Journal of the Science of Food and Agriculture J Sci Food Agric 86:2010–2037 (2006) Review Unravelling the conundrum of tannins in animal nutrition and health † Irene Mueller-Harvey ∗ NSRU Chemistry & Biochemistry Laboratory, Agriculture Department, Reading University, PO Box 236, Reading RG6 6AT, UK Abstract: This paper examines the nutritional and veterinary effects of tannins on ruminants and makes some comparisons with non-ruminants. Tannin chemistry per se is not covered and readers are referred to several excellent reviews instead: (a) Okuda T et al . Heterocycles 30:1195–1218 (1990); (b) Ferreira D and Slade D. Nat Prod Rep 19:517–541 (2002); (c) Yoshida T et al . In Studies in Natural Product Chemistry. Elsevier Science, Amsterdam, pp. 395–453 (2000); (d) Khanbabaee K and van Ree T. Nat Prod Rep 18:641–649 (2001); (e) Okuda et al . Phytochemistry 55:513–529 (2000). The effects of tannins on rumen micro-organisms are also not reviewed, as these have been addressed by others: (a) McSweeney CS et al . Anim Feed Sci Technol 91:83–93 (2001); (b) Smith AH and Mackie RI. Appl Environ Microbiol 70:1104–1115 (2004). This paper deals first with the nutritional effects of tannins in animal feeds, their qualitative and quantitative diversity, and the implications of tannin–protein complexation. It then summarises the known physiological and harmful effects and discusses the equivocal evidence of the bioavailability of tannins. Issues concerning tannin metabolism and systemic effects are also considered. Opportunities are presented on how to treat feeds with high tannin contents, and some lesser-known but successful feeding strategies are highlighted. Recent research has explored the use of tannins for preventing animal deaths from bloat, for reducing intestinal parasites and for lowering gaseous ammonia and methane emissions. Finally, several tannin assays and a hypothesis are discussed that merit further investigation in order to assess their suitability for predicting animal responses. The aim is to provoke discussion and spur readers into new approaches. An attempt is made to synthesise the emerging information for relating tannin structures with their activities. Although many plants with high levels of tannins produce negative effects and require treatments, others are very useful animal feeds. Our ability to predict whether tannin-containing feeds confer positive or negative effects will depend on interdisciplinary research between animal nutritionists and plant chemists. The elucidation of tannin structure–activity relationships presents exciting opportunities for future feeding strategies that will benefit ruminants and the environment within the contexts of extensive, semi-intensive and some intensive agricultural systems. 2006 Society of Chemical Industry Keywords: ammonia; analysis; anthelmintic; bloat; browse; diarrhoea; environmental effects; metabolism; methane; nutritional effects; parasites; physiological effects; proteins; silage; structure–activity relationships; sugars; tannins; toxicity; treatments NUTRITIONAL EFFECTS OF TANNINS Lowry et al . 1 have pointed out the surprising longevity of some commonly held perceptions – even if erro- neous – of the harmful effects of tannins. They out- lined how the first reviews in the 1960s and 1970s, which covered the effects of plant phenolics on mam- mals, had treated phenolics as toxic xenobiotics in general, despite reports demonstrating that many phenolics had very low toxicities. Nevertheless, gener- alisations persist that tannins are harmful or toxic to animals, which is at odds with the fact that humans have enjoyed tannins for thousands of years in drinks and foods. 2–4 Beneficial nutritional effects Tannins occur in many feeds such as fodder legumes, browse leaves and fruits. 5–9 Some of these feeds (species of Acacia, Dichrostachys, Dorycnium, Hedysarum, Leucaena, Lotus, Onobrychis, Populus, Rumex and Salix) can produce useful benefits in ruminants, such as better utilisation of dietary protein, faster growth rates of liveweight or wool, higher milk yields, increased fertility, and improved animal welfare and health through prevention of bloat and lower worm burdens. 1,10–15 Although tannin structures are chemically very diverse, they have one unifying property: tannins bind proteins. This characteristic is exploited in the production of leather, where tannins crosslink hide proteins. The main benefit of tannins in ruminant nutrition stems from their effect on protein digestion. Some, but not all, tannins can reduce the amount of protein that is digested in the rumen and enhance the amount of protein that is available for digestion in the small intestine. 16 This shift in ∗ Correspondence to: Irene Mueller-Harvey, NSRU Chemistry & Biochemistry Laboratory, Agriculture Department, Reading University, PO Box 236, Reading RG6 6AT, UK E-mail: [email protected]† This review is based in part on a keynote lecture presented at the XXII International Conference on Polyphenols, Helsinki, Finland, 25–28 August 2004 (Received 23 June 2005; revised version received 15 April 2006; accepted 25 May 2006) Published online 7 August 2006; DOI: 10.1002/jsfa.2577 2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00
Transcript
Journal of the Science of Food and Agriculture J Sci Food Agric 86:2010–2037 (2006)
ReviewUnravelling the conundrum of tannins inanimal nutrition and health†
Irene Mueller-Harvey∗NSRU Chemistry & Biochemistry Laboratory, Agriculture Department, Reading University, PO Box 236, Reading RG6 6AT, UK
Abstract: This paper examines the nutritional and veterinary effects of tannins on ruminants and makes somecomparisons with non-ruminants. Tannin chemistry per se is not covered and readers are referred to severalexcellent reviews instead: (a) Okuda T et al. Heterocycles 30:1195–1218 (1990); (b) Ferreira D and Slade D. NatProd Rep 19:517–541 (2002); (c) Yoshida T et al. In Studies in Natural Product Chemistry. Elsevier Science,Amsterdam, pp. 395–453 (2000); (d) Khanbabaee K and van Ree T. Nat Prod Rep 18:641–649 (2001); (e) Okudaet al. Phytochemistry 55:513–529 (2000). The effects of tannins on rumen micro-organisms are also not reviewed,as these have been addressed by others: (a) McSweeney CS et al. Anim Feed Sci Technol 91:83–93 (2001); (b) SmithAH and Mackie RI. Appl Environ Microbiol 70:1104–1115 (2004). This paper deals first with the nutritional effectsof tannins in animal feeds, their qualitative and quantitative diversity, and the implications of tannin–proteincomplexation. It then summarises the known physiological and harmful effects and discusses the equivocalevidence of the bioavailability of tannins. Issues concerning tannin metabolism and systemic effects are alsoconsidered. Opportunities are presented on how to treat feeds with high tannin contents, and some lesser-knownbut successful feeding strategies are highlighted. Recent research has explored the use of tannins for preventinganimal deaths from bloat, for reducing intestinal parasites and for lowering gaseous ammonia and methaneemissions. Finally, several tannin assays and a hypothesis are discussed that merit further investigation in orderto assess their suitability for predicting animal responses. The aim is to provoke discussion and spur readers intonew approaches. An attempt is made to synthesise the emerging information for relating tannin structures withtheir activities. Although many plants with high levels of tannins produce negative effects and require treatments,others are very useful animal feeds. Our ability to predict whether tannin-containing feeds confer positive ornegative effects will depend on interdisciplinary research between animal nutritionists and plant chemists. Theelucidation of tannin structure–activity relationships presents exciting opportunities for future feeding strategiesthat will benefit ruminants and the environment within the contexts of extensive, semi-intensive and some intensiveagricultural systems. 2006 Society of Chemical Industry
NUTRITIONAL EFFECTS OF TANNINSLowry et al.1 have pointed out the surprising longevityof some commonly held perceptions – even if erro-neous – of the harmful effects of tannins. They out-lined how the first reviews in the 1960s and 1970s,which covered the effects of plant phenolics on mam-mals, had treated phenolics as toxic xenobiotics ingeneral, despite reports demonstrating that manyphenolics had very low toxicities. Nevertheless, gener-alisations persist that tannins are harmful or toxic toanimals, which is at odds with the fact that humanshave enjoyed tannins for thousands of years in drinksand foods.2–4
Beneficial nutritional effectsTannins occur in many feeds such as fodderlegumes, browse leaves and fruits.5–9 Some of these
feeds (species of Acacia, Dichrostachys, Dorycnium,Hedysarum, Leucaena, Lotus, Onobrychis, Populus,Rumex and Salix) can produce useful benefits inruminants, such as better utilisation of dietary protein,faster growth rates of liveweight or wool, higher milkyields, increased fertility, and improved animal welfareand health through prevention of bloat and lowerworm burdens.1,10–15 Although tannin structuresare chemically very diverse, they have one unifyingproperty: tannins bind proteins. This characteristic isexploited in the production of leather, where tanninscrosslink hide proteins. The main benefit of tannins inruminant nutrition stems from their effect on proteindigestion. Some, but not all, tannins can reduce theamount of protein that is digested in the rumenand enhance the amount of protein that is availablefor digestion in the small intestine.16 This shift in
∗ Correspondence to: Irene Mueller-Harvey, NSRU Chemistry & Biochemistry Laboratory, Agriculture Department, Reading University, PO Box 236, ReadingRG6 6AT, UKE-mail: [email protected]†This review is based in part on a keynote lecture presented at the XXII International Conference on Polyphenols, Helsinki, Finland, 25–28 August 2004(Received 23 June 2005; revised version received 15 April 2006; accepted 25 May 2006)Published online 7 August 2006; DOI: 10.1002/jsfa.2577
2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00
Tannins in animal nutrition and health
Kidney
Liver
Rumen
Small intestine
Tannins + proteinsin feeds
Colon
Lower urinary N Higher faecal N
Result: better absorption of amino acids
higher N-flowlower N flow
Figure 1. Tannins that bind to dietary protein increase the nitrogenflux from the rumen to the small intestine. This process has beenreferred to as ‘ruminal escape protein’.
the site of protein digestion has been referred to as‘ruminal escape protein’ (Fig. 1).17 Ruminants giventhe tanniniferous feeds listed above tend to excrete lessurinary N and only slightly more faecal N. As a result,they absorb more of the dietary amino acids from thesetannin-containing feeds than from iso-nitrogenous,non-tannin-containing feeds.
