Jean-Pierre Jouany

Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil1

This experiment studied the effect of 3 forms of presentation of linseed fatty acids (FA) on methane output using the sulfur hexafluoride tracer technique, total tract digestibility, and performance of dairy cows. Eight multiparous lactating Holstein cows (initial milk yield 23.4 +/- 2.2 kg/d) were assigned to 4 dietary treatments in a replicated 4 x 4 Latin square design: a control diet (C) consisting of corn silage (59%), grass hay (6%), and concentrate (35%) and the same diet with crude linseed (CLS), extruded linseed (ELS), or linseed oil (LSO) at the same FA level (5.7% of dietary DM). Each experimental period lasted 4 wk. All the forms of linseed FA significantly decreased daily CH(4) emissions (P < 0.001) but to different extents (-12% with CLS, -38% with ELS, -64% with LSO) compared with C. The same ranking among diets was observed for CH(4) output expressed as a percentage of energy intake (P < 0.001) or in grams per kilogram of OM intake (P < 0.001). Methane production per unit of digested NDF was similar for C, CLS, and ELS but was less for LSO (138 vs. 68 g/kg of digested NDF, respectively; P < 0.001). Measured as grams per kilogram of milk or fat-corrected milk yield, methane emission was similar for C and CLS and was less for ELS and LSO (P < 0.001), LSO being less than ELS (P < 0.01). Total tract NDF digestibility was significantly less (P < 0.001) for the 3 supplemented diets than for C (-6.8% on average; P < 0.001). Starch digestibility was similar for all diets (mean 93.5%). Compared with C, DMI was not modified with CLS (P > 0.05) but was decreased with ELS and LSO (-3.1 and -5.1 kg/d, respectively; P < 0.001). Milk yield and milk fat content were similar for LSO and ELS but less than for C and CLS (19.9 vs. 22.3 kg/d and 33.8 vs. 43.2 g/kg, on average, respectively; P < 0.01 and P < 0.001). Linseed FA offer a promising dietary means to depress ruminal methanogenesis. The form of presentation of linseed FA greatly influences methane output from dairy cows. The negative effects of linseed on milk production will need to be overcome if it is to be considered as a methane mitigation agent. Optimal conditions for the utilization of linseed FA in ruminant diets need to be determined before recommending its use for the dairy industry.

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum, a co‐culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium, was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum. Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.

Role of rumen protozoa in nitrogen digestion in sheep given two isonitrogenous diets.

1. The effect of protozoa on digestion in the rumen was studied using either defaunated or faunated sheep. 2. Six wethers, each fitted with rumen and simple duodenal cannulas, were given two isonitrogenous diets containing either lucerne (Medicago sativa) hay (diet L) or sodium hydroxide-treated wheat straw (diet S). The diets were given in eight equal portions per day at 3-h intervals. The mean intake of dry matter, 53 g/kg body-weight0.75 per d, was similar for the two diets and each diet had a similar digestible organic matter content. Diet L promoted a large protozoal population and was rich in nitrogen sources of low rumen-degradability, while diet S supported a smaller protozoal population and was rich in rumen-degradable N. 3. Digesta flow at the duodenum was estimated by means of a dual-marker technique using chromium-mordanted lucerne hay and polyethylene glycol as markers. The microbial flow at the duodenum was estimated using diaminopimelic acid (DAPA), nucleic-acid purine bases (PB) and 35S incorporation simultaneously. The different microbial markers were compared in the defaunated sheep. Protozoal N contribution was estimated in faunated sheep. 4. Defaunated sheep had lower rumen ammonia concentrations and molar proportions of butyric acid than faunated sheep, but they had higher molar proportions of propionic acid. 5. Rumen organic matter digestion was reduced by defaunation, but this decrease was compensated for by increased intestinal digestion. 6. There was a net increase of N flow (approximately 10 g/d) between mouth and duodenum in defaunated sheep. This was explained by increases in both microbial and dietary N flows from the rumen compared with faunated sheep. 7. The influence of protozoa on solid- and liquid-phase retention times in the rumen is discussed, as well as the protozoal contribution to microbial N flow in the duodenum of faunated sheep.

Methane production associated with rumen‐ciliated protozoa and its effect on protozoan activity

K. USHIDA AND J.P JOUANY. 1996. Methane production by methanogenic bacteria associated with ciliated protozoa in the rumen, and its effect on the metabolic activity of the protozoa, were measured in vitro. Apparent daily methane emission per protozoan cell ranged from a trace amount to 2 nmol. Enhanced substrate disappearance accompained methanogensis.

Changes in methane emission and rumen fermentation parameters induced by refaunation in sheep

Elimination of protozoa or defaunation is known to reduce methane emission by ruminants. However, the long-term efficacy of this practice is not well documented. In this work methane and other end products of fermentation were monitored in five adult, rumen-cannulated wethers that had been kept defaunated for two years (Def1) and successively refaunated (Fau, 12 weeks) and defaunated (Def2) during a 6-month-long experiment. Methane was measured for periods of 4 days at about 4-weekly intervals using the SF6 tracer technique, and rumen content samples were taken regularly throughout the experiment. Refaunation was performed by single administration of a mixed protozoal inoculum (103 cells). A noticeable protozoal population was observed at 12 days after inoculation, which was followed by an exponential increase that peaked at 12 ± 3 × 105 cells/mL rumen fluid at 4 weeks and decreased thereafter to 8 ± 4 × 105 cells/mL at the end of the 12-week Fau period. Production of methane, which was 33 ± 8 L/animal.day in Def1, increased (P   0.05). Even though protozoal numbers were comparable to conventional animals 20 days after inoculation, the increase in methane production was observed later, at 8 weeks, suggesting that protozoa are not the only microbial factor favouring methanogenesis and that the ecosystem needs a relatively long adaptation time following protozoal introduction to optimise this function. The presence of protozoa did not affect total volatile fatty acids and acetate concentration in the rumen but increased butyrate, while decreasing propionate concentration (Fau v. Def1, Def2, P < 0.05). These results show that the decrease in methane emissions induced by defaunation in sheep was stable for a period of up to 2 years. In the absence of protozoa, methane decreased by ~20% in both short- and long-term defaunated animals.

