Effect of dietary fiber on the methanogen community in the hindgut of Lantang gilts.

The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (Methanobrevibacter ruminantium, Methanobrevibacter wolinii, Methanosphaera stadtmanae and Methanobrevibacter smithii), with about 89% of the methanogens belonging to the genus Methanobrevibacter. The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher (P0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (P<0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up in vitro fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the in vitro fermentation confirmed that the predominant methanogens belonged to the genus of Methanobrevibacter, and about 23% methanogens was found to be distantly related (90%) to Thermogymnomonas acidicola. In vitro fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of Methanobrevibacter species was positively correlated with CH4 production in both in vivo (P<0.01, r=0.737) and in vitro trials (P<0.05, r=0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH4 emission from gilts fed HFD.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

Board-invited review: Rumen microbiology: leading the way in microbial ecology.

Robert Hungate, considered the father of rumen microbiology, was the first to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal flora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were first described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques (such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were first made in the rumen, which have not been recognized in a broader sense.

Rumen fermentation and microbial population in lactating dairy cows receiving diets containing oilseeds rich in C-18 fatty acids.

Sixteen Holstein rumen-cannulated primiparous milking dairy cows were fed a control diet (CN) based on maize silage and soyabean meal during a 4-week period before the start of a 21-d experiment with oilseeds containing high concentration of linoleic acid (Linola™) or linolenic acid (NuLin™). Thereafter, four cows received ad libitum one of each of four dietary treatments comprising of CN, Linola (LN), NuLin (NL) and LN/NL (50/50 % combination). Each LN, NL and LN/NL treatment contained 6 % oil of DM. Rumen digesta samples were collected on days 6, 11, 16 and 21 and milk samples on days 13, 15 and 17. There were no effects (P>0.05) of the oilseeds on pH and concentrations of NH3-N and total volatile fatty acids, while the acetate:propionate ratio was decreased (P< 0.05). The oilseeds also decreased (P< 0.05) protozoa and increased (P< 0.1) total cellulolytic bacteria in rumen fluid, especially when containing high dietary linoleic acid (P< 0.05). The milk protein concentration was increased (P< 0.1) by the dietary linoleic acid, which produced most beneficial results. It was concluded that supplements of linoleic acid in diets of ruminants might contribute to better digestion of dietary fibre and increased quality of milk.

Diversity of gut methanogens in herbivorous animals.

The digestion of plant biomass by symbiotic microbial communities in the gut of herbivore hosts also results in the production of methane, a greenhouse gas that is released into the environment where it contributes to climate change. As methane is exclusively produced by methanogenic archaea, various research groups have devoted their efforts to investigate the population structure of symbiotic methanogens in the gut of herbivores. In this review, we summarized and compared currently available results from 16S rRNA gene clone library studies, which cover a broad range of hosts from ruminant livestock species to wild ruminants, camelids, marsupials, primates, birds and reptiles. Although gut methanogens are very diverse, they tend to be limited to specific phylogenetic groups. Overall, methanogens related to species of the genus Methanobrevibacter are the most highly represented archaea in the gut of herbivores. However, under certain conditions, archaea from more phylogenetically distant groups are the most prevalent, such as methanogens belonging to either the genus Methanosphaera, the order Methanomicrobiales or the Thermoplasmatales-Affiliated Lineage C Comparisons not only highlight the strong influence of host species and diet in the determination of the population structure of symbiotic methanogens, but also reveal other complex relationships, such as wide differences between breeds, as well as unexpected similarities between unrelated species. These observations strongly support the need for high throughput sequencing and metagenomic studies to gain further insight.

Modeling the distribution of ciliate protozoa in the reticulo-rumen using linear programming.

The flow of ciliate protozoa from the reticulo-rumen is significantly less than expected given the total density of rumen protozoa present. To maintain their numbers in the reticulo-rumen, protozoa can be selectively retained through association with feed particles and the rumen wall. Few mathematical models have been designed to model rumen protozoa in both the free-living and attached phases, and the data used in the models were acquired using classical techniques. It has therefore become necessary to provide an updated model that more accurately represents these microorganisms and incorporates the recent literature on distribution, sequestration, and generation times. This paper represents a novel approach to synthesizing experimental data on rumen microorganisms in a quantitative and structured manner. The development of a linear programming model of rumen protozoa in an approximate steady state will be described and applied to data from healthy ruminants consuming commonly fed diets. In the model, protozoa associated with the liquid phase and protozoa attached to particulate matter or sequestered against the rumen wall are distinguished. Growth, passage, death, and transfer of protozoa between both pools are represented. The results from the model application using the contrasting diets of increased forage content versus increased starch content indicate that the majority of rumen protozoa, 63 to 90%, are found in the attached phase, either attached to feed particles or sequestered on the rumen wall. A slightly greater proportion of protozoa are found in the attached phase in animals fed a hay diet compared with a starch diet. This suggests that experimental protocols that only sample protozoa from the rumen fluid could be significantly underestimating the size of the protozoal population of the rumen. Further data are required on the distribution of ciliate protozoa in the rumen of healthy animals to improve model development, but the model described herein does indicate that the attached protozoal population is a significant component of the total rumen protozoal community.

Supplementation with whole cottonseed causes long-term reduction of methane emissions from lactating dairy cows offered a forage and cereal grain diet.