However, the precise mechanism by which tanninsreduce ruminal protein digestion and improve itsutilisation is not fully understood. In vitro experimentsby Jones and Mangan18 led to the hypothesis thattannin–protein complexes are formed at the pHprevailing in the rumen (pH 6–7) and that post-ruminal pH shifts in the abomasum (pH < 3.5)and the small intestine (pH > 7) release proteinfrom these complexes, thus making it availablefor gastric or pancreatic digestion. This mechanismoperates in Leucaena leucocephala (Lam) de Wit,where tannins release protein post-ruminally.19 Incontrast, the tannins in Lotus corniculatus L., whichalso yields ruminal escape protein, are apparently notdissociated post-ruminally. They result, nevertheless,in useful amino acid absorption over the entirelength of the small intestine.12 It is unclear whathappens to the tannins when they are released post-ruminally and whether they bind to endogenousanimal proteins instead.10 For example, quebracho(Schinopsis lorentzii (Gris.) Engl.) tannins, which donot generate ruminal escape protein, reduced proteinabsorption dramatically in the small intestine.20 Thismay have been caused by quebracho tannins bindingto endogenous rather than feed protein.
It would be useful to investigate the origin ofthe increased faecal N levels when tanniniferousplants are fed. Faecal N could originate from tanninscomplexing with proteins from the diet, bacterialcell walls or secretions, animal tissues or saliva(see also ‘Nutritional implications of tannin–proteincomplexes’). Dietary protein would represent a greaterloss to ruminants than salivary protein, for example,because dietary protein tends to contain higher levelsof essential amino acids.
The extent to which protein digestion is affecteddepends on the source of the tannins. For example,Lotus pedunculatus Cav. tannins reduced the in vitrodegradation of the major protein in green plants(Rubisco) much more than L. corniculatus tannins.21,22
It is likely that the binding strength in tannin–proteincomplexes is an important factor in ruminal proteindigestion.23–25 However, the effect of tannins onbacteria, both in the rumen and in the intestine, maybe another factor.26,27 In the presence of L. corniculatustannins, lower concentrations of bacteria were foundin the rumen and in the digesta flowing out of therumen.28 Furthermore, the L. corniculatus tanninshad a direct effect specifically on the proteolyticbacteria in the rumen.29 However, pre-incubation ofL. corniculatus tannins with rumen bacteria resultedin more Rubisco degradation than pre-incubationwith L. pedunculatus tannins.30 It is possible thatL. pedunculatus tannins are not useful in ruminantnutrition because they inhibit the ruminal degradationof dietary protein too strongly.
Detrimental nutritional effectsAlthough it is timely to concentrate on the usefuleffects that can be derived from tannin-containingfeeds, it must be emphasised that some tanninscan also have harmful nutritional effects (N.B.‘Harmful and toxic effects’ deals with the toxic effectsseparately). In fact, tannins were often describedin the past as antinutritional factors because theycan impact negatively on animal production.1,31–33
Typical responses by ruminants and non-ruminantsare lower feed intake, protein and dry matterdigestibilities, liveweight gains, milk yield and woolgrowth.32,34,35
Tannins can render feed constituents less digestibleby binding to them. Protein digestibility tends tobe reduced most, but carbohydrate, starch and cellwall digestibilities can also be affected.34,36 Typicalexamples of tannin-containing feeds with low proteindigestibilities are Acacia aneura F. Muell., Acaciaangustissima (P. Mill.) Kuntze, Acacia cyanophyllaLindl., Acacia karoo Hayne Arzneyk, carob pulp(Ceratonia siliqua L.), fireweed flowers (Epilobiumangustifolium L.), L. pedunculatus, Robinia pseudoacaciaL. and Sesbania sesban L. Merr. (see also Table 1 in‘Appendix’).1,2,12,34,35,37–41 Some tannin-containingfeeds can result in rather high faecal N excretions,e.g. A. karoo and Leucaena pallida Britton & Rose.19,41
The origin of this faecal N has not yet been establishedand may stem from either dietary or endogenousprotein sources. Inhibition of digestive enzymes orintestinal micro-organisms could be another cause oflower protein and dry matter digestibilities.37,42
The effects of tannins on lipid digestion werecontradictory and may be due to altered ratios oflipolysis to lipogenesis.43 For example, carcass fatnessin growing lambs (Ovis aries L.) was reduced with alow-tannin Lotus diet (20 g condensed tannins (CTs)kg−1),44 while lipid digestibility in adult cockerels
(Gallus gallus L.) was enhanced by bean hulls(160–210 g CTs kg−1) but only in high-protein diets.45
Rats (Rattus rattus L.) consuming black locust (R.pseudoacacia) tannins had lower intestinal trypsin andα-amylase activities but higher lipase activities.37 Incontrast, chickens developed fatty livers after beingfed sorghum (Sorghum bicolor (L.) Moench) grains.46
Therefore some of the above observations are perhapsexplained by the effects of tannins on digestiveenzymes, but effects on growth hormone levels havealso been recorded.47
Structural diversity of tannins in feedsThe study of the nutritional effects of tannins iscomplicated by their great structural diversity, whichhas unfortunately been ignored or not sufficientlyappreciated in many feeding trials and has ledto considerable confusion in the literature.1,43,48
Attempts to attribute nutritional effects to generictannin classes, e.g. condensed versus hydrolysabletannins (CTs vs HTs), have not proved useful(Table 1), but the perception is still widespread thatHTs are toxic in comparison with CTs.12,49 Theprocyanidins and prodelphinidins (i.e. CTs; Fig. 2)in some species of Lotus, Onobrychis and Calliandraand the ellagitannins (i.e. HTs; Fig. 2) in chestnutwood extract (Castanea sativa Mill.) are beneficial,as demonstrated by higher liveweight gains or milkyields.12,48,50,51 However, the profisetinidins in S.lorentzii (i.e. CTs; Fig. 8(a)), the mixtures of CTsand phenolics in sorghum and the mixtures of CTsand HTs (gallo- plus ellagitannins) in some speciesof Acacia, Quercus and Terminalia can be harmful oreven toxic to ruminants and result in liver and kidneylesions or death.1,43,52
There is some evidence that slight changes intannin structures can also produce measurable effectson intakes. For example, snowshoe hares (Lepusamericanus Macfarlani) preferred bitterbrush (Purshiatridentata (Pursh) D.C.) or oatmeal treated withbitterbrush tannins (30 g kg−1) rather than blackbrush(Coleogyne ramosissima Torr.) or oatmeal treated withblackbrush tannins.53 Bitterbrush tannins had highercatechin/epicatechin ratios (55:45) than blackbrushtannins (20:80). However, no other information wasprovided on the nutritional composition of these twofeeds, such as total fibre, lignin or protein contents,which can also affect intakes. In another examplea commercially available tannic acid preparationconsisting mostly of tri- and tetragalloylglucoses didnot decrease protein digestibility in deer (Cervuselaphus L.) or sheep, and no gallic acid was detectedin the faeces.40 However, fireweed flowers containingmostly octa- and nonagalloylglucoses reduced proteindigestibility, and 27% of the fireweed tanninswere found as gallic acid in the faeces.40 Werecently investigated two provenances of Calliandracalothyrsus Meissn. Patulul was nutritionally superiorto San Ramon; its tannins also had a higherprocyanidin/prodelphinidin (PC/PD) ratio (85:15)
than San Ramon (30:70 to 37:63)48,51 and a lowerdegree of galloylation than San Ramon tannins(unpublished data). Similarly, L. corniculatus hasconsistently produced better in vivo protein digestionthan L. pedunculatus.12 Lotus corniculatus tannins yieldhigher PC/PD ratios (67:33 to 79:21)9 than L.pedunculatus (18:81 to 23:77).54 However, beforewe conclude that PC/PD ratios are therefore likelyto affect the nutritive value of tannin-containingfeeds, let us consider sainfoin (Onobrychis viciifoliaScop.), which has a very high nutritive value.55–57
The PC/PD ratios in sainfoin tannins58 range from7:93 to 38:62 and are comparable to the PC/PDratios of L. pedunculatus, which has a poor nutritivevalue. This suggests either that the differencesin the tannin structures from sainfoin and L.pedunculatus have not yet been identified or thatother components in these plants modulate the tannineffects (see also ‘Colorimetric assays’ and ‘Isothermaltitration calorimetry (ITC) and tannin solubilities(Kow values)’).
