver higher total yields [START_REF] Nyfeler | Strong mixture effects among four species in fertilized agricultural grassland led to persistent and consistent transgressive overyielding[END_REF][START_REF] Döring | Using legume-based mixtures to enhance the nitrogen use efficiency and economic viability of cropping systems[END_REF][START_REF] Finn | Ecosystem function enhanced by combining four functional types of plant species in intensively managed grassland mixtures: A 3-year continental-scale field experiment[END_REF][START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF][START_REF] Hunt | Seeding rate, oat companion crop, and planting season effects on irrigated organic birdsfoot trefoil stands in the mountain West United States[END_REF]Malisch et al., 2017), more nitrogen fixed per hectare [START_REF] Nyfeler | Grass-legume mixtures can yield more nitrogen than legume pure stands due to mutual stimulation of nitrogen uptake from symbiotic and non-symbiotic sources[END_REF][START_REF] Vasileva | Changes in some parameters in mixtures of sainfoin with subterranean clover[END_REF] and improved dry matter and nitrogen digestibilities of coensiled mixtures [START_REF] Wang | Effects of mixing sainfoin with alfalfa on ensiling, ruminal fermentation and total tract digestion of silage[END_REF], but success depends on the agronomic compatibility of the species, appropriate treatments, and development of new sainfoin cultivars with a more competitive canopy [START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF]Mora-Ortiz and Smith, 2016;[START_REF] Kölliker | Promising options for improving performance and proanthocyanidins of the forage legume sainfoin (Onobrychis viciifolia Scop.)[END_REF]Malisch et al., 2017).

Traditional mixtures for sainfoin establishment in the United Kingdom have included grasses such as timothy (Phleum L.) and meadow fescue [Schedonorus pratensis (Huds.) P. Beauv.] or undersowing with spring barley (Hordeum L.) as companions [START_REF] Liu | Establishment and production of common sainfoin (Onobrychis viciifolia Scop.) in the UK. 2. Effects of direct sowing and undersowing in spring barley on sainfoin and sainfoin-grass mixtures[END_REF][START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF]Mora-Ortiz and Smith, 2016). However, new investigations showed that chicory (Cichorium intybus L.)-which also has antiparasitic properties-and oat (Avena sativa L.) can be cocultivated with sainfoin for a short period (i.e., two agronomic cycles; Mora-Ortiz, 2015; Mora-Ortiz and Smith, 2016), but chicory was found to be a very aggressive partner and to suppress sainfoin [START_REF] Häring | Tanniferous forage plants: Agronomic performance, palatability and efficacy against parasitic nematodes in sheep[END_REF]. North American researchers have also explored oat and alfalfa as companion crops for irrigated birdsfoot trefoil and sainfoin [START_REF] Wang | Condensed tannins in sainfoin: Composition, concentration, and effects on nutritive and feeding value of sainfoin forage[END_REF][START_REF] Hunt | Seeding rate, oat companion crop, and planting season effects on irrigated organic birdsfoot trefoil stands in the mountain West United States[END_REF], and this involved developing a new sainfoin population for cocultivation with alfalfa. The novelty of this work lies in the fact that this new sainfoin population has good competitiveness against alfalfa compared with previous sainfoin accessions, where seedlings first produced a long taproot and could become outgrown by weeds and many companion species (Hayot Carbonero et al., 2011).

Weed Control

Weed control in sainfoin can be achieved [START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF]Mora-Ortiz and Smith, 2016;Malisch et al., 2017) through appropriate choice of partner species, sowing densities, and cutting frequencies and can lead to stable sainfoin percentages (i.e., ~40% of the sward was sainfoin), which suffice to reduce the incidence of bloat [START_REF] Wang | Comparison of alfalfa and mixed alfalfa-sainfoin pastures for grazing cattle: Effects on incidence of bloat, ruminal fermentation, and feed intake[END_REF]Malisch et al., 2017). This was also demonstrated for several other legume species in a Pan-European experiment [START_REF] Finn | Ecosystem function enhanced by combining four functional types of plant species in intensively managed grassland mixtures: A 3-year continental-scale field experiment[END_REF][START_REF] Connolly | Weed suppression greatly increased by plant diversity in intensively managed grasslands: A continentalscale experiment[END_REF][START_REF] Suter | Weed suppression enhanced by increasing functional trait dispersion and resource capture in forage ley mixtures[END_REF]. Other strategies for weed control can include application of preemergence, postemergence, and maintenance herbicides [START_REF] Sheldrick | Management and utilization of sainfoin (Onobrychis sativa)[END_REF][START_REF] Moyer | Effect of herbicide treatments on dandelion, alfalfa and sainfoin yields and quality[END_REF][START_REF] Frame | Temperate forage legumes[END_REF][START_REF] Amiri | Evaluation of post-emergence herbicides to control weeds of newly planted sainfoin (Onobrychis sativa)[END_REF][START_REF] Mora-Ortiz | Pre-breeding, functional genomics and agronomic improvement of the tanniniferous forage crop Onobrychis viciifolia[END_REF]. In the case of sainfoin, weed suppression is directly correlated with sainfoin establishment and yields.

Current understanding of CT expression in sainfoin suggests that it should be possible to breed new forage legumes with good yields and consistent CT profiles, which is important, as farmers need varieties with predictable CT traits.

Effects of Drought on Yield and Condensed Tannin Content in Sainfoin Accessions

However, instead of using irrigation to boost yield, others have focused on exploiting the taproot of sainfoin, as sainfoin can remain productive on dry, marginal soils and can continue to grow during unseasonably dry weather. To test this drought tolerance, 30 sainfoin accessions were subjected to drought for 18 wk, with the mean soil water potential of the upper 40 cm being below -2 MPa, and were compared with the dry matter yields of rainfed controls that received additional irrigation when the soil water potential was below -0.6 MPa. The results were also compared with six other forage species (legumes, nonleguminous forbs, and grasses) of known drought tolerance. This showed that some sainfoin accessions were as tolerant under severe drought as alfalfa, and a few accessions even exceeded its drought tolerance. Most sainfoin accessions also had lower drought losses than chicory [START_REF] Malisch | Phenotypic variation among and within thirty accessions of Onobrychis viciifolia examined under climate change scenarios[END_REF]. In addition, the CT traits were assessed under drought and control conditions for five of these accessions. Although CT composition was hardly affected by drought, the CT concentrations increased at the vegetative, but not the reproductive, stage [START_REF] Malisch | Drought effects on proanthocyanidins in sainfoin (Onobrychis viciifolia Scop.) are dependent on the plant's ontogenetic stage[END_REF].

Moreover, there were no interactions between drought and accession for CT traits, and it remains to be seen whether the ranking of sainfoin accessions according to their CTs is maintained across other environments [START_REF] Malisch | Drought effects on proanthocyanidins in sainfoin (Onobrychis viciifolia Scop.) are dependent on the plant's ontogenetic stage[END_REF]. Another study with 100 sainfoin genotypes from 10 ecotypes showed that drought tolerance was, however, correlated with leaf proline content [START_REF] Irani | Response to drought stress in sainfoin: Within and among ecotype variation[END_REF]. This correlation is in accordance with previous studies, and although the cause-and-effect relationship between proline and drought tolerance is not yet fully understood, proline can act as an osmoprotectant, thus stabilizing membranes and maintaining cell turgor. Additionally, there is some indication that it might contribute to upregulation of drought tolerance genes [START_REF] Szabados | Proline: A multifunctional amino acid[END_REF][START_REF] Per | Approaches in modulating proline metabolism in plants for salt and drought stress tolerance: Phytohormones, mineral nutrients and transgenics[END_REF]. Therefore, the current understanding of CT expression in sainfoin suggests that it should be possible to breed new cultivars with good yields, drought tolerance, and consistent CT profiles, which is important, as farmers need forage legumes with predictable CT traits.

