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Condensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence degradation and rumen function. The aim of the study was to explore the rumen degradation of tropical tannins-rich plants and elucidate their relationship with the disappearance of condensed tannins fractions. The effects on fermentation parameters and microbial communities colonising plant particles in the rumen was also assessed. We used in situ and in vitro approaches to study four leguminous: leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, Acacia nilotica pods and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. Plants were analysed to quantify levels of hydrolysable tannins, free condensed tannins, protein-bound condensed tannins and fibre-bound condensed tannins. Rumen dry matter, nitrogen and fibre (NDF) degradability, rumen disappearance of tannin fractions and microbial colonisation of plants was assessed in situ. The methane-mitigation potential of tannin-rich plants compared to a tropical forage without tannins was assessed in vitro. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was more variable with Gliricidia sepium showing the highest proportion (93%), Manihot esculenta and Musa spp. showed intermediate values of disappearance, and no disappearance was observed from Calliandra calothyrsus leaves. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. Dry matter and nitrogen degradation were similar for all plants except Calliandra calothyrsus and Musa spp. that showed lower values. Calliandra and Acacia nilotica had also a lower NDF degradation. Methane production was also lower for these plants and for Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important.

The present study was carried out to determine the effect of Acacia nilotica, a tropical plant rich in hydrolyzable tannins (HT), on rumen fermentation and methane (CH4) production in vitro. We used leaves and pods from A. nilotica alone and combined. The combination of HT from A. nilotica leaves and pods and condensed tannins (CT) from Calliandra calothyrsus and Leucaena leucocephala were also evaluated to assess potential differences in biological activity between HT and CT. Four series of 24-h incubations were performed using rumen contents of 4 sheep fed a tropical grass (natural grassland based on Dichanthium spp.). A first experiment tested different levels of replacement of this tropical forage (control [CTL] without tannins) by A. nilotica leaves or pods: 0:100, 25:75, 50:50, 75:25 and 100:0. A second experiment tested the mixture of A. nilotica leaves and pods in different proportions: 100:0, 75:25, 50:50, 25:75, and 0:100. A third experiment tested the 50:50 combination of A. nilotica leaves or pods with C. calothyrsus and L. leucocephala. Acacia nilotica pods and leaves had a high content of HT (350 and 178 g/kg DM, respectively), whereas C. calothyrsus and L. leucocephala had a high content of CT (361 and 180 g/kg DM, respectively). The inclusion of HT from A. nilotica leaves and pods decreased CH4 production dose-dependently (P < 0.01). Total replacement of the CTL by A. nilotica decreased CH4 production by 64 and 55% with leaves and pods, respectively. Pods were richer in HT than leaves, but their antimethanogenic effect did not differ (P > 0.05). Although A. nilotica leaves and pods inhibited fermentation, as indicated by the lower gas production and VFA production (P < 0.01), this effect was less pronounced than for CH4. Volatile fatty acid production decreased by 12% in leaves and by 30% in pods when compared with the CTL alone. Positive associative effect was reported for VFA, when HT-rich sources and CT-rich sources were mixed. Combining the 2 sources of HT did not show associative effects on fermentation or CH4 production (P > 0.05). Hydrolyzable tannin-rich sources were more effective in suppressing methanogenesis than CT-rich sources. Our results show that HT-rich A. nilotica leaves and pods have the potential to reduce ruminal CH4 production.

The current study was carried out to determine the effect of secondary metabolites on in vitro rumen microbial fermentation and their potential to inhibit methane (CH4) production involved in the global warming and greenhouse-gases. The obtained results showed that the addition of Yucca schidigera and Quillaja saponaria reduced significantly CH4 production (P

International audience; Microbial fermentation of carbohydrates in the rumen is largely responsible for the emis- sion of methane by ruminants. Ruminants fed tropical forages usually produce more enteric methane than ruminants fed temperate forages. The relative influence of forage type, breed and temperate vs tropical environment on rumen microbial populations is not known. This experiment aimed to separate these effects. We designed two parallel experiments in sheep in two sites: temperate (France) and tropical (French West Indies), using in each site two breeds, Texel (temperate origin), and Blackbelly (tropical origin) fed the same temperate forages (C3 carbon fixation, permanent grasslands of high and low quality) and tropical for- ages (C4 carbon fixation, permanent grasslands of high and low quality). We determined diet digestibility, ruminal end-products of fermentation and microbial groups: total pro- tozoa, methanogens and bacteria, and selected fibrolytic bacteria. Dry matter digestibility coefficient was higher in tropical site (612 vs 580 g/kg on average, P = 0.004) but no differ- ence was observed between C3 and C4 forages. There was no effect of site on total VFA concentration, but the acetate:propionate ratio was higher for the tropical site (4.30 vs 3.93 on average, P = 0.007). The acetate:propionate ratio was also affected by forage type with higher values for C3 than C4 forage (4.24 vs 3.99 on average, P = 0.03). Concentration of total rumen bacteria and methanogens was determined by qPCR targeting, respectively, the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. For both groups, the number of gene copies per gram of DM rumen content was higher in the trop- ical site ( P < 0.001). For cellulolytic bacteria, higher number of rrs copies per gram of DM of rumen content were detected for Fibrobacter succinogenes in the temperate site ( P < 0.001), whereas no differences were observed for Ruminococcus flavefaciens or Ruminococcus albus numbers between sites, breeds and forage type. Protozoa numbers determined by counting did not vary between sites, forages or breeds, but a site × forage interaction was observed ( P = 0.01): there were more protozoa and R. albus in tropical sites for tropical forages. Our results suggest that rumen microbiota was mainly influenced by environment (temper- ate vs tropical) and that forage type (C3 vs C4) and breed had minor effects. However, an interaction between environment and forage type was observed for some variables