What are optimum tannin concentrations infeeds?Several authors have advised that dietary concentra-tions of <50 g CTs kg−1 are beneficial.11,17,59,60 Theserecommendations originated mainly from feeding tri-als with Lotus species and may not be applicable toother feeds. For example, sulla (Hedysarum coronar-ium L.) with 72 g CTs kg−1 and sainfoin (O. viciifo-lia) with 80 g CTs kg−1 had high nutritive values forsheep,61,62 but carob pulp with only 25 g CTs kg−1
depressed growth rates.39 The relatively low concen-trations (<30 g CTs kg−1) in L. leucocephala and S.sesban had no beneficial effects on N balances inBrazilian sheep, because Brachiaria decumbens Stapfhay apparently supplied insufficient energy.63 Dietsneed to balance the energy and protein requirementsof ruminants before tannins can exert beneficial effectsthrough ruminal escape protein.10 However, the trop-ical forage Lespedeza cuneata (Dum. Cours.) G. Donwith 5–12 g CTs kg−1 had higher nutritive values thansimilar forages without CTs.64 Many browses in thetropics and other zones can contain very high levels oftannins or total phenolics (100–500 g kg−1),1,5,6,65 andAcacia nilotica L. pods are a particularly rich source offlavanol gallate tannins (contents up to 500 g kg−1 havebeen measured).66 In some parts of the world, rumi-nants may spend most of their time on such tannin-richbrowses,67 which can be useful feeds especially duringthe dry season.1 Even tannin-rich browses can yieldpositive N balances and growth rates, which on occa-sion are comparable to those achieved with legumehay or commercial protein meals.15,38,51,68–74
When ruminants such as sheep consume the tannin-containing L. corniculatus, amino acids are absorbedalong the entire length of the small intestine.16 How-ever, if these tannins are neutralised by polyethyleneglycol (PEG), the majority of amino acids are absorbedin the proximal third. Lotus corniculatus tends to have
Figure 2. Examples of tannin structures: condensed tannins from fodder legumes (procyanidins, prodelphinidins); hydrolysable tannins(ellagitannins) from chestnut wood (castalagin, vescalagin, castalin, vescalin); taratannins; procyanidin A2 from peanut skins; procyanidins B2, B3and B5; casuarictin; geraniin; a gallotannin from tannic acid, (pentagalloylglucose). Continued on next page.
lower tannin levels than L. pedunculatus; but these con-centration differences are not sufficient to explain theirdifferent nutritional effects. Lotus pedunculatus tanninsare ‘more potent’ even if diluted below the levels of
L. corniculatus tannins.75 Recently, fundamental differ-ences between these Lotus tannins were reported76 thatmay account for their contrasting nutritional effects.Lotus pedunculatus had a tannin fraction of very high
molecular weight (ca 12 300 Da), but L. corniculatustannins were less than 5300 Da.
Limited evidence indicates that some tannins maydisappear in the digestive tract and could even functionas nutrients,70,77,78 because studies by Lowry andKennedy79 suggest that some phenolics can act asmammalian nutrients: quercetin and rutin, but notcatechin, were degraded into acetate by a mixedrumen inoculum within the time span of rumenpassage.
Effects of added tannins or tannin-richsupplementsAttempts have been made to determine the nutritionaleffects of tannins by adding them to animal diets.Tannins such as quebracho (2S profisetinidins; seeFig. 8(a) below) or tannic acid (Fig. 2) are extractedfrom wood and are commercially available but arenot necessarily representative of the tannins thatoccur in most animal feeds. Generally speaking,these particular tannins have not produced nutritionalbenefits.20,40,80 However, a small number of feedadditives such as taratannins (Caesalpinia spinosa(Mol.) Kuntze), chestnut tannins, tannin-rich peanut(Arachis hypogea L.) skins and tamarind (Tamarindusindica L.) seed husks have yielded positive nutritionaleffects (Table 1). Treatment of soybean (Glycine max(L.) Merr.) meal with 100 g taratannins kg−1 (Fig. 2)resulted in reduced proteolysis in the rumen andimproved daily weight gains, feed efficiencies and Nbalances in lambs.81 Low doses of chestnut tanninssignificantly reduced ammonia in an in vitro Rusitecfermentation system;50 they also enhanced (by 5%)non-ammonia N flow to the duodenum in growingbulls (Bos taurus L.) and resulted in higher apparentintestinal protein digestion.82 However, 21 g chestnuttannins kg−1 added to soybean meal produced nosignificant effect on voluntary intake, feed conversion,daily gain or length of fattening period when lambswere finished from 15 to 25 kg.83 Although there
were no signs of histopathological toxicity, raised γ -glutamyl-transferase and aspartate amino-transferaseactivities will require further investigation. In contrast,a higher level of chestnut tannins (80 g kg−1) sprayedonto a hay diet significantly decreased dry matter(DM) digestibility in sheep (from 63.3 to 57.7%)and goats (Capra hircus L.) (from 63.7 to 60.7%),but animal liveweight and body condition were notaffected over a 10 day collection period.84
Lactating dairy cows fed diets containing 80–160 gpeanut skins kg−1 (180 g CTs kg−1; Fig. 2) showedhigher DM intakes, milk yield and milk fat contentand had lower ruminal NH3 levels and milk proteincontent. It was concluded that diets containing160 g peanut skins kg−1 were optimal.85 Lactatingdairy cows also benefited from diets containing 75 gtamarind seed husks kg−1 (140 g CTs kg−1). Huskadditions significantly increased liveweight gains (by46%). They also increased faecal N and decreasedurinary N, but the increase in N balance was notsignificant.86
Nutritional implications of tannin–proteincomplexesSome of these seemingly discrepant results involvingadded tannins may well be due to the fact that thebinding strengths in different tannin–protein com-plexes can vary over several orders of magnitude87–90
(see also ‘Isothermal titration calorimetry (ITC) andtannin solubilities (Kow values)’). An interesting com-parison showed that tannins from three Leucaenaaccessions, i.e. L. leucocephala, Leucaena trichandra(Zucch.) Urban and L. pallida, differed markedly intheir affinity for protein and that this was relatedto their effects on protein digestion and N retentionin ruminants.24 Different binding strengths may alsoaccount for the observation that apparently lower CTlevels in A. angustissima seemed to interfere morewith protein use than the higher CT levels in L.leucocephala.38
The tannins in Rumex obtusifolius L. seem tobe particularly ‘active’ – which presumably meansstrongly protein-binding – because a concentrationas low as 2 g tannins kg−1 diet was sufficient toprecipitate 50% of the soluble dietary protein andto prevent bloat91 (see also ‘Bloat’). In contrast,the interaction between quebracho tannins and feedprotein was so weak that the protein flux to theabomasum was not increased,20 which means that noruminal escape protein was generated by these tannins(Fig. 1). Current evidence therefore suggests thattannin–protein interactions are optimal only in somefeeds, e.g. Dichrostachys cinerea L., L. leucocephala, L.corniculatus and sainfoin.12,15,24
Salivary tannin-binding proteinsTannin-binding salivary proteins comprise proline-rich proteins (PRPs) and histatins (Hsts), whichhave a strong affinity for tannins.92,93 These proteinsare probably the first line of defence by someanimal species against dietary tannins; moreover,these proteins tend to precipitate only those tanninsthat usually occur in their diets, which suggests anevolutionary link between the specificity of thesesalivary proteins and the dietary tannin structures.94
For example, salivary proteins from moose (Alcesalces L.) and beaver (Castor canadensis Kuhl) onlybound linear CTs, which are common in their diets(Salix spp., Betula spp., Populus tremula L.); muledeer (Odocoileus hemionus Rafinesque) salivary proteinsbound linear and branched CTs and gallotannins,which is consistent with their more generalised diets;and salivary proteins from the omnivorous black bear(Ursus americanus Pallas) bound linear and branchedCTs and also gallo- and ellagitannins.94 When theauthors added, to beaver, deer and sheep diets,only those tannins which were not bound by theirsalivary proteins, they measured significant reductionsin protein digestibilities.
PRPs and Hsts also appear to mediate the effectsof tannins on carbohydrate digestion by sparing theenzymes involved in their degradation. PRPs and Hstsprotected α-amylase from inhibition by tannic acid.95
However, the effects of tannins on digestive enzymesare not uniform or predictable.96 In an in vitro enzymemixture containing pepsin, trypsin, chymotrypsin andpeptidase, tannic acid reduced the hydrolysis of bovineserum albumin (BSA) but increased the hydrolysis ofcasein, pea (Pisum sativum L.) meal, soybean meal andhaemoglobin.96
If tannins are not administered orally, tissue necrosiscan result. For example, barium tannic acid enemasproduced fatal liver damage in humans.97 Liver andkidney necrosis also occurred after intra-abomasaldosing of sheep and appeared to be caused by freetannic acid in the plasma.98,99 However, if givenorally to sheep, tannic acid led to several urinarymetabolites (gallic acid, pyrogallol, 4-O-methyl gallicacid) and no detectable levels of tannic acid in therumen, plasma or urine (N.B. ruminal pyrogallol was
significantly correlated with blood methaemoglobinconcentrations98). It is likely that rumen micro-organisms protected sheep from any negative effectsof tannic acid, because sheep do not produce salivaryproteins.
PRPs are either constitutive or inducible by tannins,depending on the animal species, and occur forexample in the saliva of humans, pigs (Sus scrofaL.), rats, rabbits (Oryctolagus cuniculus L.), goats,deer and bears (Ursus spp.),77,100 but the saliva ofhamsters (Mesocricetus auratus Waterhouse), sheep orcows contains few or no PRPs. As a result, ratsare more resistant to tannins than hamsters, andmule deer and goats are more resistant than sheepand cows.77,101 It therefore follows that much lessL. corniculatus tannin could be extracted from plantmaterial that had been chewed by deer (27%) than bysheep (90%),11 which suggests that salivary proteinsfrom deer bound very strongly to these dietary tannins.Robbins et al.77 hypothesised that the presence ofPRPs maximised the amount of quebracho tannin thatwas bound per unit of protein and thus minimised Nlosses.
A little-known practice that involves the successfuluse of a tannin-rich feed comes from the Mediter-ranean region. Iberian pigs (Sus scrofa meridionalis DeBeaux & Festa) relish acorns and have traditionallybeen fed extensively during the fattening phase onacorns from several oak species,102 despite the factthat acorns have high levels of galloyl glucoses (see‘Harmful and toxic effects’).103 The resulting meat isevenly infused with fat, and products fetch very highprices. It is possible that pigs will be protected bytheir PRPs against these tannins (however, ‘Browsemixtures’ explores an alternative explanation). It maytherefore be worthwhile to investigate the potential forother tannin-rich feeds in pig nutrition.