Grazing vs. Preservation

Legume swards expressing CTs can be grazed safely, as they are nonbloating [START_REF] Wang | Forthy bloat in ruminants: Cause, occurrence, and mitigation strategies[END_REF][START_REF] Macadam | Beneficial effects of temperate forage legumes that contain condensed tannins[END_REF], but optimal use of CT forages would most likely be as a substitute for existing legumes in mixed swards, such as white clover with ryegrass (Lolium L.). This is because dry matter yields are greater from grasses than from legumes (subject to adequate nitrogen availability), and because farmers in temperate climates have identified forage species (including grasses) that enable profitable farming. In these situations, the CT concentration in the diet is diluted by the contribution of the CT-free component of the sward [START_REF] Waghorn | Effect of condensed tannins in Lotus corniculatus on the nutritive value of pasture for sheep[END_REF], so high CT concentrations of the bioactive CT legume might be most useful, as the CTs in one forage can affect digestion of protein in the companion forage [START_REF] Waghorn | Bloat in cattle 46. The potential of dock (Rumex obtusifolius) as an antibloat agent for cattle[END_REF].

Several CT forages possess antiparasitic properties, which are of special interest for sheep and goat grazing systems [START_REF] Hoste | Goat nematode interactions: Think differently![END_REF], or when there is anthelmintic resistance [START_REF] Terrill | Experiences with integrated concepts for the control of Haemonchus contortus in sheep and goats in the United States[END_REF]. Condensed tannins may also provide benefits when immunity of animals is low (i.e., around parturition or weaning), but the need to combat parasitism at such times does not necessarily coincide with the availability of fresh CT forages. Therefore, bioactivity needs to be maintained when processing CT forages into hay, pellets, or silages. Conserved forages allow out-of-season feeding, but may, especially in the case of pellets (Girard et al., 2016b), also offer opportunities for standardizing and optimizing CT traits and enable transportation to other regions.

Processing of CT forages into hay, pellets, or silages can have a marked effect on CT concentration and extractability (Fig. 4) and appears to increase proteinbound, and possibly covalently linked, CTs [START_REF] Terrill | Nitrogen and fiber digestion in sheep fed fresh-frozen and field-dried high-and low-tannin sericea lespedeza[END_REF][START_REF] Minnée | The effect of ensiling forage legumes on condensed tannins[END_REF][START_REF] Lorenz | Effect of wilting, silage additive, PEG treatment and tannin content on the distribution of N fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties[END_REF][START_REF] Vernhet | Characterization of oxidized tannins: Comparison of depolymerization methods, asymmetric flow field-flow fractionation and small-angle X-ray scattering[END_REF][START_REF] Ramsay | NaOH enhances extractability and analysis of proanthocyanidins in ensiled sainfoin (Onobrychis viciifolia)[END_REF][START_REF] Girard | Bioactive compounds in forage legumes: Structural changes during conservation, their fate along the digestive tract and their potential to impact ruminant products[END_REF][START_REF] Huang | Effects of conservation method on condensed tannin content, ruminal degradation, and in vitro intestinal digestion of purple prairie clover (Dalea purpurea Vent.)[END_REF]. Ensiling sainfoin or sulla reduced ammonia production, improved silage quality, and protected plant protein during fermentation, which improved its nutritional value relative to forages without CTs (Niezen et al., 1998b;[START_REF] Lorenz | Effect of wilting, silage additive, PEG treatment and tannin content on the distribution of N fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties[END_REF]. There is a need for feeding trials to assess the biological significance of unextractable CTs in terms of ruminal or intestinal digestion and efficacy against parasitic nematodes. These questions could perhaps be addressed through experiments that explore accession differences in terms of unextractable CTs.

ELUCIDATING RELATIONSHIPS BETWEEN CONDENSED TANNIN STRUCTURES AND ANTIPARASITIC EFFECTS

Parasitism imposes a considerable nutritional penalty on animals, and therefore controlling the parasite burden will indirectly benefit the nutritional status of animals. This is the reason for noting that CT forages can be used for nutraceutical purposes, which refers to a combined action of nutritional and antiparasitic benefits that include anthelmintic [START_REF] Terrill | Experiences with integrated concepts for the control of Haemonchus contortus in sheep and goats in the United States[END_REF][START_REF] Hoste | Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF] and anticoccidial effects [START_REF] Kommuru | Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats[END_REF][START_REF] Saratsis | The effect of sainfoin (Onobrychis viciifolia) and carob pods (Ceratonia siliqua) feeding regimes on the control of lamb coccidiosis[END_REF]. Most evidence of anthelmintic effects of CTs stems from in vitro assays, but some in vivo feeding trials under controlled experimental conditions have also yielded promising results. It is therefore timely to summarize the emerging trends and CT structure-activity relationships, which were obtained with a range of in vitro assays, to guide future feeding trials.

Antiparasitic Effects of Prodelphinidins and Galloylated Condensed Tannins

Condensed tannins with high molar percentages of PDs and galloylation have given good anthelmintic effects in vitro with parasite larvae (L). These include inhibition of L3 migration and L4 motility with Ascaris suum, as well as L1 feeding inhibition, adult motility assays of Ostertagia ostertagi and Cooperia oncophora, and larval exsheathment inhibition of Haemonchus contortus and Trichostrongylus colubriformis of L3 larvae [START_REF] Brunet | Monomers of condensed tannins affect the larval exsheathment of parasitic nematodes of ruminants[END_REF][START_REF] Quijada | Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are influenced by structural features of condensed tannins[END_REF]Desrues et al., 2016a;[START_REF] Ramsay | Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum[END_REF]. These findings could explain the excellent in vivo results with sheep or goats that were obtained when feeding sericea lespedeza (these CTs consist of almost pure PDs with high mDP values), big trefoil, sainfoin, sulla plants, or hazelnut (Corylus L.) peels (high PD percentage) and lentisk browse (galloylated compounds) [START_REF] Landau | Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse[END_REF][START_REF] Rodríguez-Pérez | A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves[END_REF][START_REF] Hoste | Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock[END_REF]. These in vivo trials found better host resilience and lower fecal egg counts of H. contortus, Teladorsagia circumcincta, and T. colubriformis and of a mixed gastrointestinal nematode infection, which were attributed to lower worm fecundity [START_REF] Niezen | Growth and gastrointestinal nematode parasitism in lambs grazing either lucerne (Medicago sativa) or sulla (Hedysarum coronarium) which contains condensed tannins[END_REF](Niezen et al., , 1998a;;[START_REF] Paolini | Effects of the repeated distribution of sainfoin hay on the resistance and the resilience of goats naturally infected with gastrointestinal nematodes[END_REF][START_REF] Landau | Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse[END_REF], and also lower Eimeria oocyst counts [START_REF] Kommuru | Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats[END_REF]. Control of T. colubriformis was reflected in higher liveweight gains by lambs fed sulla than alfalfa [START_REF] Niezen | Growth and gastrointestinal nematode parasitism in lambs grazing either lucerne (Medicago sativa) or sulla (Hedysarum coronarium) which contains condensed tannins[END_REF].

Antiparasitic Effects of the Mean Degree of Condensed Tannin Polymerization

The average size of CTs in a polymeric mixture (mDP value) can also affect their antiparasitic activity, with larger polymers being more potent against exsheathment of H. contortus L3 and larval feeding of O. ostertagi and C. oncophora L1 larvae in vitro [START_REF] Quijada | Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are influenced by structural features of condensed tannins[END_REF]Desrues et al., 2016a). However, PD-rich plants tend to have CTs with high mDP values and high CT concentrations [START_REF] Kommuru | Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats[END_REF][START_REF] Laaksonen | Proanthocyanidins and their contribution to sensory attributes of black currant juices[END_REF]Mueller-Harvey et al., unpublished data, 2016), confounding our understanding of CT structure-activity relationships. The characteristics of CTs responsible for antiparasitic effects need to be understood to identify optimal feed sources.

