Investigating the efficacy of purified tannin extracts from underutilized temperate forages in reducing enteric methane emissions in vitro.

The study investigated how the concentration and composition of purified tannin extracts, at various inclusion rates, affect the ruminal in vitro fermentation parameters. Tannin extracts were isolated from four different forage species: birdsfoot trefoil (Lotus corniculatus), sulla (Hedysarum coronarium), big trefoil (Lotus pedunculatus), and salad burnet (Sanguisorba minor). Plants extracts were purified by Sephadex LH-20 gel chromatography and analyzed by UPLC-ESI-MS/MS. The results showed a large variation among the extracts from different species in terms of tannin composition and structural features. The extracts from salad burnet were dominated by hydrolysable tannins, comprising mainly ellagitannins. The extracts derived from sulla and big trefoil contained predominantly proanthocyanidins (PA), primarily composed of prodelphinidins with high mean degree of polymerisation (mDP). Birdsfoot trefoil extracts comprised procyanidin-rich PAs with low mDP. To determine whether the combined presence of tannins and flavonoid together lead to synergistic or antagonistic effects, the tannin extracts were incubated both with or without rutin at concentrations of 10, 20, and 30 g/kg DM, using a base substrate of perennial ryegrass (Lolium perenne, control). In general, all the tannin extracts decreased methane (CH4) production compared to the control, while no significant effect of rutin was observed on both gas (GP) and CH4 production, neither pure, nor in the simultaneous presence of tannins. The highest CH4 reduction (15%, at 30 g/kg DM) was observed from sulla and big trefoil extracts compared to control, but this was also supplemented with a concomitant reduction in GP (11%) indicating a reduction in feed digestibility. The extracts from birdsfoot trefoil and salad burnet reduced CH4 by up to 12% without significantly reducing GP, indicating the importance of tannin composition on ruminal fermentation.

The feeding of heather (Calluna vulgaris) to Teladorsagia circumcincta infected lambs reduces parasitism but can detrimentally impact performance.

Gastrointestinal nematode (GIN) infections impact small ruminant health, welfare, and production across farming systems. Rising anthelmintic resistance and regulation of synthetic drug use in organic farming is driving research and development of sustainable alternatives for GIN control. One alternative is the feeding of plants that contain secondary metabolites (PSMs) e.g., proanthocyanidins (PA, syn. condensed tannins) that have shown anthelmintic potential. However, PSMs can potentially impair performance, arising from reduced palatability and thus intake, digestibility or even toxicity effects. In this study, we tested the trade-off between the antiparasitic and anti-nutritional effects of heather consumption by lambs. The impact of additional feeding of a nematophagous fungus (Duddingtonia flagrans) on larval development was also explored. Lambs infected with Teladorsagia circumcincta or uninfected controls, were offered ad libitum heather, or a control chopped hay for 22 days during the infection patent period. Eight days into the patent period, parasitised lambs were supplemented (or remained unsupplemented) with D. flagrans for a 5-day period. Performance and infection metrics were recorded, and polyphenol levels in the heather and control hay were measured to investigate their association with activity. The lambs consumed heather at approximately 20% of their dry matter intake, which was sufficient to exhibit significant anthelmintic effects via a reduction in total egg output (P = 0.007), compared to hay-fed lambs; the magnitude of the reduction over time in heather fed lambs was almost 10-fold compared to control lambs. Negative effects on production were shown, as heather-fed lambs weighed 6% less than hay-fed lambs (P < 0.001), even though dry matter intake (DMI) of heather increased over time. D. flagrans supplementation lowered larval recovery in the faeces of infected lambs by 31.8% (P = 0.003), although no interactions between feeding heather and D. flagrans were observed (P = 0.337). There was no significant correlation between PA, or other polyphenol subgroups in the diet and egg output, which suggests that any association between heather feeding and anthelmintic effect is not simply and directly attributable to the measured polyphenols. The level of heather intake in this study showed no antagonistic effects on D. flagrans, demonstrating the methods can be used in combination, but provide no additive effect on overall anthelmintic efficacies. In conclusion, heather feeding can assist to reduce egg outputs in infected sheep, but at 20% of DMI negative effects on lamb performance can be expected which may outweigh any antiparasitic benefits.

Structural Features of Hydrolyzable Tannins Determine Their Ability to Form Insoluble Complexes with Bovine Serum Albumin.