However, not all reports concur with the abovehypothesis that salivary proteins are responsible forresistance to tannins. For example, there were nosignificant differences in protein and carbohydratedigestion between sheep and goats that had been fedtannin-containing Desmodium intortum (Mill.) Urb. orC. calothyrsus.104
Another interesting experiment compared theinteractions of CTs from D. intortum and L.pedunculatus and tannic acid with BSA, plant leafproteins and salivary mucoproteins from sheepand goats.105 Insoluble tannin–protein complexesresulted with BSA and leaf proteins, but solublecomplexes with the salivary proteins. This study raisedseveral questions: what effect do other compoundsin the digestion matrix have on tannin–proteincomplexation; is protein digestibility affected bythe solubility of the tannin–protein complexes; aretannins in the soluble complexes more readilywashed out of the rumen than those in insolublecomplexes and how does this affect their post-ruminaldigestion?
PHYSIOLOGICAL EFFECTS OF TANNINSHarmful and toxic effectsThe harmful effects of tannins in ruminant andmonogastric animals can range from producingchronic or systemic effects (subacute intoxication), e.g.A. angustissima and Quercus coccifera L. leaves,78,106,107
to causing occasional deaths, e.g. A. nilotica fruits,Terminalia oblongata F. Muell., Clidemia hirta (L.) D.Don, Quercus spp. leaves and green acorns.1,52,108–111
Many monogastric animal species appear to be moresensitive to tannins than ruminants, but caution isneeded when making such generalisations, becausethe presence or absence of salivary tannin-bindingproteins cuts across ruminants and non-ruminants(see ‘Nutritional implications of tannin–proteincomplexes’).1,34,106 For example, the Iberian pigs inMediterranean countries ingest per day up to 20 gof gallic and ellagic acid esters and preferentially eatQuercus rotundifolia Lam. acorns, which have highertannin concentrations than Quercus suber L. acorns,without suffering any organ damage.102,103
Responses to tannic acid were generally negativeand resulted in gastric ulceration and hypersecretionof mucosa.112 It is, however, worth bearing in mindthat feeding trials have been carried out with only alimited number of animal species or diets and mayalso have been too short-term for adaptation to takeplace.1,34,45,113 Furthermore, in some experiments theeffects of tannins could have been confounded with theeffects of other compounds; for example, high-tanninsorghums contain many co-occurring phenolics, suchas the relatively rare 3-deoxyanthocyanidins andflavan-4-ols,114 and faba beans (Vicia faba L.) containlectins and trypsin inhibitors. This might explainwhy lactating pigs fed sorghum/soybean meal had apoor N balance,115 whereas Iberian pigs thrive onacorns.102,103
Bioavailability of tanninsThe evidence for absorption of CTs is still equivocaland is based on findings of higher plasma antioxidantcapacity in rabbits which had been fed grapeseed (Vitisvinifera L.) tannins116 and on higher radioactivity inproline-rich tissues of rats given 14C-labelled CTs.117
Substantial disappearances of 14C-labelled CTs fromD. intortum were observed in the gastrointestinaltract of sheep and goats.118 However, it is not clearwhat contribution any monomeric phenolics or low-molecular-weight (low-MW) tannins could have madeto these disappearances, or indeed whether the 14Cdisappearances could have been due to bound galloylor glycosyl groups that could easily have been cleavedby intestinal micro-organisms. Bound galloyl andglycosyl groups have been detected in several tanninsby matrix assisted laser desorption time-of-flight massspectrometry (MALDI-TOF-MS).119,120
Whilst it is accepted that monomers such as cate-chin, epicatechin, epigallocatechin, etc. are absorbed,the direct bioavailability of oligomeric tannins is stillin question.3,121,122 The dietary procyanidin dimer B3
(Fig. 2) and grapeseed oligomers were neither cleavedinto monomers nor absorbed in rats.123 However,small quantities (<1%) of two purified procyanidindimers, B2 and B5 (Fig. 2), were transferred to theserosal side of an isolated small intestine.124 It is alsoworth noting that dimeric to hexameric procyanidinsfrom cocoa were cleaved at pH 2 in sodium phosphatebuffer solution and also in simulated gastric juice (pH2) over 3.5 h at 37 ◦C and that the higher oligomersdegraded much faster than the lower oligomers.121
These results may help to dispel the view that only‘hydrolysable’ tannins are readily degraded. It remainsto be seen whether these reactions are nutritionallyimportant, as gastric pH values are rarely that lowafter a meal.
After intragastric administration of oligomericCTs from apple (Malus domestica Borkh.), dimersto tetramers could be detected within 30 min inrat plasma by liquid chromatography/tandem massspectrometry (LC/MS/MS).125 It is likely that PRPswould have bound to these tannins had they been fedorally, but not when given intragastrically, and thiscould have affected their bioavailability. As mentionedabove (‘Nutritional implications of tannin–proteincomplexes’), the fatal liver damage that resultedfrom barium tannic acid enemas97 could have beencaused by direct absorption from the colon of eithertannic acid or its metabolites, as they would nothave been bound by PRPs or Hsts. To date, thereis no evidence for the direct absorption of higher-MWcondensed tannins across the intestinal membrane ineither ruminants or monogastrics, but evidence forthe absorption of punicalagin, an HT, is contradictory(see ‘Metabolism of tannins and phenolics’). It is worthremembering though that only a few studies have sofar investigated the bioavailability of a limited range oftannins.
It is interesting that all quebracho tannins could berecovered from the faeces of deer and bears (probablythese tannins were complexed by salivary proteins; see‘Salivary tannin-binding proteins’) but only 75% fromsheep faeces.77 The authors presumed that the quebra-cho tannins had been metabolised in sheep, possiblyby the rumen micro-organisms. Indeed, there is con-siderable evidence that points to tannin degradation byintestinal micro-organisms, and the resulting metabo-lites might be more bioavailable than the originaltannin compounds.126–128 Tannin degradation doesnot only depend on the presence of micro-organisms.Some tannins are also degraded at physiologically rel-evant pH values: the procyanidins (CTs) from cocoa(Theobroma cacao L.) and casuarictin, an ellagitannin(ET) (Fig. 2), from strawberries (Fragaria ananassaDuch.) degraded at pH 2 and pH 7–8 respectivelyinto their monomeric constituents.121,129
Metabolism of tannins and phenolicsSeveral studies have shown that tannins can disappearin the intestinal tract of ruminants and non-ruminants:
(1) PerezMaldonado and Norton118 detected substan-tial losses of 14C-labelled CTs from D. intortum in thegastrointestinal tract of sheep and goats; (2) Deprezet al.127 found that 14C-labelled polymeric procyani-dins which had been isolated from the leaves of Salixcaprea L. and were free of catechin monomers orproanthocyanidin dimers and trimers were catabolisedby human intestinal micro-organisms into phenolicacids; and (3) Abia and Fry130 concluded that 14C-labelled carob CTs were not inert in the gut ofrats – approximately 18% of the CTs had under-gone degradation such as depolymerisation and/ormetabolism. In contrast, Nakamura and Tonogai122
concluded that the oligomers of grape seed extractwere not absorbed or metabolised by rats.
It is well known that micro-organisms can degradegallic acid and tannic acid into 4-O-methyl gal-lic acid, pyrogallol, phloroglucinol and resorci-nol (Fig. 3)1,98,131 and that catechin and epicat-echin are methylated or conjugated to sulfateesters or glucuronides.132,133 Ring fission of cate-chins produces valerolactones and phenylpropionicacids.134 Similar degradation products have beenreported from Pycnogenol, which is a commer-cial procyanidin-rich medicinal pine (Pinus mar-itima Mill.) bark extract. This product yieldedtwo urinary metabolites after oral administrationto humans: glucuronic acid or sulfate conjugatesof δ-(3,4-dihydroxyphenyl)-γ -valerolactone and δ-(3-methoxy-4-hydroxyphenyl)-γ -valerolactone.135 Simi-larly, 3H-labelled epigallocatechin gallate fed to ratsresulted in 5-(5′-hydroxyphenyl)-γ -valerolactone 3′-O-β-glucuronide as the main urinary metabolite and5-(3′, 5′-hydroxyphenyl)-γ -valerolactone as a faecalmetabolite (Fig. 4).136 Ellagitannins from strawber-ries, raspberries and walnuts were also apparentlydegraded by human intestinal micro-organisms andexcreted as urolithin B-glucuronide (Fig. 5).137
Figure 4. Urinary and faecal metabolites of epigallocatechingallate.136
Sheep fed tannic acid had glucuronides of resorcinoland 2-carboxy-2′,4′,4,6-tetrahydroxy diphenyl 2,2′-lactone as urinary metabolites; leaves of T. oblongata(containing ETs) also produced these glucuronidesplus punicalagin138 (Fig. 5), which was considered tobe a toxic ET degradation product.139 However, theacute hepatotoxicity and nephrotoxicity in sheep thatwere ascribed to punicalagin were surprising, because(1) it had been thought that this large compound(1083 Da) would not be absorbed from the gut,(2) relatively large dose rates were required to producetoxic effects in mice (1 mg g−1 liveweight)140 and(3) a diet containing 60 g punicalagin kg−1 was alsonot toxic when fed to rats for 37 days, despite thefact that punicalagin and related metabolites such asgallagic acid (Fig. 5) were found in plasma, liver andkidney.141 Moreoever, Cerda et al.141 pointed out thatpomegranate (Punica granatum L.) juice contains ≥2 gpunicalagin L−1 and is consumed by humans.
HO
OH
OH
pyrogallol
OH
OH
resorcinol
OH
OHHO
phloroglucinol
HO
OH
OH
COOH
HO
OH
OH
OCH3O
4-O-methyl gallic acid
gallic acid
Figure 3. Microbial degradation products of gallic acid.
Systemic effectsThe toxic effects observed in chickens that hadbeen fed high-tannin sorghum grains were causedby an inhibition of the post-digestive metabolism.100
The authors concluded that absorption of low-MWpolyphenols, which are associated with the tanninsin sorghum grain, rather than tannins themselveswas responsible for the effects on body tissues andshould be viewed as a systemic effect. This caused legabnormalities in chickens, induction of liver enzymes,higher concentrations of urinary glucuronides, andanimal deaths that were too rapid to be caused byreduced nutrient digestibility.142 A recent review onthe health benefits of plant phenolics134 also suggestedthat the epidemiology of flavonols and catechinspoints to a systemic effect, which results from directabsorption of these compounds or their metabolites.