Apart from the examples above, very few other plants with high molar PD or galloyl percentages or high mDP values (>15) have been evaluated in vivo to determine their efficacy against gastrointestinal parasites. The plant kingdom remains a rich and underexplored resource of such promising CTs (Table 3), and it would be timely to test these in vitro results by feeding plants, browse, or agroindustrial byproducts with differing CT traits to parasitized livestock. As traits vary across accession and environment, it is important to analyze the dietary CTs to establish their relationships with antiparasitic effects. This would enable guidelines to be formulated for development of new plant cultivars for optimal bioactivities.

One other aspect also needs investigation: are CT traits that are optimal for antiparasitic effects compatible with nutritional benefits, or would farmers require plants with different CTs for either antiparasitic or nutritional purposes? Given the encouraging results with sericea lespedeza and panicledleaf ticktrefoil [Desmodium paniculatum (L.) DC.], especially in parasitized animals, anthelmintic and nutritional benefits may not be mutually exclusive [START_REF] Terrill | Experiences with integrated concepts for the control of Haemonchus contortus in sheep and goats in the United States[END_REF][START_REF] Cherry | Feeding panicled tickclover to growing goats reduces Haemonchus contortus infection without negative effects on growth[END_REF], but other options could include sacrificing nutrition for a short period, enabling an appropriate period of CT feeding to achieve parasite control.

Mechanisms of Antiparasitic Condensed Tannin Action

A key question concerns the mechanisms by which CTs exert their effects. It is thought that the ability of CTs [START_REF] Sivakumaran | Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria[END_REF][START_REF] Tibe | Condensed tannins and flavonoids from the forage legume sulla (Hedysarum coronarium)[END_REF][START_REF] Mechineni | Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections, skin and carcass microbial load, and meat quality of growing goats[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF]Ropiak et al., 2016b Galloylated CTs Shea (Vitellaria paradoxa C. F. Gaertn.) nuts, persimmon (Diospyros kaki Thunb.) fruits, lentisk (Pistacia lentiscus L.) leaves, carob (Ceratonia siliqua L.) fruits, grape (Vitis vinifera L.) seeds, great water dock (Rumex hydrolapathum Huds.) roots, dock (Rumex obtusifolius L.) leaves [START_REF] Papagiannopoulos | Identification and quantification of polyphenols in carob fruits (Ceratonia siliqua L.) and derived products by HPLC-UV-ESI/MSn[END_REF][START_REF] Spencer | Isolation and characterisation of procyanidins from Rumex obtusifolius[END_REF][START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF][START_REF] Rodríguez-Pérez | A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves[END_REF][START_REF] Ramsay | Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum[END_REF]Ropiak et al., 2016b High mean degree of polymerization values Erect canary clover plant, persimmon fruits, apple (Malus domestica Borkh. spp., cider cultivars); leaves of Persian ironwood, London plane, black currant, black locust; white clover flowers; sericea lespedeza plant Guyot et al., 2001a;[START_REF] Sivakumaran | Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria[END_REF][START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF][START_REF] Mechineni | Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections, skin and carcass microbial load, and meat quality of growing goats[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF]Ropiak et al., 2016b to suppress gastrointestinal nematodes stems from their ability to bind with proteins [START_REF] Hoste | Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections[END_REF]. Thus, CTs could act by inhibiting key parasite enzymes, and indeed inhibition of parasite glutathione-S-transferases, which play an important role in the detoxification of xenobiotic compounds, has been confirmed in in vitro experiments [START_REF] Hansen | Proanthocyanidins inhibit Ascaris suum glutathione-Stransferase activity and increase susceptibility of larvae to levamisole in vitro[END_REF]. Prodelphinidins have more phenolic groups capable of forming hydrogen bonds with proteins than PCs (Fig. 1), but it has also been established that the mDP value is the most important factor for CT protein aggregation and precipitation (Zeller et al., 2015b;[START_REF] Ropiak | Identification of structural features of condensed tannins that affect protein aggregation[END_REF]. Taken together, these observations could explain why PDs, which generally have larger mDP values than PCs, have better anthelmintic properties (Hoste et al., 2016;[START_REF] Kommuru | Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats[END_REF][START_REF] Kommuru | Effect of sericea lespedeza leaf meal pellets on adult female Haemonchus contortus in goats[END_REF]. By using electron microscopy, a number of studies have revealed physical deformations of H. contortus adult worms that had been exposed to CTs in either in vitro experiments or collected after feeding sericea lespedeza or sainfoin plants or tzalam [Lysiloma latisiliquum (L.) Benth.] leaves [START_REF] Martínez-Ortíz-De-Montellano | Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions[END_REF][START_REF] Kommuru | Effect of sericea lespedeza leaf meal pellets on adult female Haemonchus contortus in goats[END_REF]. The shriveled surfaces and plaque formations around orifices could account for inhibition of feeding and lowering of fecundity and are thought to stem from CTs interacting directly with proteins on parasite surfaces (Ropiak et al., 2016a). Scanning and transmission electron microscopy detected not only external but also internal damage to the cuticle and sensilla of the lip region of young and adult Caenorhabditis elegans by different CT types, and to the cuticle and underlying tissue (i.e., muscle cells) and intestinal cells of H. contortus L3 larvae and adults and of T. colubriformis L3 larvae by sainfoin and tzalam CTs [START_REF] Brunet | Ultrastructural changes in the third-stage, infective larvae of ruminant nematodes treated with sainfoin (Onobrychis viciifolia) extract[END_REF][START_REF] Martínez-Ortíz-De-Montellano | Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions[END_REF]Ropiak et al., 2016a). Given the low uptake (bioavailability) of polymeric CTs in mammalian tissues in comparison with monomeric flavonoids [START_REF] Li | Interactions between plasma proteins and naturally occurring polyphenols[END_REF], it would be worth exploring whether the internal tissue damage arises from the CTs or from other compounds that may be present in plant extracts [START_REF] Brunet | Monomers of condensed tannins affect the larval exsheathment of parasitic nematodes of ruminants[END_REF][START_REF] Williams | Anthelmintic activity of transcinnamaldehyde and A-and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum)[END_REF][START_REF] Klongsiriwet | Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins[END_REF]Desrues et al., 2016a;[START_REF] Mengistu | The in vitro anthelmintic properties of browse plant species against Haemonchus contortus is determined by the polyphenol content and composition[END_REF]. It also raises the question whether mixtures of CTs plus co-occurring smaller plant compounds (such as quercetin, luteolin, cinnamaldehyde, etc.) can cause both external and internal damage, or whether internal damage may be due to a disruption of nematode metabolism, in turn causing necrosis of cells and tissues. Condensed tannins together with a flavone (luteolin), a flavonol (quercetin), or cinnamaldehyde can act synergistically against parasitic nematodes in vitro [START_REF] Barrau | Effect of bioactive compounds from sainfoin (Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus: Role of tannins and flavonol glycosides[END_REF][START_REF] Klongsiriwet | Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF]Ropiak et al., 2016a). These in vitro findings are yet to be tested in feeding trials using combinations of feeds with different CTs and such monomeric compounds, but such combinations should hopefully lead to future on-farm applications.