Effect of live yeast culture supplementation on hindgut microbial communities and their polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber or high-starch diet

Four cecum and right ventral colon-fistulated horses were assigned in a 4 x 4 Latin square design and fed a high-fiber (HF) or a high-starch (HS) diet with or without 10 g of Saccharomyces cerevisiae (SC; CBS 493.94) containing 4.5 x 10(9) cfu/g. The HF and HS diets consisted of pelleted feeds and long wheat straw (18.0 and 3.5 g of DM.kg(-1) of BW.d(-1), respectively) given in 2 equal meals to provide an NDF:starch ratio of 3.5 and 1.0, respectively. After a 21-d adaptation period intestinal contents were collected 4 h after the morning meal on d 23 and 25 to determine bacterial and SC concentrations. Polysaccharidase activities (CMCase, xylanase, amylase) and activities of glycoside hydrolases (alpha-l-arabinosidase, beta-d-cellobiosidase, beta-d-glucosidase, beta-d-xylosidase) were determined in liquid-associated bacteria (LAB) and solid-adherent bacteria (SAB) isolated from both compartments. Lactobacilli were increased in the cecum (P = 0.012) and colon (P = 0.086) when starch intake increased, whereas total anaerobes, cellulolytics, and streptococci did not change in either compartment. In yeast-supplemented horses, SC concentrations were greater in cecum (4.4 x 10(6) cfu/mL) than in right-ventral colon (5.6 x 10(4) cfu/mL) and did no change with diet. Concentrations of lactobacilli and lactic-acid utilizers were greater (P = 0.099 and 0.067, respectively) in the cecum but remained similar in the colon of SC-supplemented horses. The CMCase activities of SAB were not affected by diet. Colonic xylanase activities of SAB were reduced (P = 0.046) by starch addition, but no change was seen in the cecum. All SAB glucoside hydrolase activities in the cecum and colon, except beta-d-xylosidase in the cecum, were decreased when starch intake was increased. The LAB CMCase (P = 0.049 in the colon) and xylanase (P = 0.021 in the cecum; P < 0.001 in the colon) activities decreased with starch intake. No effect of starch on LAB or SAB amylase activity was observed. Addition of SC improved SAB CMCase in the cecum (P = 0.019) and colon (P = 0.037) as well as beta-d-cellobiosidase (P = 0.002) and beta-d-glucosidase (P = 0.041) in the cecum. Only xylanase in the cecum (P = 0.015) and beta-d-xylosidase in the cecum (P = 0.028) were improved with SC, whereas colonic LAB alpha-amylase activity was significantly decreased (P = 0.046). Most enzymes involved in plant cell wall digestion were increased after SC addition. This fact may contribute to explain a better digestion of fiber that has been previously reported in SC-supplemented horses.

Characterization of XYN10B, a modular xylanase from the ruminal protozoan Polyplastron multivesiculatum, with a family 22 carbohydrate-binding module that binds to cellulose

A new xylanase gene, xyn10B, was isolated from the ruminal protozoan Polyplastron multivesiculatum and the gene product was characterized. XYN10B is the first protozoan family 10 glycoside hydrolase characterized so far and is a modular enzyme comprising a family 22 carbohydrate-binding module (CBM) preceding the catalytic domain. The CBM22 was shown to be a true CBM. It showed high affinity for soluble arabinoxylan and is the first example of a CBM22 that binds strongly to celluloses of various crystallinities. The enzymic properties of XYN10B were also analysed. Its optimal temperature and pH for activity were 39 degrees C and 7.0 respectively; these values being close to those of the ruminal ecosystem. The phylogenetic relationships between the XYN10B CBM22 or catalytic domain and related sequences from ruminal and non-ruminal bacteria and eukaryotes are reported. The xyn10B gene is shown to lack introns.

Effect of defaunation on protein and fibre digestion in sheep fed on ammonia-treated straw-based diets with or without maize.

Using a defaunating method which preserved bacteria and fungi in the rumen, the effect of protozoa on protein and fibre digestion was studied in six adult wethers in relation to the nature of the diet. Sheep were given daily, 42 g dry matter (DM)/kg metabolic body-weight (W0.75), one of two isonitrogenous diets: one contained ammonia-treated wheat straw as the only energy source (diet S) and the other was supplemented with maize grain pellets (diet SM). Mean daily intakes (g/d) of nitrogen, neutral-detergent fibre and acid-detergent fibre were respectively 22, 573 and 373 for diet S and 23, 450 and 334 for diet SM. Elimination of protozoa increased duodenal non-ammonia-nitrogen flow. This result was mainly due to an increase in microbial protein flow and, to a lesser extent, to a higher dietary protein flow. Defaunation markedly increased the efficiency of microbial protein synthesis. Maize-grain supplementation had a net positive effect on this variable in defaunated sheep, but not in faunated sheep. Cell-wall carbohydrates were less well digested in the defaunated rumen, and the negative effect of defaunation was greatest with the diet SM. Intestinal fibre digestion increased in the defaunated sheep especially in those fed on diet SM, but not enough to compensate for the decrease in rumen digestion.

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

Aims:  To assess the effect of protozoal species on rumen fermentation characteristics in vitro.

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid‐soluble 14C from 14C leucine‐labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non‐ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoal population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non‐methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell‐free rumen fluid and thzis effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage‐induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.