It has been hypothesised that animals can reducetheir intakes in order to allow time to detoxify suchcompounds.143 Iason and Murray144 conducted twointeresting experiments with compounds that occurnaturally in ericaceous plants. They injected orcinol(3,5-dihydroxytoluene; Fig. 6) intravenously at likelyphysiological concentrations into sheep and foundthat energy expenditure increased significantly by5%. When orcinol and quinol (p-hydroxybenzene)were infused together into the rumen at likely dietaryconcentrations, they observed an increase in urinaryenergy excretions and a reduction in digestible energyintake, which they attributed to chronic toxic effectsin the tissues. Herbivores browsing plants with highconcentrations of secondary plant compounds tend tohave well-developed detoxification mechanisms, e.g.a large induction response of liver enzymes.145 Foleyet al.78 suggested the use of detoxification indices suchas glucuronic acid or hippuric acid excretions in orderto assess the costs of detoxifying different types ofphenolic-rich feeds. Although these urinary excretionindices would need to be calibrated against dietaryintakes of known plant phenolics and their excretionrates, it would be worth pursuing such a line of enquiry,especially for unconventional feed resources.
Further studies will be needed to elucidate theeffects of tannins and their associated low-MWphenolics in a range of feeds, as there are many more
CH3
HO OH
3,5-Dihydroxytoluene (syn. orcinol)
HO OH
p-Hydroxybenzene (syn. quinol)
Figure 6. Phenolic compounds from ericaceous plants.144
compounds than have been investigated so far and thevarious consumers possess different digestive systems.It is worth bearing in mind also that not all phenolicsproduced systemic effects, as some phenolics can actas nutrients.1
TREATMENTS AND FEEDING METHODS TOOVERCOME NEGATIVE EFFECTS OF TANNINSAddition of supplementsThe liveweight losses associated with feeding livestockCT-rich mulga (A. aneura) during drought periodsin Australia have traditionally been overcome withmineral supplements containing N, P and S.146
These may assist in the detoxification processand also provide additional nutrients to rumenmicro-organisms, as mulga tannins reduced proteindigestibility.1
Additions of methionine and choline to chick dietsalleviated the toxicity of tannic acid and also thetoxicity of CTs or possibly the low-MW phenolicsin sorghum (see ‘Systemic effects’),100 presumablybecause they functioned as methyl donors in thedetoxification process and facilitated the excretionof compounds such as 4-O-methyl gallic acid.46
However, methionine additions did not overcome thetoxic effects of faba bean tannins in chick diets.147
These discrepancies between sorghum and faba beandiets have not yet been explored.
Addition of tannin-binding polymersSeveral polymers such as PEG and polyvinylpoly-pyrrolidone (PVPP) (Fig. 7) bind strongly to tan-nins and can reduce their antinutritional or toxiceffects in vitro and in vivo in rats, rabbits andsheep.1,106,148,149 However, given the fact that sometannins have positive nutritional effects, it is not sur-prising that there are instances where PEG effects werespecies-dependent and removed the beneficial tannineffects.15,62,107,150–152 Furthermore, whilst PEG waseffective against quebracho tannins, it failed to reversethe negative effects of tannic acid in an in vitro fer-mentation system (rate and extent of fermentation).153
PEG also failed to correct the negative N balances insheep fed C. calothyrsus leaves.150
It is therefore not justified to promote thewidespread use of a relatively expensive feed additive,
HOCH2[-CH2-O-CH2-]nCH2OH
Polyethylene glycol (PEG)
NO
CH CH2
n
H H
Polyvinylpolypyrrolidone (PVPP)
Figure 7. Two tannin-binding polymers.
as has happened during a drought in the 1990s insouthern Africa (personal observation), without priorfeeding trials.
Alkaline treatmentsAlkaline treatments such as Ca(OH)2, NaOH or woodash can be very effective in preventing the toxic orantinutritional effects of tannins and/or associatedphenolics in the leaves of Quercus stellata Wangenh.,Quercus incana Bartr. or C. hirta or in sorghumgrain.49,66,108,154,155 The fact that ammonia is just aseffective as these metal hydroxides demonstrated thathigh OH− concentrations, rather than chelating metalions, were responsible for improving the nutritivevalue.156 Interestingly, NaOH treatment was moreeffective with whole than with ground sorghumgrain,157 which could be due to the fact that most of thesorghum phenolics are located in the outer pericarp.The nutritive value of L. leucocephala, however, wasnot improved by urea or wood ash treatments,158
which is not surprising, as these tannins appear tobe particularly suited for generating ruminal escapeprotein.10 It may be concluded that alkaline treatmentsare probably most useful for overcoming the acutetoxic effects of certain – as yet unknown – tannin orphenolic compounds.
Browse mixturesLowry159 observed that farmers usually minimiseantinutritional problems by feeding leaf mixtures,which dilute or reduce toxic effects. Recent researchby just a few authors has indeed shown that mixturesproduced less deleterious effects than tanniniferousbrowses fed as sole feeds.41,160–163 This supports thehypothesis derived from ecological studies78 that thebest strategy for herbivores would be to mix diets inorder to minimise the energy costs of detoxification.The benefit that may be derived from such an approachto utilising tannin-rich feeds has hardly been exploredin animal nutrition.
SilagesSilages prepared from tannin-containing plants tendto have reduced soluble N contents, which improvestheir feeding value.164–166 Lotus corniculatus silagescompared favourably with alfalfa (Medicago sativa L.)and red clover (Trifolium pratense L.) silages for feedingdairy cows.167 Dry matter intakes were similar amongthese diets, but milk and protein yields were higher forthe Lotus silages. Lotus corniculatus silage also achieveda significantly higher N balance in lambs than redclover or alfalfa silage.168
Up to now, few studies have investigated themerits of conserving tannin-rich fodder for dry seasonfeeding in tropical countries. In one study, mixtures oftropical grasses and tree leaves from L. leucocephala orGliricidia sepium (Jacq.) Walp. were ensiled, resultingin high-quality silages which promoted weight gainin sheep.169 It is worth noting that a significantproportion of the ‘ruminal escape protein’ apparently
survived the silage fermentation process.169 Similarly,Titterton170 found that silages containing maize (Zeamays L.) stover mixed with tree leaves from speciesof Calliandra, Acacia, Gliricidia or Leucaena could beused to replace commercial feed supplements for dryseason feeding and, in a good year, into lactationwithout loss of milk yield from dairy cows. Ensilinggreen tea (Camellia sinensis (L.) O. Kuntze) wastewith whole-crop oat (Avena sativa L.) increased Nretention in goats, and the authors also concludedthat feed proteins survived the ensiling process andwere digested post-ruminally, as the added N did notincrease ruminal ammonia, urinary or faecal N.171,172
Contribution of sugars or energyAs mentioned in ‘Systemic effects’, the detoxificationof intravenously injected phenolics carried an energycost.144 It is therefore interesting that high levels ofsugars can alleviate the antinutritional effects of sometannins. The following tannin-containing feeds havehigh levels of soluble sugars and/or starch and alsoa high nutritive value for both ruminants and non-ruminants: fruits from Gleditsia triacanthos L. andPiliostigma thonningii (Schum.) Milne-Redh., acornsfrom Quercus spp., Prosopis cineraria (L.) Druce leavesand sainfoin.58,74,102,103,173–176
Although even low concentrations of carob tannins(25 g kg−1) produced growth-depressing effects, thehigh concentrations of sugars (470 g kg−1) in carobpulp may be responsible for the low but positivelamb growth rates of 48 g day−1.39 A good sourceof energy is likely to assist in the detoxification ofsome tannins, co-occurring lower-MW phenolics ortheir metabolites. This could also explain why sugar-rich cactus (Opuntia ficus-indica (L.) P. Mill.) fruits,cactus pads and molasses removed the toxic effects ofkermes oak (Q. coccifera) and increased the nutritivevalue of several browses.107,177–179 The addition ofmaize grain to browse leaves may have increasedthe weight gain of sheep either by balancing energyand protein requirements or by supplying energyfor the detoxification process.180 This hypothesis isalso supported by observations that European roedeer (Capreolus capreolus L.) selected diets containinghigh concentrations of both phenolics (including BSAprotein-binding tannins) and soluble sugars.181
ANIMAL HEALTH AND WELFAREParasitesAlthough farmers have traditionally used plants for de-worming animals,182,183 the evidence that animals useplants for self-medicating purposes is still equivocal.184
Much work remains to be done so that traditionalknowledge can aid the development of plant-based anthelmintic products that yield consistentresults. Samples from different geographical regionshave produced variable results,183 as the synthesisof secondary plant products can be affected byenvironmental growing conditions.185 Moringa oleifera
Lam K. represents an excellent example where thecombination of traditional knowledge and scientificstudy resulted in an improved product for humannutrition.186
Several tropical legumes have also shown somepromising results. Grazing of L. cuneata forage(50 g CTs kg−1) achieved remarkably high reductions(57–100%) in faecal egg counts (FECs), totalfaecal egg output and the numbers of parasiticnematodes (species of Haemonchus, Teladorsagia andTrichostrongylus) in goats.64 High levels of dried A.karoo leaves, which contain ca 240 g CTs kg−1,41 alsosignificantly reduced FECs and Haemonchus contortusRud. worm burdens in goats.187 In comparison, A.nilotica leaves had hardly any effect on FECs despitevery high concentrations (ca 400 g kg−1) of catechingallate tannins.5,6,187,188 However, weight gains onboth browse diets were comparable to a commercialgoat meal diet after 7 weeks, which illustrates just someof the complexities of this type of research.187 Feedingof Acacia polyacantha Willd. reduced the FECsof a mixed nematode population by approximately30% and the Oesophagostomum columbianum Curticeworm burden by 13% in goats.189 The authors alsonoted that quebracho extracts appeared to be muchmore anthelmintic than wattle (Acacia mearnsii DeWild) extracts in vivo. This could stem from eitherconcentration or slight structural differences betweenthe tannins in these commercial extracts (see ‘Methaneemissions from ruminants’; Fig. 8). Pregnant goatsgiven free access to tannin-rich browse in Ugandanrangelands over a 6 month period had significantlylower FECs and worm burdens and gained moreweight than goats given daily PEG drenches thatneutralised the tannins.190 Unfortunately, no detailswere given of the browse plants consumed. Mixtures ofbrowse diets might therefore be useful both to controlintestinal parasites and to reduce the antinutritionaleffects of high-tannin diets (see ‘Browse mixtures’).