Tissue and Host Responses to Condensed Tannins

In addition to direct anthelmintic effects against parasites, more work is warranted on how CTs may influence host responses to parasites. [START_REF] Niezen | The effect of feeding sulla (Hedysarum coronarium) or lucerne (Medicago sativa) on lamb parasite burdens and development of immunity to gastro intestinal nematodes[END_REF] measured higher antibody titers against antigens to adult T. circumcincta and adult and larval T. colubriformis when lambs were fed with sulla compared with alfalfa. This may be a result of increased intestinal flow of proteins and amino acids associated with ruminal protection of protein degradation, which can contribute to the host's ability to maintain growth, immune function, and regulation of worm populations (Ríos-De [START_REF] Álvarez | The effect of dietary sainfoin (Onobrychis viciifolia) on local cellular responses to Trichostrongylus colubriformis in sheep[END_REF][START_REF] Ramírez-Restrepo | Characterization of immune responses against gastrointestinal nematodes in weaned lambs grazing willow fodder blocks[END_REF][START_REF] Hoste | Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections[END_REF]. Moreover, recent in vitro studies have also indicated that CTs can directly modulate the activity of immune cells such as gd T cells and dendritic cells, potentially enhancing the host's innate immune response [START_REF] Tibe | Condensed tannins from Botswanan forage plants are effective priming agents of gd T cells in ruminants[END_REF][START_REF] Williams | Polymerization-dependent activation of porcine g d T-cells by proanthocyanidins[END_REF][START_REF] Williams | Co-operative suppression of inflammatory responses in human dendritic cells by proanthocyanidins and products from the parasitic nematode Trichuris suis[END_REF]. Similar to other bioactivity studies, immunemodulating activity in vitro is highly dependent on CT size, with mDP >6 eliciting a stronger response than CTs with mDP <6, and flavan-3-ol monomers have little or no effect [START_REF] Williams | Polymerization-dependent activation of porcine g d T-cells by proanthocyanidins[END_REF][START_REF] Williams | Co-operative suppression of inflammatory responses in human dendritic cells by proanthocyanidins and products from the parasitic nematode Trichuris suis[END_REF].

Effects of Fermentation on Condensed Tannin Activity

Work has started on determining how fermentation affects CT concentrations and bioactivities. According to results from the HCl-butanol-acetone or thiolysis assays, fermentation can reduce "apparent" CT concentrations by 30 or 85%, respectively, in silages [START_REF] Mena | Bioactivation of high-molecular-weight polyphenols by the gut microbiome[END_REF][START_REF] Ramsay | NaOH enhances extractability and analysis of proanthocyanidins in ensiled sainfoin (Onobrychis viciifolia)[END_REF] and the gut [START_REF] Desrues | Condensed tannins in the gastrointestinal tract of cattle after sainfoin (Onobrychis viciifolia) intake and their possible relationship with anthelmintic effects[END_REF][START_REF] Quijada | Condensed tannin changes along the digestive tract in lambs fed with sainfoin pellets or hazelnut skins[END_REF]. However, despite these apparent losses, sericea lespedeza and sainfoin silage extracts and silages still exerted anthelmintic effects in vitro by inhibiting the exsheathment of H. contortus L3 larvae and in vivo by lowering adult worm burden and fecal egg counts [START_REF] Heckendorn | Effect of sainfoin (Onobrychis viciifolia) silage and hay on established populations of Haemonchus contortus and Cooperia curticei in lambs[END_REF][START_REF] Manolaraki | Propriétés anthelminthiques du sainfoin (Onobrychis viciifoliae): Analyse des facteurs de variations et du rôle des composés phénoliques impliqués[END_REF][START_REF] Terrill | Effect of ensiling on anti-parasitic properties of sericea lespedeza[END_REF]. This may either be due to sufficient quantities of undegraded CTs or hydrolysis of anthelmintic flavonoids from inactive glycosides [START_REF] Manolaraki | Propriétés anthelminthiques du sainfoin (Onobrychis viciifoliae): Analyse des facteurs de variations et du rôle des composés phénoliques impliqués[END_REF], or to proteinbound CTs surviving ruminal fermentation and exerting anthelmintic activity in the abomasum, where a lower pH facilitates dissociation of the CT-protein complex [START_REF] Jones | Complexes of the condensed tannins of sainfoin (Onobrychis viciifolia Scop.) with Fraction 1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH[END_REF]. A high activity against O. ostertagi nematodes of the abomasum contrasted with no activity against intestinal Cooperia oncophora nematodes and was associated with 2.3% CTs (g 100 g -1 dry matter by thiolysis) in the abomasum vs. 0.02% CTs in the intestine (Desrues et al., 2016a(Desrues et al., , 2016b[START_REF] Desrues | Condensed tannins in the gastrointestinal tract of cattle after sainfoin (Onobrychis viciifolia) intake and their possible relationship with anthelmintic effects[END_REF]. However, both nematode species were affected by CTs in the in vitro larval feeding inhibition assay (Desrues et al., 2016a).

EFFECTS ON RUMINAL FERMENTATION AND CONSEQUENCES FOR RUMINANT NUTRITION AND GREENHOUSE GAS EMISSIONS Effects on Nutrition and Dietary Protein Use

Nutritional effects of CTs are currently understood in general terms, and information concerning the ways that CTs affect specific processes are only now being defined by determining the CT traits of forages used for in vivo and in vitro measurements and their effects on the microbiome [START_REF] Grosse Brinkhaus | Effect of exchanging Onobrychis viciifolia and Lotus corniculatus for Medicago sativa on ruminal fermentation and nitrogen turnover in dairy cows[END_REF], 2017). Binding to dietary proteins and reduction in rumen proteolysis, resulting in higher proportions of dietary protein passing to the small intestine, have been measured [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF], but details and mechanisms are not understood. The findings by [START_REF] Kariuki | The digestion of dietary protein bound by condensed tannins in the gastro-intestinal tract of sheep[END_REF] indicated that the release of dietary protein between the abomasum and terminal ileum was correlated with the protein precipitation capacity of CTs, which depends on the structures of both the CTs and the proteins [START_REF] Hagerman | The specificity of proanthocyanidin-protein interactions[END_REF][START_REF] Dobreva | Interaction of sainfoin (Onobrychis viciifolia) condensed tannins and proteins[END_REF]. The measurements of [START_REF] Wang | Effect of condensed tannins in Lotus corniculatus upon the digestion of methionine and cysteine in the small intestine of sheep[END_REF] are especially important in this regard, because they showed that in sheep fed birdsfoot trefoil with polyethylene glycol (PEG, which inactivated the CTs), ~80% of amino acid absorption occurred in the first half of the intestine, but the entire length of the intestine was required for absorption when CTs were active. Hence, the unknowns concerning CT interactions with rumen function, microbial growth, and intestinal absorption need to be evaluated in terms of dietary CT traits.

The current evidence suggests that only four forage legumes-birdsfoot trefoil, sainfoin, sulla and crownvetch [Securigera varia (L.) Lassen]-support higher ruminant growth rate or milk yield when fed as a sole diet, compared with CT-free diets [START_REF] Burns | Responses of dairy heifers to crownvetch, sericea lespedeza, and alfalfa forages[END_REF][START_REF] Waghorn | Condensed tannins and the nutritive value of herbage[END_REF]Waghorn et al., , 1997;;[START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Rochfort | Plant bioactives for ruminant health and productivity[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF][START_REF] Patra | A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen[END_REF][START_REF] Macadam | Case study: Average daily gain and blood fatty acid composition of cattle grazing the nonbloating legumes birdsfoot trefoil and cicer milkvetch in the Mountain West[END_REF][START_REF] Naumann | Condensed tannins in the ruminant environment: A perspective on biological activity[END_REF][START_REF] Piluzza | Tannins in forage plants and their role in animal husbandry and environmental sustainability: A review[END_REF][START_REF] Macadam | Beneficial effects of temperate forage legumes that contain condensed tannins[END_REF]. However, other CT forage legumes or some CT browse species may be beneficial when fed as part of a diet (e.g., lotuses; [START_REF] Ayres | Birdsfoot trefoil (Lotus corniculatus) and greater lotus (Lotus uliginosus) in perennial pastures in eastern Australia 2. Adaptation and applications of lotus-based pasture[END_REF]. A universal consequence of dietary CTs is a reduction in urinary nitrogen excretion (because of reduced rumen proteolysis) and an increase in fecal excretion of nitrogen [START_REF] Mueller-Harvey | Unravelling the conundrum of tannins in animal nutrition and health[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF].