The ability of 32 purified and characterized hydrolyzable tannins to form insoluble complexes with model protein bovine serum albumin was investigated with a turbidimetric 96-well plate reader method. The results showed a clear relationship between the hydrolyzable tannin structure and the intensity of haze that formed during the tannin-protein complexation. In addition to molecular weight, structural features such as number of galloyl groups, degree of oxidative coupling between the galloyls, positional isomerism, and cyclic vs acyclic glucose core were the major structural features that affected the ability of the monomeric hydrolyzable tannins to form insoluble complexes with bovine serum albumin. While oligomers were superior to monomers in their capability to precipitate the model protein, their activity depended less on the functional groups, but mostly on their size and overall flexibility. These results allowed us to construct an equation that predicted the protein precipitation capacity of the studied hydrolyzable tannins with high accuracy.

The in vitro anthelmintic properties of browse plant species against Haemonchus contortus is determined by the polyphenol content and composition.

The aims of the present study were to (a) evaluate the anthelmintic activity of 10 East African browse plant extracts, (b) examine their role in inhibition of Haemonchus contortus larval exsheathment, (c) establish relationship between inhibition of larval exsheathment and browse plant extract polyphenol composition. Acetone/water (70/30%) extracts of air dried leaves of Acacia etbaica, Cadaba farinosa, Capparis tomentosa, Dichrostachys cinerea, Dodonaea angustifolia, Euclea racemosa, Maerua angolensis, Maytenus senegalensis, Rhus natalensis and Senna singueana were used. The larval exsheathment inhibition assay (LEIA) was applied using H. contortus third stage larvae (L3) and browse plant extract concentrations of 0, 150, 300, 600, 1200µg/ml in phosphate buffered saline (PBS). Data were analysed using the PROC MIXED procedure of SAS. Polyvinylpolypyrrolidone (PVPP) was used to evaluate whether polyphenols were involved in L3 exsheathment inhibition. All browse plant extracts significantly (P≤0.001) inhibited larval exsheathment in a dose dependent manner. The dose required to inhibit 50% of the larvae (EC50) was highest in C. farinosa and lowest in E. racemosa and M. senegalensis. Significant differences (P<0.001) between the control and PVPP treated A. etbaica, C. tomentosa, M. angolensis, R. natalensis and D. cinerea indicates that larval inhibition was largely due to non-phenol compounds. For E. racemosa, M. senegalensis, D. angustifolia and S. singueana, PVPP treatment reversed inhibition activity and in these extracts, inhibition was mostly attributable to tannin and other polyphenols (kaempferol, quercetin and myricetin based glycosides). Overall, the browse plant extracts have anthelmintic property against H. contortus and larval inhibition resulting from the presence of phenolic and non-phenolic compounds.

Hydrolysable tannin-based diet rich in gallotannins has a minimal impact on pig performance but significantly reduces salivary and bulbourethral gland size.

Tannins have long been considered 'anti-nutritional' factors in monogastric nutrition, shown to reduce feed intake and palatability. However, recent studies revealed that compared with condensed tannins, hydrolysable tannins (HT) appear to have far less impact on growth performance, but may be inhibitory to the total activity of caecal bacteria. This in turn could reduce microbial synthesis of skatole and indole in the hindgut of entire male pigs (EM). Thus, the objective of this study was to determine the impact of a group of dietary HT on growth performance, carcass traits and boar taint compounds of group housed EM. For the study, 36 Swiss Large White boars were assigned within litter to three treatment groups. Boars were offered ad libitum one of three finisher diets supplemented with 0 (C), 15 (T15) or 30 g/kg (T30) of HT from day 105 to 165 of age. Growth performance, carcass characteristics, boar taint compounds in the adipose tissue and cytochrome P450 (CYP) isoenzymes CYP2E1, CYP1A2 and CYP2A19 gene expression in the liver was assessed. Compared with C, feed efficiency but not daily gain and daily feed intake was lower (P<0.05) in T15 and T30 boars. Except for the percentage carcass weight loss during cooling, which tended (P<0.10) to be greater in T30 than C and T15, carcass characteristics were not affected by the diets. In line with the numerically lower androstenone level, bulbourethral and salivary glands of T30 boars were lighter (P<0.05) than of T15 with intermediate values for C. Indole level was lower (P<0.05) in the adipose tissue of T30 than C pigs with intermediate levels in T15. Skatole levels tended (P<0.10) to be lower in T30 and C than T15 pigs. Hepatic gene expression of CYP isoenzymes did not differ between-treatment groups, but was negatively correlated (P<0.05) with androstenone (CYP2E1 and CYP1A2), skatole (CYP2E1, CYP2A) and indole (CYP2A) level. In line with the numerically highest androstenone and skatole concentrations, boar taint odour but not flavour was detected by the panellists in loins from T15 compared with loins from C and T30 boars. These results provide evidence that HT affected metabolism of indolic compounds and androstenone and that they affected the development of accessory sex glands. However, the effects were too small to be detected by sensory evaluation.