Recent research demonstrated that quebrachotannins had a direct anthelmintic effect on theeconomically important nematode Trichostrongyluscolubriformis (Giles) Loos in sheep.191 Apparently,the direct inhibitory effect of CTs is dependent ontheir species of origin. The in vitro migration of T.colubriformis decreased in the following order whenexposed to CTs: Dorycnium pentaphyllum Scop. >
O. viciifolia > L. pedunculatus > Dorycnium rectum(L.) Ser. > R. obtusifolius > H. coronarium > L.corniculatus.192 This may explain why L. pedunculatusand H. coronarium were able to minimise the effects ofheavy parasite burdens on animal performance and/orreduce the numbers of parasites in sheep and deermore effectively than L. corniculatus.11,12
The diet of lambs had a significant impact on egghatching and on the development of T. colubriformislarvae both in the laboratory and in the field:193
larval recoveries from faeces were significantly lowerfrom lambs fed D. rectum and L. corniculatus ratherthan white clover (Trifolium repens L.), alfalfa or H.
coronarium. Parasitic nematodes go through severaldevelopmental stages in their life cycles, and an in vitromigration assay, whilst useful as a screening tool, maynot predict the more complex outcomes in vivo andamongst different animal species. Ramırez-Restrepoet al.194 evaluated whether L. corniculatus could beused in an organic sheep-farming system to controlintestinal parasites. Lotus corniculatus swards resultedin significantly lower FECs in ewes and lambs, togetherwith higher liveweight gains and wool production,than perennial ryegrass (Lolium perenne L.)/whiteclover pastures. They concluded that L. corniculatuseliminated the need for pre-lambing anthelmintictreatments. However, FECs increased in lambs afterweaning, and the authors therefore suggested furtherresearch on integrating other CT-containing foragesthat are highly active against parasites, e.g. sulla, intothe grazing system.
These are important findings, because the sheep andgoat industries are encountering serious nematoderesistance to synthetic anthelmintics, which alreadycauses considerable production losses in SouthAmerica, South Africa and Australia and is startingto cause problems in the USA.195,196 A recent reviewtherefore concluded that forages with CTs could beused to control gastrointestinal parasites and mightreduce the need for anthelmintic drenches.64
Two lesser-known examples of the use of tanninsto combat health problems have also been reported.Diet change can bring about diarrhoea, which wassuccessfully treated in pigs and cattle with a castalagin-based product from chestnut and in calves with a greentea extract.197,198 Green tea polyphenols stimulatedthe growth of beneficial species of Bifidobacteriumand Lactobacillus but suppressed harmful Clostridiumperfringens (Veillon & Zuber) Hauduroy bacteria.
BloatBloat is caused by the formation of a stable foam inthe rumen, which, if untreated, can result in animaldeaths. It tends to occur when animals are allowed tograze young lush forages such as clover or alfalfa.12,17
However, tannin-containing plants, e.g. Lotus spp.,sainfoin and several others, never cause bloat, and CTconcentrations as low as 1–5 g kg−1 are sufficient toprevent it.199 The precise mechanism by which tanninsprevent bloat is still unclear; both growth inhibition ofslime-producing ruminal bacteria and a destabilisingeffect on the proteinaceous frothy foam have beenproposed as possible mechanisms.12,17,200
Animal deaths from bloat were also reduced ona mixed pasture of alfalfa/sainfoin (9:1), whichamounted to 2–5 g CTs kg−1 in the diet.17 Similarly,a grass/alfalfa pasture with 10% dock (R. obtusifolius),or 2 g tannins kg−1 in the diet, also prevented bloat.201
It is therefore suggested that the contributions bytannin-containing ‘weeds’ to ruminant welfare andbloat prevention should be considered.
However, some tannin-containing fodder legumessuch as sainfoin are currently not widely used by
farmers, as their agronomic performance tends to beinferior to that of alfalfa or clover.202,203 Nevertheless,modern North American sainfoin varieties have showngood yields, and current research is aimed at improvingtheir agronomic performance in order to develop aneconomically viable forage legume that benefits animalhealth.17,55,204
The discovery that BAN genes encode a newenzyme, anthocyanidin reductase, has led to asignificant breakthrough in our understanding of CTsynthesis in plants.205 This enzyme converts cyanidinto (−)epicatechin, which is an important chainextension unit in many CTs. Transgenic expressionof BAN in tobacco resulted in CT accumulation inthe petals. CT biosynthesis occurred also in alfalfaforage after transformation with a maize anthocyaninregulatory gene.206 Therefore it should now bepossible to develop new CT-containing plants thatbenefit animal health and welfare by preventing bloatand reducing the worm burdens in ruminants.207
Although it is not yet known which particulartannin molecules or what molecular properties areresponsible for preventing bloat, reducing nematodemobility or lowering parasitic worm burdens, plantswith defined tannin compounds will be most usefulfor elucidating the structure–activity relationships.208
Molecular biology could play a useful role bycreating designer plants to probe these relationships.Such information will be of great use in futuretransformation studies of tannin-containing plantswith improved nutritional and veterinary properties.
ENVIRONMENTAL EFFECTSRuminants are important producers of greenhousegases such as ammonia and methane.209,210 Theproposed imposition of tariffs to mitigate greenhousegas emissions will affect the agricultural industry, andtherefore dietary options to reduce such emissions arecurrently being investigated.
Nitrogen emissions from ruminantsThe digestion of high-quality forages is relativelyinefficient in ruminants and results in large ammonialosses from the rumen (20–35% of dietary N), mostof which is excreted as urea in urine (Fig. 1).210–213
However, some tannins can improve the utilisation ofdietary N by rendering plant proteins less susceptibleto ruminal degradation (see ‘Beneficial nutritionaleffects’). As a result, such tannin-containing foragescreate significantly lower urinary N losses.12,38,68
Although faecal N outputs tend to be slightly higherowing to the formation of tannin–protein complexes,it is considered to be an environmentally safer form ofN than urinary N.12,15 In fact, Grabber et al.214 usedthe term ‘time-release nitrogen fertiliser’ for manurefrom tannin-containing feeds and predicted with theaid of a simulation model that new alfalfa varieties with10–20 g CTs kg−1 would increase the profitability of a
100-cow dairy farm by reducing nitrate and ammonialosses from 96 to 71 lb acre−1 (from 108 to 80 kg ha−1).
A recent report215 found that very low levels ofdietary ellagitannins (ca 1 g kg−1) from chestnut woodalso cut N losses from stored manure of lactatingdairy cows by 50% over an 8 week period. Much workremains to be done in terms of evaluating differenttypes of tannins for their effectiveness in improvingamino acid absorption in ruminants and reducing Nlosses from urine and faeces.
Methane emissions from ruminantsIt is well known that low-quality feeds account for ca90% of the world’s methane production by ruminants.Van Soest210 therefore pointed out that manipulationof cattle diets with high nutritive value in intensivefarming systems would have little effect on worldwidemethane outputs. Recent research, however, hasprovided some interesting data on how tannin-containing diets can reduce methane emissions fromruminants. Freshly fed L. cuneata (180 g CTs kg−1)
resulted in lower methane emissions, expressed aseither quantity per day (7.4 vs 10.6 g day−1) or relativeto dry matter intake (6.9 vs 16.2 g kg−1 DM intake),compared with a diet containing Digitaria ischaemum(Schreb.) Schreb. ex Muhl and Festuca arundinaceaSchreb.216 Similar results were obtained with freshtannin-containing L. pedunculatus and goat willow (S.caprea), which reduced methane emissions from therumen by 16–20% per unit intake.217,218 Silage fromL. corniculatus also reduced methane emissions perunit intake (g CH4 kg−1 DM intake) by 23% and perunit production (g CH4 kg−1 milk solids) by 13%.209
The question remains whether lower methane lossesalso result in lower energy losses from ruminants.12
Roth et al.219 compared quebracho (CTs), mimosa(A. mearnsii; CTs) and chestnut (ETs) tannins fortheir effects on in vitro gas production, methaneproduction and digestibility. They chose relativelyhigh concentrations of tannins (90–440 g tanninskg−1), and their results suggested that appropriatedoses of tannins would reduce methane productionbut not digestibility. It remains to be seen whetherthe small effect of the quebracho tannins was dueto a low tannin concentration in the extract orto the structural differences that exist between theprofisetinidins in quebracho and those in mimosaextracts. Mimosa profisetinidins are characterised by2R stereochemistry, a preponderance of 4 → 8 linksto the terminal catechin and a 3,4-trans configuration(Fig. 8); quebracho profisetinidins are characterisedby 2S stereochemistry, a higher proportion of 4 →6 links to the terminal catechin and a 3,4-cisconfiguration (Fig. 8).220,221 Furthermore, mimosatannins have a higher degree of hydroxylation andquebracho tannins have a lower water solubility.222
A greater use of traditional tannin-containing feedssuch as species of Lotus, Onobrychis, Salix and Gleditsia,Quercus spp. acorns, etc. could therefore assist in themove from intensive to more extensive feeding systems
in temperate countries, not only by improving theefficiency of nutrient utilisation but also by reducingthe need for anthelmintics.12,102,223
ANALYSIS OF TANNIN MIXTURES ANDPREDICTION OF ANIMAL RESPONSESColorimetric assaysTannins can be detected after producing a colourwith several different reagents. The Prussian blue,Folin–Ciocalteu, HCl/butanol and vanillin methodsare the most widely employed, but the use of 4-dimethylaminocinnamaldehyde and rhodanine hasalso been advocated.224–226 The Folin–Ciocalteureagent reacts with phenolic groups in generalto produce a blue colour, but is therefore notspecific for tannins. The HCl/butanol assay cleavescondensed tannins oxidatively and yields brightlycoloured anthocyanidins. However, this reaction isnot quantitative and colour yields are dependent oninter-flavanoid linkages (4 → 8 vs 6 → 8 bonds; A- vsB-type tannins) and the presence or absence of 5-OHgroups.227 This explains for example why quebrachotannins (no 5-OH groups) yield less colour than Lotustannins (with 5-OH groups). Schofield et al.225 andMueller-Harvey226 reviewed in detail several methodsfor quantifying condensed and hyrolysable tanninsand suggested that, given the limitations of currenttannin assays, more than one assay be employedconcurrently. Moreover, feeds can contain severaldifferent types of tannins (CTs, GTs, ETs), whichcannot be measured adequately by a single assay.The statement by Lowry et al.1 that ‘the simplicityof absorbance measurements masks the problems ofextracting meaningful data’ cannot be emphasisedstrongly enough. Not surprisingly, results from simplecolorimetric assays have not proved particularly usefulfor predicting the nutritional responses to tannin-containing feeds.