Evaluation of nutritional benefits can be complicated, especially if both feed quality (digestibility) and voluntary feed intake are important. Variations in intake are likely to confound comparisons of digestibility, but digestible matter intake is a recognized indicator of performance. There are a number of ways that the effects of CTs can be determined, and several studies (Table 4) have fed a CT forage to two groups of animals, with one receiving daily doses (or intraruminal infusions) of PEG to bind and deactivate the CTs. This ensures that the majority of the diet is the same and enables the effect of the CTs to be evaluated. An option used by some researchers to compare a CT diet with a similar non-CT diet is fraught with difficulties. No species are optimal for comparisons, and any differences in composition, digestion, or intake will compromise the evaluation of the CTs. It is also important to distinguish the CT effect from a legume effect when evaluating a CT forage fed with grasses, for example, and if the animals are parasitized, any benefits of CTs could arise from direct or indirect impacts on the parasites [START_REF] Hoste | Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock[END_REF][START_REF] Hoste | Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants[END_REF].

Furthermore, because CTs reduce the digestion of protein in the rumen and over the entire gastrointestinal tract [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF], it is unlikely that any nutritional benefits will result when dietary crude protein is insufficient. Benefits are more likely when dietary protein is in excess of requirements. However, if in the presence of enough protein (or amino acids) other nutrients are limiting (e.g., energy intake or phosphorus), providing additional protein will not improve production [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF][START_REF] Pagán-Riestra | Phosphorus and other nutrient disappearance from plants containing condensed tannins using the nylon bag technique[END_REF]. Hence, the methods by which animals are fed and their physiological state (e.g., lactating, growing, or at maintenance) when evaluating the nutritional effects of CTs can contribute to inconsistencies in findings. In addition, comparative measures of digestion may be confounded by variation in intakes, because increasing intakes may reduce digestibility [START_REF] Tyrrell | Effect of intake on digestive efficiency[END_REF], but on other occasions have no effect [START_REF] Hammond | Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages[END_REF].

A number of studies with sainfoin and birdsfoot trefoil have also yielded contradictory results, often with lower or no production benefits measured when compared with CT-free controls [START_REF] Thomson | The digestion of dried lucerne (Medicago sativa, L) and dried sainfoin (Onobrychis viciifolia, Scop) by sheep[END_REF]Waghorn et al., 1997;[START_REF] Theodoridou | Effects of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep[END_REF][START_REF] Aufrère | Mixing sainfoin and lucerne to improve the feed value of legumes fed to sheep by the effect of condensed tannins[END_REF]Azuhnwi et al., 2013b;[START_REF] Copani | Silages containing bioactive forage legumes: A promising protein-rich feed source for growing lambs[END_REF]Girard et al., 2016aGirard et al., , 2016b)). In addition to the constraints mentioned above (dietary crude protein concentration, or whether other nutrients are limiting production), variations in CT traits within germplasms may also affect animal responses [START_REF] Grabber | Protein and drymatter degradability of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental USA[END_REF]. For example, the UK Hampshire Common and Cotswold Common sainfoin accessions had higher PC/PD ratios (>30:70) than the continental European Visnovsky accession (<19:81) [START_REF] Stringano | Analysis of sainfoin (Onobrychis viciifolia) proanthocyanidins by complementary and newly developed techniques[END_REF][START_REF] Stringano | Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (Onobrychis viciifolia) germplasm collection[END_REF] and could have accounted for these contradictory reports. Birdsfoot trefoil has delivered good growth rates in the United States and New Zealand, and the PC/PD ratios were ~80:20 [START_REF] Meagher | Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry[END_REF][START_REF] Macadam | Beneficial effects of temperate forage legumes that contain condensed tannins[END_REF]. However, when two sainfoin cultivars with PC/PD ratios of 24:76 (Visnovsky) and 37:63 (Perly) were fed to lambs infected with H. contortus, concentrations of essential amino acids in plasma were higher than when both diets had been treated with PEG to inactivate CTs (Azuhnwi et al., 2013b); it is not known whether the higher plasma concentrations were a direct result of CTs on protein digestion or an anthelmintic effect against H. contortus. Variable results could be investigated by considering CT traits in conjunction with diet composition (e.g., protein, fiber, water-soluble carbohydrate, starch, and amino acid contents, as these can be affected by environment; [START_REF] Grabber | Protein and drymatter degradability of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental USA[END_REF], forage harvesting, or preservation methods. This means that nutritional evaluations of CT plants need to be undertaken under documented and controlled conditions, with information on harvesting and preservation methods (grazed, dried as hay or pellets, or ensiled).

Another potentially important issue could be how animals are fed, because CT traits also vary within plants. In Spain, it is traditional to take the first sainfoin cut as hay and then leave animals to graze the regrowth (Dr S.F. Demdoum, personal communication, 2010). Under light stocking regimes in the United Kingdom, sheep will only eat the sainfoin tops (flowers and younger leaves) and leave older leaves and stems intact (Mueller-Harvey, personal observation). Therefore, feeding whole plants as pellets, hay, or silage could result in forages with very different nutritive values than grazed forages, and also because conservation affects CT concentrations; these facts could be another reason for the contradictory results mentioned above. One of the very few feeding trials that compared two birdsfoot trefoil cultivars with similar nutritional composition-apart from their CT concentrations-found that the 'Maitland' cultivar (3.5 g CTs 100 g -1 dry matter) achieved better nitrogen retention in sheep than the 'Empire' cultivar (0.5 g CTs 100 g -1 dry matter) [START_REF] Waghorn | Nutritive value of Lotus corniculatus L. containing low and medium concentrations of condensed tannins for sheep[END_REF]. Greater retention of dietary protein suggested that the Maitland CTs protected more soluble protein from digestion in the Waghorn et al., 1997;[START_REF] Min | The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: A review[END_REF][START_REF] Meagher | Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF] Birdsfoot trefoil (Goldie [indoor trials], Norcen, Oberhaunstadter [grazed]) 84:16 ND Enhanced absorption of essential amino acids, positive effects on livestock production (increased cattle and sheep growth, cow and sheep milk yield, sheep fertility and wool growth) [START_REF] Waghorn | Effect of condensed tannins in Lotus corniculatus on the nutritive value of pasture for sheep[END_REF]Waghorn et al., 1997;[START_REF] Meagher | Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: Progress and challenges[END_REF][START_REF] Macadam | Case study: Average daily gain and blood fatty acid composition of cattle grazing the nonbloating legumes birdsfoot trefoil and cicer milkvetch in the Mountain West[END_REF]MacAdam and Villalba, 2015 Crownvetch Chemung, Penngift (field cured, windrowed, baled) 26:74 >13 Larger daily gain of cattle and sheep under grazing or stall feeding compared with alfalfa or sericea lespedeza. [START_REF] Burns | Responses of dairy heifers to crownvetch, sericea lespedeza, and alfalfa forages[END_REF], 1977 (Note: our CT data are unpublished and not from the plants that were fed) † With information, if available, on variety or accession and method of feeding. ‡ PC/PD, procyanidin:prodelphinidin ratio; ND, not determined. § mDP, mean degree of polymerization.