Chemical Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and Motility of Haemonchus contortus Nematodes.

The use of synthetic drugs against gastrointestinal nematodes of ruminants has led to a situation where resistance to anthelmintics is widespread, and there is an urgent need for alternative solutions for parasite control. One promising approach is to use polyphenol-rich bioactive plants in animal feeds as natural anthelmintics. In the present work, the in vitro activity of a series of 33 hydrolyzable tannins (HTs) and their hydrolysis product, gallic acid, against egg hatching and motility of L1 and L2 stage Haemonchus contortus larvae was studied. The effect of the selected compounds on egg and larval structure was further studied by scanning electron microscopy. The results indicated clear relationships between HT structure and anthelmintic activity. While HT size, overall flexibility, the types and numbers of functional groups, together with the linkage types between monomeric HTs affected the activity differently, the optimal structure was found with pentagalloylglucose.

Evolutionary potential of root chemical defense: genetic correlations with shoot chemistry and plant growth.

Root herbivores can affect plant fitness, and roots often contain the same secondary metabolites that act as defenses in shoots, but the ecology and evolution of root chemical defense have been little investigated. Here, we investigated genetic variance, heritability, and correlations among defensive phenolic compounds in shoot vs. root tissues of common evening primrose, Oenothera biennis. Across 20 genotypes, there were roughly similar concentrations of total phenolics in shoots vs. roots, but the allocation of particular phenolics to shoots vs. roots varied along a continuum of genotype growth rate. Slow-growing genotypes allocated 2-fold more of the potential pro-oxidant oenothein B to shoots than roots, whereas fast-growing genotypes had roughly equivalent above and belowground concentrations. Phenolic concentrations in both roots and shoots were strongly heritable, with mostly positive patterns of genetic covariation. Nonetheless, there was genotype-specific variation in the presence/absence of two major ellagitannins (oenothein A and its precursor oenothein B), indicating two different chemotypes based on alterations in this chemical pathway. Overall, the presence of strong genetic variation in root defenses suggests ample scope for the evolution of these compounds as defenses against root herbivores.

Heritability, covariation and natural selection on 24 traits of common evening primrose (Oenothera biennis) from a field experiment.

This study explored genetic variation and co-variation in multiple functional plant traits. Our goal was to characterize selection, heritabilities and genetic correlations among different types of traits to gain insight into the evolutionary ecology of plant populations and their interactions with insect herbivores. In a field experiment, we detected significant heritable variation for each of 24 traits of Oenothera biennis and extensive genetic covariance among traits. Traits with diverse functions formed several distinct groups that exhibited positive genetic covariation with each other. Genetic variation in life-history traits and secondary chemistry together explained a large proportion of variation in herbivory (r(2) = 0.73). At the same time, selection acted on lifetime biomass, life-history traits and two secondary compounds of O. biennis, explaining over 95% of the variation in relative fitness among genotypes. The combination of genetic covariances and directional selection acting on multiple traits suggests that adaptive evolution of particular traits is constrained, and that correlated evolution of groups of traits will occur, which is expected to drive the evolution of increased herbivore susceptibility. As a whole, our study indicates that an examination of genetic variation and covariation among many different types of traits can provide greater insight into the evolutionary ecology of plant populations and plant-herbivore interactions.

Seasonal variation in the content of hydrolysable tannins in leaves of Betula pubescens.

The contents of 13 hydrolysable tannins in the leaves of white birch (Betula pubescens L.) trees were analysed at twelve stages throughout the growing season. All individual galloylglucoses, from 1-O-galloyl-beta-D-glucopyranose to 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, accumulated in young leaves, while ellagitannins showed significantly variable seasonal trends. The major ellagitannin during the whole growing season was pedunculagin while 2,3-(S)-HHDP-glucopyranose. the end product of the proposed ellagitannin pathway, accumulated in mature leaves. Relationships between the characteristics of seasonal variation in the contents of individual ellagitannins and their chemical structures were used to unravel the biogenesis of ellagitannins in birch leaves. Evidence of degradation of ellagitannins through hydrolysis during leaf growth and development is presented and implications for herbivory are discussed.

Characterization of the polyphenolic composition of purple loosestrife (Lythrum salicaria).

Phenolic compounds of purple loosestrife (Lythrum salicaria L.) were analysed by the use of liquid chromatography-mass spectrometry (LC/MS) equipped with atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI). The presence of vitexin and orientin as well as their isomers, isovitexin and isoorientin, were confirmed using ion trap multiple stage LC/MS3 analysis. Several phenolic acids and tannins were also detected. Ellagitannins, vescalagin and pedunculagin, are reported from the plant for the first time.