One of the most important steps in quantifying tan-nins depends on the use of appropriate standards.228
Stewart et al.48 showed that the best approach is to pre-pare a separate crude tannin standard for each plantaccession. Even the use of a standard prepared from adifferent accession of the same species can be mislead-ing: a standard based on tannins from C. calothyrsusof San Ramon provenance would have significantlyunderestimated the tannins in C. calothyrsus of Pat-ulul provenance. Simple solvent extractions are onepossible approach for isolating crude tannin mixturesfrom plants.229 Chromatography on Sephadex LH20is another widely used method for preparing crudetannin mixtures, which can then be used as stan-dards in colorimetric assays.48 However, two recentpapers76,230 demonstrated that the usual procedureof eluting tannins from Sephadex LH20 with ace-tone/water (7:3) missed the higher-MW fractions ofL. pedunculatus and Dorycnium spp. tannins, whichcould be eluted instead with water or methanol/water(1:1). It would appear that the ability of Sephadex
Figure 8. (a) 2S profisetinidins from quebracho (Schinopsis spp.) and (b) 2R profisetinidins from mimosa (Acacia mearnsii).208,210
LH20 to operate partially in a size exclusion mode hasbeen overlooked for many years.230,231
The composition of these tannin mixtures canbe characterised by MALDI-TOF-MS9,119,120,232,233
or electrospray ionisation mass spectrometry (ESI-MS).76,230,234,235 Normal phase, reverse phase and gelpermeation high-performance liquid chromatography(HPLC) are suitable for separating and quantifying
individual compounds.9,121,236,237 These techniquesare invaluable for identifying the main featuresof different tannin mixtures and are a necessarycomplement to colorimetric assays when attemptingto relate the effects of tannins to in vivo responses.
Protein precipitation assaysSeveral techniques have been devised to determinetannin concentrations by measuring the amount ofprotein they precipitate.24,238–242 Another assay, theso-called radial diffusion assay, is also based onprotein precipitation and is suitable for screening largenumbers of samples.243 Tannins are placed in thecentre of an agar plate which contains BSA as theprotein. As tannins diffuse outwards, they precipitateBSA, and the diameter of the resulting ring is measuredand compared with diameters obtained with knowntannin standards.
Much work remains to be done in order to unravelwhich particular factors influence most the digestionof proteins in tannin-containing plants. Relevant fac-tors could be (1) the total quantity of protein thatis precipitated by tannins during digestion, (2) theastringency of a tannin, i.e. unit of protein boundper unit of tannin, or (3) the binding strength in pro-tein–tannin complexes. Tannins and proteins can bindvia two distinctly different mechanisms, i.e. hydropho-bic interactions and hydrogen bonding.244,245 Bothtypes of interactions are likely to occur in dietary tan-nin–protein complexes, but their relative importancehas not yet been elucidated. Hagerman et al.244 discov-ered two model tannins, each of which bound predom-inantly via one of these two mechanisms to BSA: thenon-polar pentagalloylglucose (PGG; Fig. 2) formed ahydrophobic coat around the protein, whereas a polarepicatechin polymer, epicatechin16 (4 → 8) catechin,formed hydrogen-bonded crosslinks between proteinmolecules. The epicatechin polymer was more effi-cient than PGG at precipitating BSA on a molar ormass basis, and the epicatechin polymer–BSA com-plex was also much more stable than the PGG–BSAcomplex. Similarly, the profisetinidin CTs from que-bracho interacted with salivary histatins by a differentmode than PGG.246
Although it has been known for some time that thebinding strengths in different tannin–protein com-plexes can vary over orders of magnitude,87 surpris-ingly few studies have related the protein-bindingabilities of tannins to in vivo protein digestibilities.Robbins et al.143 concluded that the reduction in pro-tein digestibility in wild ruminants was proportionalto the BSA protein-precipitating capacity of planttannins. Osborne and McNeill24 came to a similarconclusion, as L. leucocephala tannins have a weakerBSA-binding capacity than L. pallida tannins andare nutritionally superior.10,24 An interesting recentstudy by Andrabi et al.247 compared two techniqueswhich used either 15N- or 125I-labelled protein–CTcomplexes for in vivo assessments of post-ruminal pro-tein digestibilities. The 125I technique proved more
precise and was capable of detecting differences inprotein digestibilities. These digestibilities were cor-related with CT astringency values, which measurethe amount of protein precipitated by CTs on a g g−1
basis.It remains to be seen (1) whether such distinctly
different interaction mechanisms also occur betweennaturally occurring mixtures of tannins and nutrition-ally relevant proteins and (2) whether these two typesof binding interactions (hydrophobic versus hydro-gen bonds) and the binding strengths in the resultingcomplexes have any bearing on protein digestion,tannin toxicity or bioavailability in ruminant and non-ruminant feeds. An interesting proposition was madeby Wroblewski et al.,93 who suggested that the muchweaker interaction of epigallocatechin gallate (com-pared with pentagalloyl glucose) with salivary Hstsmay allow its uptake into the body and be related tothe reported health benefits of tea.
It is questionable whether the widely used BSAis a suitable model for the fraction I protein(Rubisco), which accounts for 32–40% of proteinin forage leaves.248 A protein precipitation techniquethat used Rubisco instead of BSA was unable todifferentiate between the nutritional properties ofLotus, sainfoin, sulla and dock tannins.249 The authorsconcluded that measurements of Rubisco degradationrather than precipitation may be more relevant toruminant nutrition. Rubisco consists of large andsmall subunits (LSUs, SSUs) and, in the absenceof CTs, rumen micro-organisms degraded the LSUsmuch more rapidly than the SSUs. CTs inhibited LSUdegradation much more than SSU degradation.211,250
Interestingly, Tanner et al.250 also found that therewere only small differences between the inhibitoryeffects of CTs from species of Dorycnium, Hedysarum,Lotus and Onobrychis. However, if the CTs were firstpre-incubated with rumen fluid, LSU degradationwas affected much more by the L. pedunculatusthan the L. corniculatus CTs, which suggested thatthe L. pedunculatus tannins were more inhibitory toproteolytic bacteria.30 Differential binding of tanninsto rumen micro-organisms could well be anotherimportant factor in protein digestion.27 Tannin-resistant bacteria tend to secrete an extracellularpolysaccharide and form a thick glycoprotein, whichhas a high affinity for tannins.126
Tannins from several tropical forages (Arachispintoi Krapov. & W.C. Greg., Desmodium ovalifolium(Schum.) Walp., G. sepium and Manihot esculentaCrantz) precipitated different amounts of BSAcompared with alfalfa leaf protein in a radial diffusionassay which had been modified to mimic rumenconditions (pH 6.8, 39 ◦C).251 In the same assay,quebracho tannins and tannic acid precipitated BSAbut not alfalfa leaf protein. The authors concluded thatmeasures of ‘biological tannin activity’ based on thisparticular BSA precipitation assay might not reflectthe ability of tannins to precipitate plant proteins fromherbivore diets.251
Another point to remember is that complexationbetween tannins and proteins can lead to both solubleand insoluble complexes.93,252,253 Soluble complexesare likely to be washed out of the rumen morequickly than insoluble complexes,248 and thereforeboth types of complex formation will be relevant toanimal nutrition, but only insoluble complexes will beestimated in an assay based on protein precipitation.
Isothermal titration calorimetry (ITC) and tanninsolubilities (Kow values)We recently probed tannin–protein interactions byisothermal titration calorimetry (ITC).25,89 ITCmeasures the amount of energy that is released whentannins are slowly titrated into a protein solution.The ITC protein-binding curves exhibited higheraffinities for tara- and D. cinerea tannins and loweraffinities for myrabolan (Terminalia chebula Willd.)and A. nilotica tannins. The binding curve of D. cinereatannins with gelatin was sigmoidal, suggesting a highlyspecific interaction. In contrast, the A. nilotica tanninsbound via a two-stage process: the first stage wascharacterised by cooperative, ligand-enhanced bindingand the second stage by non-specific absorption.