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rumen but did not interfere with protein digestion and absorption in the abomasum and small intestine. More research is needed to establish the precise fate of dietary protein in the abomasum and intestine in the presence of CTs. Estimates based on their potentially beneficial protein protection effects suggest that if alfalfa had just 1% CTs in its dry matter, this could achieve a 12% increase in net returns for US dairy farmers [START_REF] Mccaslin | New strategies for forage quality improvement in alfalfa[END_REF]. It is unfortunate that few studies describe the composition of CTs in plants that have been fed to ruminants, and the LegumePlus program has attempted to address this issue by encouraging communication and collaboration among chemists and plant and animal scientists. However, we still do not know which CT traits plant breeders should be targeting (i.e., dietary CT concentration, PC/PD ratio, or mDP values or a combination of these) to increase livestock production and product quality. Apart from a few in vitro studies (Azuhnwi et al., 2013b;[START_REF] Hatew | Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methane production and fermentation characteristics[END_REF][START_REF] Huyen | Sainfoin (Onobrychis viciifolia): A forgotten crop for dairy cows with future potential[END_REF]Huyen et al., 2016b), no feeding trials have attempted to resolve the relationships between animal production and CT traits. Interdisciplinary research is needed to uncover the mechanisms by which CTs exert their effects on rumen (microbial) digestion and utilization of dietary nutrients, and this will require compositional (rather than colorimetric) analyses of the dietary CTs.

The size of CTs is the key parameter that controls CT protein aggregation and precipitation. This has been illustrated using bovine serum albumin (BSA) and gelatin, where aggregation increased markedly as the mDP values increased from 3 to 8 (corresponding to CT molecular weights of ~1000 to 2400 Da), but there were only small differences in the efficacies of larger CTs with mDP >9 to aggregate the proteins [START_REF] Ropiak | Identification of structural features of condensed tannins that affect protein aggregation[END_REF]. It would be worth testing whether CT size also affects the mechanism by which CTs interact with dietary or endogenous animal proteins, as Zeller et al. (2015b) showed that relatively more alfalfa protein was precipitated by CTs than BSA protein, which could be due to the major leaf protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) being much larger (560 kDa) than BSA (67 kDa). It will be necessary to determine if CTs with different mDP values affect dietary protein degradation and digestion, especially as the major proteins in forages (RuBisCo), beans (albumins, prolamins), and intestinal gut tissue and mucoproteins have quite different structures, which will influence their interactions with CTs [START_REF] Hagerman | The specificity of proanthocyanidin-protein interactions[END_REF]. As an aside, we have included bean proteins in this context, as several papers from the 1960s and 1970s on the nutritional effects of dietary tannins originated from feeding CT-containing beans [START_REF] Jansman | Tannins in faba beans (Vicia faba L.)[END_REF]Lowry et al., 1996). It would therefore be timely to review some of these results in the light of new knowledge on CT and protein structures.

Effects on the Quality of Animal Products

There are some effects of sainfoin diets on milk and meat quality and on the animal's physiological response to dietary CTs. Cattle fed a grass-sainfoin (mixture of Zeus and Esparcette accessions) silage or sheep grazed on big trefoil ('Maku') partitioned energy towards protein synthesis, rather than lipid synthesis, compared with grass-corn (Zea mays L.) silage and white clover diets, respectively [START_REF] Purchas | Fatness of lambs grazed on 'Grasslands Maku' lotus and 'Grasslands Huia' white clover[END_REF]Huyen et al., 2016a). Feeding dairy cows with sainfoin pellets lowered milk and blood urea concentrations compared with alfalfa and birdsfoot trefoil pellets (Girard et al., 2016b;[START_REF] Grosse Brinkhaus | Effect of exchanging Onobrychis viciifolia and Lotus corniculatus for Medicago sativa on ruminal fermentation and nitrogen turnover in dairy cows[END_REF] because of the reduction in rumen proteolysis and ammonia absorption.

Sainfoin and birdsfoot trefoil diets reduced bacterial biohydrogenation in the rumen, increased unsaturated fatty acid contents in milk, cheese, and meat products, and reduced indole and skatole in lamb meat [START_REF] Priolo | Carcass and meat quality of lambs fed fresh sulla (Hedysarum coronarium L.) with or without polyethylene glycol or concentrate[END_REF][START_REF] Schreurs | Concentration of indoles and other rumen metabolites in sheep after a meal of fresh white clover, perennial ryegrass or Lotus corniculatus and the appearance of indoles in the blood[END_REF][START_REF] Macadam | Beneficial effects of temperate forage legumes that contain condensed tannins[END_REF]Girard et al., 2016aGirard et al., , 2016b;;[START_REF] Huyen | Sainfoin (Onobrychis viciifolia): A forgotten crop for dairy cows with future potential[END_REF]Huyen et al., 2016a). Skatole and indole were associated with "fecal" flavor characteristic of pasture-fed products and originated from amino acid degradation by Clostridium aminophilum [START_REF] Attwood | Production of indolic compounds by rumen bacteria isolated from grazing ruminants[END_REF], which was relatively sensitive to CTs [START_REF] Sivakumaran | Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria[END_REF]. More recently, [START_REF] Campidonico | Fatty acid composition of ruminal digesta and longissimus muscle from lambs fed silage mixtures including red clover, sainfoin, and timothy[END_REF] reported that CTs and polyphenol oxidases in a sainfoin-red clover silage mixture generated additive effects that increased the intramuscular unsaturated fatty acid contents of lambs compared with a pure grass diet.

Effects on Nitrogen and Methane Emissions

The reduction in urinary nitrogen and increase in fecal nitrogen excretion seem to be universal consequences of dietary CTs fed to ruminants and are important because a greater proportion of nitrogen is lost from urine than feces. Thus, CTs can improve soil nitrogen status, lower emissions of the potent greenhouse gas nitrous oxide (N 2 O), and lessen nitrogen leachate into waterways and groundwater [START_REF] Kingston-Smith | Plant-based strategies towards minimising 'livestock's long shadow[END_REF][START_REF] Theodoridou | Effects of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep[END_REF]. A shift from urinary to fecal nitrogen could redue nitrogen losses by 25% and achieve savings on nitrogen fertilizers based on preliminary estimates from the Integrated Farm System model for dairy farms (Zeller and Grabber, 2015). Other opportunities for reducing the environmental impact of nitrogen emissions from ruminant livestock include the use of galloylated CTs and epigallocatechin gallate (a galloylated flavan-3-ol monomer), because they are urease inhibitors [START_REF] Huynh-Ba | Anti-urease cosmetic or dermatological composition[END_REF][START_REF] Powell | Tannin extracts abate ammonia emissions from simulated dairy barn floors[END_REF][START_REF] Takeuchi | Natural products and food components with anti-Helicobacter pylori activities[END_REF]. Urease inhibition reduces ammonia emissions from urine and subsequent N 2 O production [START_REF] Kingston-Smith | Plant-based strategies towards minimising 'livestock's long shadow[END_REF]. Grape seeds and some agroindustrial residues are sources of galloylated CTs and epigallocatechin gallate [START_REF] Li | High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: A highly galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin[END_REF][START_REF] Lee | Quantitative analysis of major constituents in green tea with different plucking periods and their antioxidant activity[END_REF][START_REF] Ramsay | Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum[END_REF], and application to the barn floor could lessen ammonia and N 2 O emissions from intensive systems.

