Warren C. McNabb

The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants

New methodology for measuring forage condensed tannin (CT) content is described and the effects of CT upon forage feeding and nutritive value for ruminant animals are reviewed. CT react with forage proteins in a pH-reversible manner, with reactivity determined by the concentration, structure and molecular mass of the CT. Increasing concentrations of CT in Lotus corniculatus and Lotus pedunculatus reduce the rates of solubilization and degradation of fraction 1 leaf protein in the rumen and increase duodenal non-NH3 N flow. Action of medium concentrations of total CT in Lotus corniculatus (30-40 g/kg DM) increased the absorption of essential amino acids from the small intestine and increased wool growth, milk secretion and reproductive rate in grazing sheep without affecting voluntary feed intake, thus improving the efficiency of food conversion. High concentrations of CT in Lotus pedunculatus (75-100 g/kg DM) depressed voluntary feed intake and rumen carbohydrate digestion and depressed rates of body and wool growth in grazing sheep. The minimum concentration of CT to prevent rumen frothy bloat in cattle is defined as 5 g/kg DM and sheep grazing CT-containing legumes were shown to better tolerate internal parasite infections than sheep grazing non CT-containing forages. It was concluded that defined concentrations of forage CT can be used to increase the efficiencies of protein digestion and animal productivity in forage-fed ruminants and to develop more ecologically sustainable systems of controlling some diseases under grazing.

Regulation of Tight Junction Permeability by Intestinal Bacteria and Dietary Components

The human intestinal epithelium is formed by a single layer of epithelial cells that separates the intestinal lumen from the underlying lamina propria. The space between these cells is sealed by tight junctions (TJ), which regulate the permeability of the intestinal barrier. TJ are complex protein structures comprised of transmembrane proteins, which interact with the actin cytoskeleton via plaque proteins. Signaling pathways involved in the assembly, disassembly, and maintenance of TJ are controlled by a number of signaling molecules, such as protein kinase C, mitogen-activated protein kinases, myosin light chain kinase, and Rho GTPases. The intestinal barrier is a complex environment exposed to many dietary components and many commensal bacteria. Studies have shown that the intestinal bacteria target various intracellular pathways, change the expression and distribution of TJ proteins, and thereby regulate intestinal barrier function. The presence of some commensal and probiotic strains leads to an increase in TJ proteins at the cell boundaries and in some cases prevents or reverses the adverse effects of pathogens. Various dietary components are also known to regulate epithelial permeability by modifying expression and localization of TJ proteins.

Polyphenols and agriculture : beneficial effects of proanthocyanidins in forages

Abstract Proanthocyanidins (PA), also known as condensed tannins, belong to the oldest of plant secondary metabolites. These compounds are widespread in woody plants, but are also found in certain forages. Proanthocyanidins can exert effects in organisms because of their ability to complex with proteins. Forages containing moderate concentrations of PA (2–4% DM) can exert beneficial effects on protein metabolism in sheep, slowing degradation of dietary protein to ammonia by rumen micro-organisms and increasing protein outflow from the rumen, thus increasing absorption of amino acids in the small intestine of the animal. This was shown to result in increases in lactation, wool growth and liveweight gain, without changing voluntary feed intake. Dietary PA can also contribute to improved animal health by reducing the detrimental effects of internal parasites in sheep and the risk of bloat in cattle. In contrast, high dietary PA concentrations (6–12% DM) depress voluntary feed intake, digestive efficiency and animal productivity. Temperate forages containing PA, such as Lotus corniculatus and L. pedunculatus , will not persist in intensive grazing systems if continuously grazed (i.e. set stocked), especially in mixtures with perennial ryegrass and white clover and need to be grown as pure species and rotationally grazed. Nevertheless, inputs of these ‘special purpose’forages can increase sustainability and productivity in intensive grazing systems through increasing the efficiency of animal production, reducing urinary nitrogen (N) excretion and reducing chemical inputs as anthelmintics and as detergents used to control rumen bloat in cattle. Proanthocyanidins are derived from the flavonoid biosynthesis pathway, and knowledge is rapidly increasing about the molecular control of PA biosynthesis. These recent investigations may ultimately enable the expression by genetic engineering of increased levels of PA in the leaves of agriculturally important forage plants such as white clover and perennial rye grass, which will withstand continuous defoliation under grazing and currently contain only trace amounts of PA. This could potentially lead to the widespread use of PA in temperate grazing systems. Further consideration, should now be given to long-term adaptations in the animal, and to ecological effects on the soil ecosystem by PA and their degradation products excreted in animal faeces. More information is required in particular on the effects of PA on soil nitrification, ammonia volatilisation from soil, and nitrogen levels in the groundwater.

