Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine. However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood. The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne)-white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins). The bacterial populations were quantified using a competitive polymerase chain reaction. Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined. When sheep fed on ryegrass-white clover, populations of Clostridium proteoclasticum B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-1), respectively. When the diet was changed to Lotus corniculatus, the average populations (after 8-120 h) of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis decreased (P < 0.001) to 2.4 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-1), respectively. When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis were higher (P < 0.01-0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8-120 h) of 4.9 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(6), respectively. Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05-0.001) than sheep that were fed Lotus corniculatus plus PEG. The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum. The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus. Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen.

Three experiments were undertaken to determine the effect of condensed tannin (CT) in Lotus pedunculutus (45-55 g extractable CT/kg DM) on the digestion of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39; Rubisco; fraction 1 leaf protein). In two of the experiments Lotus pedunculutus was fed to sheep, with one group receiving a continuous intraruminal infusion (per fistulum) of PEG (molecular weight 3500) to bind and inactivate the CT (PEG group). The other group, which did not receive PEG, was termed the control sheep (CT acting). Expt 3 involved in vitvo incubations of Lotus pedunculutus in buffered rumen fluid, with and without PEG added. In all experiments the results have been interpreted in terms of the effects of CT on Rubisco solubTzation and degradation. Disappearance of N and Rubisco from Lotus pedunculutus suspended in polyester bags in the rumen was used as a measure of solubilization. Degradation was defined as the disappearance of Rubisco from in vitro incubations of Lotus pedunculatus in rumen fluid. In Expt 1, CT reduced the digestion of Rubisco in the rumen from 0.96 to 0.72 of intake (P c 0.01). Rubisco digestion in the small intestine was 0.27 of intake in control sheep and 0.04 of intake in PEG sheep. In Expt 2, PEG had no effect on the loss of Rubisco from Lotus pehneulatus contained in polyester bags which were incubated in the rumen, hence CT did not affect the solubilization of Rubisco. Observations in Expt 1 were confirmed by in vitro incubations in Expt 3, where PEG addition substantially increased the rate of degradation of plant protein to NH,. Addition of PEG decreased the period of time taken to degrade 50% of the Rubisco from about 13.8 h to about 3.0 h. It was concluded that the action of CT reduced the digestion of Rubisco in the rumen of sheep fed on fresh Lotus pedunculutus, and that this was primarily due to the ability of CT to slow its degradation by rumen micro-organisms, without affecting its solubilization. Both fresh-minced, and freeze-dried and ground lotus were used for in smco and in vitro incubations; however, fresh-minced lotus was more suitable for the evaluation of protein solubilization and degradation in fresh forages.Condensed tannin : Rnmen degradation: Ribulose-1,5-bisphosphate carboxylase When ruminants fed on fresh forage were given abomasal infusions of protein or fed on protein protected from rumen degradation, production responses included increased wool growth (Reis, 1979), milk production in dairy cattle (Rogers et al. 1980;Hamilton et al. 1992) and sheep (Penning et al. 1988), and live-weight gain (Barry, 1981;Stock et al. 1981 ;Hogan, 1982; Poppi et al. 1988;Fraser et al. 1990). These results suggest that productivity is reduced because insufficient essential amino acids (EAA) are absorbed from the small intestine relative to energy. The low absorption of amino acids (AA) is due in part to the

A series of in vitro experiments was undertaken to determine the extent to which Sephadex LH‐20 treated extracts from a range of temperate forages precipitated ribulose‐1,5‐bisphosphate carboxylase (Rubisco) and affected the enzymatic hydrolysis of Rubisco protein by trypsin and chymotrypsin at a range of pH values. Rubisco was chosen because it represents the principal dietary protein for ruminants fed fresh forages. Condensed tannins (CT) or proanthocyanidins (PA) are routinely purified by chromatography using Sephadex LH‐20 as a matrix. However, these extracts contained non‐CT phenolics together with PA so the term ‘CT extract’ was preferred to ‘PA’ to describe the extracts. The in vitro precipitation of Rubisco provided a means to compare the reactivity of the CT extracts. The amount of CT extract required to precipitate all the Rubisco in 10 μg of total soluble leaf protein from white clover (Trifolium repens) when this protein was incubated with CT extracts of Lotus corniculatus, L pedunculatus and sainfoin (Onobrychis viciifolia) was similar, with between 25 and 50 μg of extract required. The CT extract of sulla (Hedysarum coronarium) also precipitated all the Rubisco, however this only occurred with 50 μg of the extract. The CT extract of dock (Rumex obtusifolius) precipitated all the Rubisco when 5 μg of extract or greater was incubated with total soluble leaf protein. However, the differences between the reactivity of all these CT extracts at a range of pH values appeared to be small. Condensed tannin extracts of L corniculatus and L pedunculatus partially inhibited the hydrolysis of Rubisco by trypsin and chymotrypsin to a similar extent, but the extent of the inhibition was affected by pH. The inhibition was greater at pH 6·0 than 7·0, whilst at pH 8·0, CT extracts had little or no affect on trypsin and chymotrypsin. It was concluded that, although the precipitation of Rubisco provided an ideal method for comparing CT extracts, reactivity alone was unlikely to account for the differences in nutritive value that occur with forages containing CT. © 1998 SCI.

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can inýuence the digestion of plant protein in the rumen. We compared the eþ ects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens) was incubated with fresh rumen ýuid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantiüed by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and imaging densitometry. To deduce the eþ ects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG ; MW 3350). The two CT extracts diþ ered markedly in their eþ ects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg-1 total soluble leaf protein, the CT extract from L pedunculatus was more eþ ective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg-1 total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which aþ ects the activity of the CT needs to be considered when assessing the eþ ects of CT on protein metabolism in ruminants.

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