International audience; An in vivo trial was conducted in sheep to investigate the effect of three tropical tannin-rich plants (TRP) on methane emission, intake and digestibility. The TRP used were leaves of Glyricidia sepium, Leucaena leucocephala and Manihot esculenta that contained, respectively, 39, 75 and 92 g condensed tannins/kg DM. Methane was determined with the sulphur hexafluoride tracer technique. Eight rumen-cannulated sheep of two breeds (four Texel, four Blackbelly) were used in two 4 × 4 Latin square designs. Four experimental diets were tested. They consisted in a tropical natural grassland hay based on Dichanthium spp. fed alone (C) or in association with G. sepium (G), L. leucocephala (L) or M. esculenta (M) given as pellets at 44% of the daily ration. Daily organic matter intake was higher in TRP diets (686, 984, 1054 and 1186 g/day for C, G, L and M respectively; p < 0.05) while apparent organic matter total tract digestibility was not affected (69.9%, 62.8%, 65.3% and 64.7% for C, G, L and M respectively; p > 0.05). Methane emission was 47.1, 44.9, 33.3 and 33.5 g/kg digestible organic matter intake for C, G, L and M, respectively, and was significantly lower (p < 0.05) for L and M than for G and C. Our results confirm the potential of some TRP to reduce methane production. The strong decrease in methane and the increase in intake with TRPs may be due to their presentation as pellets.

The objective of this work was to study nutritional strategies for decreasing methane production by ruminants fed tropical diets, combining in vitro and in vivo methods. The in vitro approach was used to evaluate the dose effect of condensed tannins (CT) contained in leaves of Gliricidia sepium, Leucaena leucocephala, and Manihot esculenta (39, 75, and 92 g CT/kg DM, respectively) on methane production and ruminal fermentation characteristics. Tannin-rich plants (TRP) were incubated for 24 h alone or mixed with a natural grassland hay based on Dichanthium spp. (control plant), so that proportions of TRP were 0, 0.25, 0.5, 0.75, and 1.0. Methane production, VFA concentration, and fermented OM decreased with increased proportions of TRP. Numerical differences on methane production and VFA concentration among TRP sources may be due to differences in their CT content, with greater effects for L. leucocephala and M. esculenta than for G. sepium. Independently of TRP, the response to increasing doses of CT was linear for methane production but quadratic for VFA concentration. As a result, at moderate tannin dose, methane decreased more than VFA. The in vivo trial was conducted to investigate the effect of TRP on different ruminal microbial populations. To this end, 8 rumen-cannulated sheep from 2 breeds (Texel and Blackbelly) were used in two 4 × 4 Latin square designs. Diets were fed ad libitum and were composed of the same feeds used for the in vitro trial: control plant alone or combined with pellets made from TRP leaves at 44% of the diet DM. Compared to TRP, concentration of Ruminococcus flavefaciens was greater for the control diet and concentration of Ruminococcus albus was least for the control diet. The methanogen population was greater for Texel than for Blackbelly. By contrast, TRP-containing diets did not affect protozoa or Fibrobacter succinogenes numbers. Hence, TRP showed potential for mitigating methane production by ruminants. These findings suggest that TRP fed as pellets could be used to decrease methane production.

International audience; Emptying the rumen of animals fitted with rumen cannulas is a regularly used experimental technique. This study in sheep aimed to evaluate whether total rumen emptying (TRE) results in disturbances in digestive parameters such as rumen pH, fermentation and microbial ecosystem, and whether it affects animal welfare. Eight adult wethers from two breeds offered hay and water ad libitum were rumen-emptied manually. Ruminal parameters were assessed 2 days before and 7 days after TRE. Welfare was assessed by measuring behavioural and cardiac responses to TRE involving human handling. Behavioural and cardiac measurements were performed (i) 2 h before, (ii) during and (iii) 2 h after the rumen emptying–filling session on wethers exposed to the same TRE operator. Results showed that TRE did not affect most of the digestive parameters analysed (ruminal pH, bacteria, methanogens) but did modify Dasytricha population and volatile fatty acid concentrations. Likewise, rumen emptying did not alter most behavioural and cardiac parameters, and we found no TRE-related change in fear reactions to the operator.

International audience; Enteric methane (CH4) produced by domestic ruminants represents approximately 15% of the global emissions of this potent greenhouse gas. For reducing rumen CH4 emission various compounds have been tested as feed additives. Among these compounds, tannins are considered a promising group of natural additives. A meta-analysis by Jayanegara et al. (2012) showed that condensed and hydrolysable tannins might reduce CH4 production. However, it is still unclear (1) whether tannin supplementation reduces rumen CH4 in every situation and (2) to which extent this is associated with adverse effects on digestibility and their potential toxicity to some rumen micro-organisms (Goel et al., 2005). In this experiment we investigated the effect of tanniniferous tropical plants on enteric CH4 production and on numbers of methanogens, protozoa, and total and main cellulolytic bacteria