It may be of note that the D. cinerea tanninsbound gelatin much more strongly (ITC data)and also had a higher water solubility than theA. nilotica tannins (see Table 2 in ‘Appendix’).25
Tannins differ greatly in their solubilities in, forexample, ethyl acetate, octanol, acetone/water andwater.76,222,254,255 Contrasting solubilities in octanoland water give rise to a wide range of so-called Kow
values (Table 2),244,255 which have been used in thepharmaceutical and agrochemical sector to predictthe toxicological properties and environmental fate ofchemicals:256,257
Kow-value = Concentration in octanolConcentration in water
Higher Kow values indicate that a molecule ismore ‘fat-soluble’ than ‘water-soluble’ and that itmay be absorbed into body tissues. Tannins withhigher Kow values are therefore more likely tobe absorbed and to exert physiological, i.e. toxicor medicinal, effects. Indeed, this seems to bethe case with the partially galloylated CTs fromChinese rhubarb (Rheum palmatum L.), which aresubstantially insoluble in water but highly solublein organic solvents.258 Tanaka et al.258 also foundthat hydrophobic interaction of these galloylated CTswith rhein 8-O-glucoside, the major anthraquinoneglycoside in rhubarb, significantly increased theirwater solubility and may assist in the transfer ofthese bioactive CTs from the food matrix to theabsorbing body tissues. These results also demonstratethat co-occurring matrix compounds will influence thenutritional or medicinal effects of tannins.
It is conceivable that toxic tannins have higherKow values and bind non-specifically and relativelyweakly, whereas tannins with lower Kow values
bind specifically and more strongly to proteins andimprove the efficiency of N utilisation in ruminants(Table 2). Kow values in the literature appear tosupport this hypothesis. PGG (Fig. 2) has higher Kow
values (ranging from 32 to 129 depending on theexperimental conditions)244,255,258 and is an importantconstituent of tannic acid, which can be toxic.31,98
However, castalagin has a lower Kow value (0.1)255
and is the major constituent of chestnut tannins, whichare approved as feed additives in Switzerland.50 It isof note that the ranges of known Kow values for HTsand CTs overlap: values for HTs range from 0.0001to >100 and values for CTs from 0.002 to ca 13. Thisfact may indicate why classifying tannins into CTs andHTs is not helpful for predicting animal responses.
Techniques based on protein precipitation, ITC andKow values allow the study of real-life, complex tanninmixtures. The author would like to propose that thesetechniques be evaluated in animal-feeding trials inorder to identify the characteristics of desirable andsafe tannins.
CONCLUSIONS AND OUTLOOKPlants tend to produce complex mixtures of tannins,and not all tannins have the same effects. Digestionof tannin-rich feeds appears to be facilitated by co-feeding with a ready source of energy to support thedetoxification of any associated phenolic compounds,or by feeding mixtures rather than single speciesof high-tannin materials. The focus of this reviewhas been on tannin–protein interactions and theireffects on protein digestion and animal health.Caution is needed when attributing physiologicaleffects to tannins: in the case of sorghum grain,other compounds rather than the tannins are probablyresponsible for the observed toxic effects. A similarsituation may exist in T. oblongata, but this has notyet been proven beyond doubt. Although not coveredin this review, one also needs to bear in mind thatthe digestive tract is a complex system which containsmicro-organisms and many different matrices that mayinteract with tannins and thus modulate their effects.
Tannins in L. corniculatus, L. leucocephala, O.viciifolia and D. cinerea are particularly effectiveat improving protein digestion in ruminants andappear to interact in an optimal fashion withfeed proteins. The underlying mechanisms for theirsuperior effects require further study. To summarise,there is some evidence that the following structuralfeatures of tannins affect the nutritive value offeeds: procyanidin/prodelphinidin ratios; degree ofgalloylation; molecular weights; and binding strengthsin tannin–protein complexes. It is also of note thattannins interact with proteins via either hydrophobicor hydrogen bonds or via a combination of these twodistinctly different binding mechanisms. It remainsto be shown which of these mechanisms is moreimportant in terms of animal nutrition or health.
It could be argued that tannins that interactpredominantly via hydrogen bonds form strongercomplexes with proteins (e.g. dietary or salivaryproteins) and are therefore more likely to generateruminal escape protein. Circumstantial evidencesuggests that there is an optimum interaction betweencertain tannins and feed proteins, i.e. neither toostrong nor too weak an interaction. Conversely,tannins that interact via hydrophobic bonds formweaker complexes with proteins and may thereforebe more easily dissociated from such complexes,resulting in their metabolism in the digestive tractor in medicinal, veterinary or toxic physiologicaleffects. This and alternative hypotheses need to bescrutinised, e.g. the differential effects of tanninson proteolytic rumen micro-organisms. One of thekeys to elucidating the biological effects of tanninsdepends on understanding which diets produce highlevels of urinary phenolics or increased faecal N.Does faecal N originate from dietary protein, bacterialcells or secreted proteins, animal tissue protein orsalivary proteins? How important is the fact thatsome tannins shift the site of amino acid absorptionalong the entire length of the small intestine? Feedswith well-defined compositions of tannins, proteinsand fermentable carbohydrates would allow us toevaluate the structure–activity relationships of thetannin–protein hypothesis and also to test alternativehypotheses.
Preliminary evidence suggests that the strength ofinteractions between tannins and proteins is relatedto their solubilities in solvents such as water andoctanol and that the resulting Kow values may beof use in predicting these interactions. It is also of notethat the Kow values do not distinguish between CTsand HTs, which could explain why the classificationinto CTs and HTs has not been helpful forpredicting animal responses. If true, this could explainwhy quebracho tannins, which have lower watersolubilities than the closely related mimosa tannins (orhigher Kow values), have only very weak interactionswith dietary proteins but have better anthelminticproperties. Similarly, it has been suggested thatthe weak interaction between epigallocatechin gallate(compared with pentagalloylglucose) and salivaryhistatins may account for the health benefits of greentea. The reader will notice from Table 2 that somecontradictions or unresolved issues still exist withthese arguments. This review has attempted to unravelseveral facets of the tannin conundrum, but furtherresearch is needed, especially at the interface betweentannin chemistry and in vivo effects.
Tannins form a highly diverse group of naturalproducts with promising nutritional, veterinary andenvironmental effects. Current research focuses inparticular on their anthelmintic properties andon their ability to improve the efficiency withwhich nutrients are used by ruminants in order toreduce environmental losses of methane and, moreimportantly, nitrogen. However, progress is hampered
by a lack of suitable analytical techniques that arecapable of predicting these biological effects. Theestablishment of structure–activity relationships andthe development of meaningful tannin assays would beaided by experimental plant models with altered tannincompositions. Any newly developed tannin assays willassist plant breeders and molecular biologists with thescreening of plant germplasm for the benefit of animalsand the environment.
ACKNOWLEDGEMENTSI am grateful to Professor PJ Van Soest and ananonymous reviewer for their very helpful comments.
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Sheep and calves: good growthrates, better N retention than onalfalfa
71,265
Arachis hypogaea (peanutskins): testa
18–24% PC: A- and B-type bonds;proanthocyanidins A-1, A-2;epicat(4β-8,2β-O-7)ent-epicat;epicat(4β-6,2β-O-7)cat;epicat(4β-6,2β-O-7)ent-cat;epicat(4β-6,2β-O-7)ent-epicat;epicat(4β-8,2β-O-7)cat;epicat(4β-8,2β-O-7)epicat;dimers to tetramers
<16% peanut skins in diet is usefulfor dairy cows: increased drymatter intake, milk yield and %milk fat, but lower % milk protein
2% tannins: no toxic effects (lambs);8% tannin sprayed onto hay: nohepatic damage; improved Nsupply to duodenum (bulls);treatment against diarrhoea (pigs,cattle); reduced ammoniaemission from urine and fromstored manure from lactatingcows
CT were modified in rat gut; 2.5%carob pulp tannins produced lowgrowth rates (lambs), negativeeffects on digestibility despitehigh sugar and pectin contents(47%); 0.9% tannin diet had nonegative effect on weight gain
39,130,174,277
Ceratonia siliqua (carob):leaves
Goats: no toxic effects 278
Desmodium ovalifolium 7–29% CT Ruminal escape protein 279–281Dichrostachys cinerea: fruits 18% CT; A- and B-type bonds Ruminal escape protein (goats) 15,232,263Dorycnium spp.: leaves 15–20% CT; PC/PD ratios 5:95 to
13:87; MW 3300–35 600Anthelmintic (in vitro and in vivo) 192,193,230,282
Lotus corniculatus PC/PD ratios 67:33 to 84:16; CE asterminal units; GE as extenderunits; mostly 4 → 8, some 4 → 6interflavan linkages; smallamounts of epiafzelechinmonomers and glucose in CT; DP2–19 (MW < 5300)
Ruminal escape pass protein, betterin vivo protein digestion than L.pedunculatus; anthelmintic
7,9,12,64,76
Lotus pedunculatus PC/PD ratios 19:81 to 23:77; CEand GE occur in terminal andextender units; small amounts ofglucose in CT; 4 → 8 interflavanlinkages; DP 2–44(MW < 12 300)
Ruminal escape protein, but lessamino acids absorbed in lowerintestines compared with L.corniculatus; anthelmintic
Up to 7% CT; mixture of CT depends onsorghum variety: PC with A- and B-typebonds, glucosylated proapigeninidinsand proluteolinidins with terminaleriodictyol
Systemic effects in rats andchickens attributed to low-MWphenolics; 14C-CT not absorbedby chickens
114,120,142
Tamarindus indica: seedhusk
15% tannins Lactating dairy cows: more Nretained; improved weight gains;no effect on intake or milk yield;higher faecal N, lower urinary N
86
Terminalia oblongata: leaves 11–29% tannins; HT, ET Hepatotoxic and nephrotoxiceffects; poisoning and deaths ofcattle and sheep; resorcinolglucuronide, 2-carboxy-2′ ,4′,4,6-tetrahydroxy diphenyl2,2′-lactone glucuronide in urine