It is clear that CTs can provide important benefits to ruminant farming; however, high dietary concentrations or CTs with the "wrong" compositional traits will lower the digestion and utilization of dietary protein and absorption of essential amino acids by ruminants [START_REF] Min | The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: A review[END_REF]. The challenge is to identify which CT traits are best able to enhance utilization of dietary protein to improve animal production, environmental sustainability, and profitability for farmers.

CONCLUSIONS AND POSSIBLE DIRECTIONS FOR THE FUTURE

Condensed tannins are the fourth largest group of secondary plant metabolites in the plant kingdom and provide opportunities for breeding forage legumes with novel CT traits. Research on CT-containing feeds has the potential to improve ruminant health by preventing bloat and mitigating effects of parasitism, as well as lowering environmental footprints and improving the sustainability of food quality and production for consumers [START_REF] Tedeschi | Developing a conceptual model of possible benefits of condensed tannins for ruminant production[END_REF]. Innovative molecular approaches have enabled alfalfa and white clover to express PC dimers and trimers in their foliages [START_REF] Hancock | Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa[END_REF][START_REF] Hancock | Progress towards developing bloat-safe legumes for the farming industry[END_REF][START_REF] Verdier | MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula[END_REF][START_REF] Mccaslin | New strategies for forage quality improvement in alfalfa[END_REF]. Apart from research by Mosjidis and colleagues, who selected for low CT concentration and grazing-tolerant sericea lespedeza for cattle production [START_REF] Mosjidis | Forage legume breeding and evaluation at Auburn University in the last 16 years[END_REF], there has been hardly any plant breeding for enhanced CT composition; however, progress is becoming feasible, as new genomic data and molecular markers for CTs have been obtained [START_REF] Kempf | Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm[END_REF]Mora-Ortiz et al., 2016). Alternatively, cultivars with specific CT traits could also be obtained by conventional selection, focusing on the compositional CT differences that already exist between plant species and between, but also within, accessions and plant parts.

This review has highlighted that interdisciplinary research is essential for developing new forages with desirable CT traits and bioactivities, and that it requires well-coordinated inputs from plant scientists, chemists, animal nutritionists, and parasitologists. Such collaborations have succeeded in identifying the large variation in CT traits and their in vitro nutritional and parasitological effects that pertain to the germplasms of sainfoin and birdsfoot trefoil [START_REF] Stringano | Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (Onobrychis viciifolia) germplasm collection[END_REF][START_REF] Grabber | Yield, morphological characteristics, and chemical composition of European-and Mediterranean-derived birdsfoot trefoil cultivars grown in the colder continental United States[END_REF][START_REF] Malisch | Large variability of proanthocyanidin content and composition in sainfoin (Onobrychis viciifolia)[END_REF], which grow in temperate climates. Such variation is likely to exist also in other species (e.g., sericea lespedeza and prairie clover, which grow in warm humid and colder regions, respectively; [START_REF] Mosjidis | Forage legume breeding and evaluation at Auburn University in the last 16 years[END_REF][START_REF] Berard | Condensed tannin concentrations found in vegetative and mature forage legumes grown in western Canada[END_REF]. It would be timely to explore these in vitro results by feeding CT forage legumes with varying CT traits to develop robust targets and tools for plant breeding.

Researchers must become aware that most colorimetric assays are not appropriate for determining CT concentrations or compositions. For example the p-dimethylaminocinnamaldehyde and HCl-vanillin reagents detect CTs, but also their monomeric flavan-3-ol precursors, and the Folin-Ciocalteu reagent detects all phenolic groups, whether in monomeric flavonoids, proteins, or condensed or hydrolyzable tannins [START_REF] Schofield | Analysis of condensed tannins: A review[END_REF]. These problems are made worse by the use of inappropriate CT material to construct calibration curves for analysis of CT concentrations, as mentioned above [START_REF] Grabber | Acetone enhances the direct analysis of procyanidin-and prodelphinidin-based condensed tannins in Lotus species by the butanol-HCl-iron assay[END_REF][START_REF] Krueger | Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health[END_REF]. The fact that a multitude of methods or standards have been chosen to measure CTs has prevented comparisons of published CT concentrations from feeding trials and the setting of optimum thresholds for CT traits. Appropriate CT analysis is crucial to progressing this field.

Laboratory studies have probed the impact of CT concentration, polymer size, and PC/PD ratios on ruminal fermentation and antiparasitic effects. The time has come for feeding trials with selected forages of similar nutritional compositions but different CT traits to test these in vitro results and establish the in vivo nutritional and antiparasitic effects associated with contrasting CT traits. This will also require comparison of forages that have been grazed or processed into hay, pellets, or silages, because CTs become less extractable on processing, but the underlying mechanisms and biological significance of these changes are yet to be explored. Such studies will help to optimize dietary protein utilization and energy partitioning and reduce the environmental footprint of livestock production.

We also need answers to the following questions: what effects do CTs exert on intestinal cells in ruminants and nonruminants in terms of nutrient absorption and cell signaling cascades? What are the mechanisms by which CTs protect dietary protein from rumen degradation and affect amino acid absorption from the intestine (e.g., in the presence of CTs, amino acid absorption takes place across the entire intestine; however, in the absence of CTs, absorption occurs in the first third of the intestine; [START_REF] Wang | Effect of condensed tannins in Lotus corniculatus upon the digestion of methionine and cysteine in the small intestine of sheep[END_REF]? What are the effects on the ruminal or colonic microbiomes? How does the impact of CTs on recycling of urea-nitrogen modify the utilization of energy and protein in dairy cows, and what is the origin of higher fecal nitrogen outputs in the presence of CTs? We need to establish the fate and bioactivity of CTs during ensiling and digestion, their mechanisms of action against parasites along the digestive tract, and their effects on the in vivo immune response and establish relationships with CT concentration and composition. This review has also highlighted the need for analytical methods and CT standards that are fit for purpose so that published CT values can be compared between research groups and experiments. Although considerable progress has been achieved over recent years, questions remain on how to translate the research results into practice, such as:

• How should we use CT forages? Is it better to graze forages with low CT concentrations and use high-CT forages as supplements?

• Can high-CT and CT-free forages be grazed together in strips, and could this improve utilization of dietary protein?

• Can the competitiveness and persistency of CTcontaining legumes be increased so they can be sown together with other crops and achieve successful weed suppression?

• What are the ecological implications of intake and selectivity by different ruminant species on plant persistence in the field?

• Does an optimum CT concentration and composition exist that can deliver nutritional plus antiparasitic effects? According to [START_REF] Cherry | Feeding panicled tickclover to growing goats reduces Haemonchus contortus infection without negative effects on growth[END_REF], it may be possible to achieve both, or do we need forages with different CT compositions (i.e., cultivars that either improve protein utilization or possess antiparasitic effects)?

• Will increased use of CT forages generate resistance of parasites to CTs, and how can feeding regimes mitigate against this?

• Is it best to use a short-term supply of high-CT forages for reducing parasite burdens at strategic times (e.g., pregnancy, parturition, weaning), or should a longer-term supply of low-CT forages be used to boost the immune response?

• What agronomic, harvesting, or processing measures can best ensure that CT plants deliver consistent results? Which cultivars should be grazed and processed into hay, pellets, or silage, and at what times of the year?

From these questions we need to develop practical solutions through collaboration with farmers and veterinarians. Producers and consumers alike are looking for sustainable innovations that produce high-quality foods profitably while also maintaining soil fertility and the quality of our environment.

  www.crops.org crop science, vol. 59, may-june 2019

Fig. 2 .

 2 Fig. 2. The LegumePlus project-an interdisciplinary European Union-funded research and training network on sainfoin (http:// legumeplus.eu).

Fig. 3 .