Effects of condensed tannins in Lotus pedunculatus on its nutritive value for sheep. 1. Non-nitrogenous aspects

Lotus pedunculatus was grown under high fertility conditions and its nutritive value was determined in a feeding trial with sheep at Palmerston North, New Zealand in 1989. The condensed tannins (CT) accounted for 5.5% of lotus dry matter (DM) and its effect on digestion was evaluated by giving an intraruminal infusion of polyethylene glycol (PEG) to six of the sheep (PEG group). PEG preferentially binds with CT so that the lotus becomes essentially CT-free. The experiment was carried out with 14 sheep (six PEG and eight «Tannin») held in metabolism crates indoors and given freshly cut lotus hourly, for 32 days. This paper presents data relating to carbohydrate and mineral digestion, together with aspects of rumen function [...]

Consequences of plant phenolic compounds for productivity and health of ruminants

Plant phenolic compounds are diverse in structure but are characterised by hydroxylated aromatic rings (e.g. flavan-3-ols). They are categorised as secondary metabolites, and their function in plants is often poorly understood. Many plant phenolic compounds are polymerised into larger molecules such as the proanthocyanidins (PA; condensed tannins) and lignins. Only the lignins, PA, oestrogenic compounds and hydrolysable tannins will be considered here. Lignins slow the physical and microbial degradation of ingested feed, because of resilient covalent bonding with hemicellulose and cellulose, rather than any direct effects on the rumen per se. The PA are prevalent in browse and are expressed in the foliage of some legumes (e.g. Lotus spp.), but rarely in grasses. They reduce the nutritive value of poor-quality diets, but can also have substantial benefits for ruminant productivity and health when improved temperate forages are fed. Beneficial effects are dependent on the chemical and physical structure, and concentration of the PA in the diet, but they have been shown to improve live-weight gain, milk yield and protein concentration, and ovulation rate. They prevent bloat in cattle, reduce gastrointestinal nematode numbers, flystrike and CH4 production. Some phenolic compounds (e.g. coumestans) cause temporary infertility, whilst those produced by Fusarium fungi found in pasture, silage or stored grains can cause permanent infertility. The HT may be toxic because products of their metabolism can cause liver damage and other metabolic disorders.

Assay and digestion of 14C-labelled condensed tannins in the gastrointestinal tract of sheep

Three experiments were conducted to determine the fate of condensed tannins (CT) during digestion in sheep. CT were measured as extractable, protein-bound and fibre-bound fractions using the butanol-HCl procedure. In Expt 1, purified CT were added to digesta from different parts of the digestive tract obtained from a pasture-fed sheep. Recoveries of CT after 0 and 4 h of anaerobic incubation at 39 degrees averaged: rumen 78.9 and 57.5%; abomasum 50.9 and 49.0%; duodenum 64.4 and 46.0% and ileum 43.4 and 38.8%. In Expt 2, [14C]CT was given per abomasum over a 6.5 h period at 15 min intervals to a sheep previously fed on Lotus pedunculatus (which contains CT). The sheep was killed at the end of the period and 92.4% of the label was recovered. Virtually all of the label was in the digesta, and none was detected in the blood, so that the CT-carbon appeared not to be absorbed from the small intestine. In Expt 3, rumen, abomasal and ileal digesta and faeces samples from sheep fed on Lotus pedunculatus were analysed for CT and CT flow along the digestive tract calculated from reference to indigestible markers. Values were low in all digesta samples, indicating disappearance of CT across the rumen and small intestine, and CT recovery in faeces was only about 15% of intake. However, the 14C results from Expt 2 suggested that little if any CT-carbon was absorbed and the low recoveries in Expt 1 are considered to be a consequence of either conformational changes to the CT molecule such that it is no longer detectable by colorimetric methods, an inability of the analytical method to release bound CT for the butanol-HCl assay, or interference from other digesta constituents. It is concluded that the butanol-HCl method of CT analysis is appropriate for quantifying CT in herbages but not in digesta or faeces, and that a substantial part of CT released during protein digestion in the small intestine may not be detectable by normal CT analytical methods.

Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation

BackgroundIntestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses.ResultsThe effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER) across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P < 0.05, fold-change > 1.2), including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions were visualised by fluorescent microscopy of immuno-stained occludin, zona occludens (ZO)-1, ZO-2 and cingulin. Caco-2 cells treated with L. plantarum MB452 had higher intensity fluorescence of each of the four tight junction proteins compared to untreated controls.ConclusionsThis research indicates that enhancing the expression of genes involved in tight junction signalling is a possible mechanism by which L. plantarum MB452 improves intestinal barrier function.

Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colubriformis in vitro

The effects of condensed tannins extracted from seven forages on the viability of the eggs and first stage (L1) larvae of the sheep nematode Trichostrongylus colubriformis were evaluated in in vitro assays. The extracts of condensed tannins were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), sulla (Hedysarum coronarium), sainfoin (Onobrychus viciifolia), Dorycnium pentaphylum (DP), Dorycnium rectum (DR) and dock (Rumex obtusifolius). Extracts containing 200 to 500,μg/ml reduced the proportion of eggs that hatched. The larval development assay was used to evaluate the effect of the extracts on the development of either eggs or LI larvae to L3 infective larvae. Development was allowed to proceed for seven days by which time the larvae in control incubations had reached the infective L3 stage. Extracts containing 200 μg/mI from LP, DP, DR or dock prevented egg development, and only 11, 8 and 2 per cent of the eggs developed to 13 larvae with extracts from LC, sulla and sainfoin, respectively. When the concentration was 400 μg/mI no eggs developed to L3 larvae. The addition of the extracts after hatching also inhibited the development of Ll to L3 larvae; 200 μg/ml extracted from LP, LC, sulla, sainfoin, DP, DR and dock resulted in only 14, 18, 17, 15, 14, 16 and 4 per cent of Ll larvae developing to the L3 stage compared with 85 per cent for controls, and 400 μg/ml further reduced the development of Li larvae. Statistical analyses showed that when the extracts were added before hatching they were significantly (P<0.001) more effective at inhibiting the larval development than when they were added after hatching. The condensed tannins from dock had the greatest inhibitory effect on egg development followed by the tannins from DR, sainfoin, DP, LP, sulla and LC.

Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function

The aim of this research was to identify bacterial isolates having the potential to improve intestinal barrier function. Lactobacillus plantarum strains and human oral isolates were screened for their ability to enhance tight junction integrity as measured by the transepithelial electrical resistance (TEER) assay. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER, and the best-performing probiotic strain, Lactobacillus rhamnosus HN001, was used as a benchmark to evaluate the isolates. One isolate, L. plantarum DSM 2648, was selected for further study because it increased TEER 135% more than L. rhamnosus HN001. The ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells was determined, and L. plantarum DSM 2648 performed better than L. rhamnosus HN001 in all the assays. Lactobacillus plantarum DSM 2648 was able to reduce the negative effect of Escherichia coli [enteropathogenic E. coli (EPEC)] O127:H6 (E2348/69) on TEER and adherence by as much as 98.75% and 80.18%, respectively, during simultaneous or prior coculture compared with EPEC incubation alone. As yet, the precise mechanism associated with the positive effects exerted by L. plantarum DSM 2648 are unknown, and may influence its use to improve human health and wellness.

The effect of condensed tannins in Lotus pedunculatus on the digestion and metabolism of methionine, cystine and inorganic sulphur in sheep

Two experiments were conducted with sheep fed on fresh Lotus pedunculatus containing 50-55 g condensed tannin (CT)/kg dry matter. Effects of CT were assessed by comparing control sheep (CT operating) with sheep receiving a continuous intraruminal infusion of polyethylene glycol (PEG) to bind and inactivate CT. Digestion of methionine and cystine was determined using a continuous intraruminal infusion of indigestible markers, whilst plasma irreversible loss (IRL) of methionine, cystine and inorganic sulphate was determined using 35S labelling. The proportion of microbial non-NH3-N (NAN) in whole rumen digesta NAN and the IRL of reducible S from the rumen were determined using a continuous intraruminal infusion of (NH4)2(35)SO4. The proportion of microbial NAN in whole rumen digesta NAN (0.44 v. 0.71) and the IRL of reducible S from the rumen (0.84 v. 2.49 g S/d) were lower in control than PEG sheep. PEG sheep lost 30% of ingested methionine and cystine across the rumen, whereas the control sheep lost no methionine and cystine across the rumen. Apparent absorption of methionine from the small intestine was 27% higher in control than PEG sheep, but both groups had a similar apparent absorption of cystine. The apparent digestibility of cystine in the small intestine was lower in control (0.42) than PEG (0.53) sheep, whereas the apparent digestibility of methionine was similar (0.78) for both groups. CT had no effect on plasma methionine IRL, but markedly increased the IRL of cystine (39.8 v. 22.4 mumol/min) and reduced the IRL of plasma inorganic sulphate (35.9 v. 50.2 mumol/min). A three-pool model comparing interconversions between the three plasma metabolites showed that CT increased the flow of cystine to body synthetic reactions (36.5 v. 17.3 mumol/min). This was due to trans-sulphuration of methionine to cystine being greater in control than in PEG sheep, whilst the oxidation of both methionine and cystine were reduced in control sheep. It was concluded that CT reduced the proteolysis of forage protein and the degradation of S amino acids to inorganic sulphide in the rumen, resulting in increased net absorption of methionine and increased utilization of cystine for body synthetic reactions in sheep with a high capacity for wool growth (and, hence, high cystine requirement).