 3 Fig. 3. Near-infrared reflectance spectroscopy (NIRS) for predicting condensed tannin (CT) composition of sainfoin plants that had been analyzed by thiolysis (Gea et al., 2011; Mueller-Harvey et al., 2011).

Fig. 4 .

 4 Fig. 4. Average percentages of extractable and unextractable condensed tannins (CTs) in fresh, pelleted, and ensiled sainfoin, sulla, and birdsfoot trefoil[START_REF] Minnée | The effect of ensiling forage legumes on condensed tannins[END_REF][START_REF] Lorenz | Effect of wilting, silage additive, PEG treatment and tannin content on the distribution of N fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties[END_REF][START_REF] Ramsay | NaOH enhances extractability and analysis of proanthocyanidins in ensiled sainfoin (Onobrychis viciifolia)[END_REF].

  www.crops.org crop science, vol. 59, may-june 2019

Table 1 .

 1 Condensed tannin (CT) concentrations (g/100 g dry matter) and compositions of forage legumes and selected pasture plants (Note: these studies used various CT assays that were applied either to whole plants, isolated extracts, or purified CT fractions).

	Plant species	CT concentration PC/PD ratio † mDP ‡	References
		g 100 g -1 dry matter
	Erect canary clover [Dorycnium rectum (L.) Ser.]	15-20	5:95-17:83	10-127	Sivakumaran et al., 2004
	Sericea lespedeza [Lespedeza cuneata (Dum.	6-13	3:97	33	Mechineni et al., 2014
	Cours.) G. Don]
	Panicledleaf ticktrefoil [Desmodium paniculatum	22	nd §	nd	Pawelek et al., 2008
	(L.) DC.]
	Sulla (Hedysarum coronarium L.)	2-12	11:89-27:73	3-46	Tibe et al., 2011
	Big trefoil (Lotus pedunculatus Cav.)	5-10	16:84-20:80	2-44	Meagher et al., 2004; Sivakumaran et al., 2006
	Sainfoin (Onobrychis viciifolia Scop.)	1-9	5:95-50:50	12-84	Berard et al., 2011; Stringano et al., 2012;
					Azuhnwi et al., 2013a; Malisch et al., 2015
	Prairie clover (Dalea purpurea Vent.)	4-9	nd	nd	Berard et al., 2011
	Birdsfoot trefoil (Lotus corniculatus L. var.	0-5	60:40-84:16	9	Meagher et al., 2004; Sivakumaran et al., 2006;
	corniculatus)				Berard et al., 2011; Grabber et al., 2014
	† PC/PD, procyanidin:prodelphinidin ratio.
	‡ mDP, mean degree of polymerization.
	§ nd, not determined.

Table 2 .

 2 Overview of techniques for determining extractable or unextractable condensed tannins (CTs) and their composition.

		Extractable	Unextractable	Information on CT
	Analytical technique †	CTs	CTs	composition	Literature
	HCl-butanol-acetone	X	X	Limited	Grabber et al., 2013
	Thiolysis, phloroglucinolysis	X	X	X	Guyot et al., 2001b; Gea et al., 2011; Hixson et al., 2015,
					2016; Ramsay et al., 2016
	NMR	X	X	X	Grabber et al., 2013; Zeller et al., 2015a
	NIRS	X	X	X	Petersen et al., 1991; Larkin et al., 1997; Dykes et al.,
					2014; Grabber et al., 2014; Klongsiriwet 2016
	MALDI-TOF MS	X	-

X Stringano et al., 2011;[START_REF] Feliciano | Deconvolution of matrixassisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins[END_REF]

; Krueger et al., 2013 UPLC-MS/MS X -X Engström et al., 2014 www.crops.org crop science, vol. 59, may-june 2019

Table 3 .

 3 Plant materials containing condensed tannins (CTs) with structural characteristics that are of interest for conferring antiparasitic activities.

	CT traits	Plant species and part	Reference
	Prodelphinidin-rich CTs	Peels of hazelnut (Corylus avellana L.) seeds; leaves of Persian ironwood
		[Parrotia persica (DC.) C.A. Mey.], London plane (Platanus ´ hispanica Mill.
		ex Münchh. [occidentalis ´ orientalis]), black or red currant (Ribes nigrum
		L., R. rubrum L.) bushes, black locust (Robinia pseudoacacia L.); sainfoin
		(Onobrychis viciifolia Scop.), sulla (Hedysarum coronarium L.), sericea
		lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don] plants; white clover
		(Trifolium repens L.) flowers; erect canary clover [Dorycnium rectum (L.) Ser.]

Table 4 .

 4 Variation of nutritional effects achieved with different sainfoin accessions and feeding regimes in comparison with two trefoil species and crownvetch forage legumes.

	References		Scharenberg et al., 2007; Stringano et al., 2012;	Azuhnwi et al., 2013b	Grosse Brinkhaus et al., 2016	Copani et al., 2016	Theodoridou et al., 2012		Maughan et al., 2014; Villalba et al., 2015	Karnezos et al., 1994	Bermingham et al., 2001		Huyen et al., 2016b	Thompson et al., 1971; Stringano et al., 2012		Egan and Ulyatt, 1980
	Nutritional effects		Lambs: lower apparent organic matter and fiber digestibility, increased plasma	concentration of essential amino acids, no effect on body N retention	No effect on N retention in dairy cows	Lower growth rate of lambs than on red clover mixtures	Condensed tannins (CTs) had no effect on N retention compared with the	+polyethylene glycol (PEG) control	Comparable weight gains by beef calves grazing sainfoin and alfalfa diets	Growth rate and slaughter data were the same for sainfoin-and alfalfa-fed lambs	CTs in sulla, but not in sainfoin, improved amino acid absorption compared	with the +PEG control	Dairy cows: higher milk yield compared with grass silage	Farmers report high growth rates of lambs fed UK sainfoin accessions (but	no accession details were provided in feeding trial)	Positive effect on N retention compared with rye grass and clover, greater	recycling and degradation of urea, improved utilization of recycled N	Reduced growth rate, lower apparent digestibility of essential amino acids
	mDP §		16-29		ND	ND	ND		ND	ND	ND		ND	12-32		ND		ND
	PC/PD ‡		19:81-24:76		ND	ND	ND		ND	ND	ND		ND	29:71-33:67		ND		20:80
	Plant species †	Sainfoin accessions:	Visnovsky (dried or ensiled)		Perly (pellets, 20% of basal diet)	Perly (ensiled with timothy grass)	Perly (ensiled)		Shoshone (grazed)	Renumex (grazed)	No details (fresh forage)		Zeus/Esparcette (ensiled)	UK accessions (Sombourne,	Hampshire, Cotswold Common)	Fakir-cut and stall-fed	Lotus spp.	Big trefoil (Maku-indoor trials)

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† NMR, nuclear magnetic resonance; NIRS, near-infrared reflectance spectroscopy; MALDI TOF MS, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass spectrometry (for further information, see[START_REF] Zeller | Activity, purification, and analysis of condensed tannins: Current state of affairs and future endeavors[END_REF].

Acknowledgments

This work was supported by a European Union Marie Curie training network ("LegumePlus", PITN-GA-2011-289377), the Danish Council for Independent Research (Technology and Production Sciences, Grant no. 12-126630), the Institut National de la Recherche Agronomique, France (Drastic and Sustainable Treatment Reduction against Parasitism in Livestock project from the 'Gestion Intégrée de la Santé Animale' Metaprogramme), and in part by a USDA-ARS Specific Cooperative Agreement no. 58-3655-0-155F with the University of Reading, UK. We are grateful to the USDA-AR GRIN germplasm database for providing seeds of the sainfoin accessions.

Conflict of Interest

The authors declare that there is no conflict of interest.