The objective of our work was to supplement a forage and cereal diet of lactating dairy cows with whole cottonseed (WCS) for 12 wk and to determine whether the expected reduction in CH(4) would persist. A secondary objective was to determine the effect of supplementing the diet with WCS on milk yield and rumen function over the 12-wk feeding period. Fifty lactating cows were randomly allocated to 1 of 2 diets (control or WCS). The 2 separate groups were each offered, on average, 4.2 kg of DM/cow per day of alfalfa hay (a.m.) and 6.6 kg of DM/cow per day of ryegrass silage (p.m.) on the ground in bare paddocks each day for 12 wk. Cows in each group were also individually offered dietary supplements for 12 wk in a feed trough at milking times of 5.4 kg of DM/cow per day of cracked wheat grain and 0.5 kg of DM/cow per day of cottonseed meal (control) or 2.8 kg of DM/cow per day of cracked wheat grain and 2.61 kg of DM/cow per day of WCS. The 2 diets were formulated to be similar in their concentrations of CP and ME, but the WCS diet was designed to have a higher fat concentration. Samples of rumen fluid were collected per fistula from the rumen approximately 4 h after grain feeding in the morning. Samples were taken from 8 cows (4 cows/diet) on 2 consecutive days in wk 2 of the covariate and wk 3, 6, 10, and 12 of treatment and analyzed for volatile fatty acids, ammonia-N, methanogens, and protozoa. The reduction in CH(4) emissions (g/d) because of WCS supplementation increased from 13% in wk 3 to 23% in wk 12 of treatment. Similarly, the reduction in CH(4) emissions (g/kg of DMI) increased from 5.1% in wk 3 to 14.5% in wk 12 of treatment. It was calculated that the average reduction in CH(4) emissions over the 12-wk period was 2.9% less CH(4) per 1% added fat, increasing from 1.5% in wk 3 to 4.4% less CH(4) in wk 12. There was no effect of WCS supplementation on rumen ammonia-N, rumen volatile fatty acids, rumen methanogens, and rumen protozoa. On average over the 12-wk period, supplementation with WCS decreased the yield of milk (10%), fat (11%), protein (14%), lactose (11%), and fat plus protein (12%) and BW gain (31%). The WCS supplementation had no effect on milk fat concentration but resulted in a decrease in concentration of protein (5%) and lactose (11%). The major finding from this study is that addition of WCS to the diet of lactating dairy cows resulted in a persistent reduction in CH(4) emissions (g of CH(4)/kg of DMI) over a 12-wk period and that these reductions in CH(4) are consistent with previous work that has studied the addition of oilseeds to ruminant diets.

Long-term monensin supplementation does not significantly affect the quantity or diversity of methanogens in the rumen of the lactating dairy cow.

A long-term monensin supplementation trial involving lactating dairy cattle was conducted to determine the effect of monensin on the quantity and diversity of rumen methanogens in vivo. Fourteen cows were paired on the basis of days in milk and parity and allocated to one of two treatment groups, receiving (i) a control total mixed ration (TMR) or (ii) a TMR with 24 mg of monensin premix/kg of diet dry matter. Rumen fluid was obtained using an ororuminal probe on day -15 (baseline) and days 20, 90, and 180 following treatment. Throughout the 6-month experiment, the quantity of rumen methanogens was not significantly affected by monensin supplementation, as measured by quantitative real-time PCR. The diversity of the rumen methanogen population was investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone gene libraries. DGGE analysis at each sampling point indicated that the molecular diversity of rumen methanogens from monensin-treated cattle was not significantly different from that of rumen methanogens from control cattle. 16S rRNA gene libraries were constructed from samples obtained from the rumen fluids of five cows, with a total of 166 clones examined. Eleven unique 16S rRNA sequences or phylotypes were identified, five of which have not been recognized previously. The majority of clones (98.2%) belonged to the genus Methanobrevibacter, with all libraries containing Methanobrevibacter strains M6 and SM9 and a novel phylotype, UG3322.2. Overall, long-term monensin supplementation was not found to significantly alter the quantity or diversity of methanogens in the rumens of lactating dairy cattle in the present study.

In vitro methane emission and acetate:propionate ratio are decreased when artificial stimulation of the rumen wall is combined with increasing grain diets in sheep.

The interaction of retention time in the rumen and concentrate diet on methane production in vitro and acetate:propionate ratio was examined. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into 4 groups. The sheep had a 5-wk acclimatization period on an oaten chaff diet, followed by two 3-wk diet phases. Two of the 4 groups were maintained on the oaten chaff diet for the duration of the experiment, with pot scrubbers added to the rumen of 1 of the 2 groups. The remaining 2 groups were offered a low-grain diet (35% grain) in the first diet phase followed by a high-grain diet (70% grain) in the second diet phase. Pot scrubbers were also added to the rumen of 1 of these 2 groups of grain-fed sheep. Pot scrubbers in combination with a low-grain diet decreased the amount of methane produced in vitro from 4.25 to 3.71 mmol/mL of digesta when compared with oaten chaff-fed sheep without pot scrubbers (P < 0.05). The acetate:propionate ratio was 1.6 in sheep fed a high-grain diet with pot scrubbers compared with 2.4 in sheep fed a high-grain diet without pot scrubbers in their rumen (P < 0.05). At high levels of grain, when employing a multivariate statistical analysis including all data, sheep given the combined treatment of grain and pot scrubbers were different from all other sheep groups in this experiment (P < 0.05). Furthermore, sheep fed a high-grain diet were different from sheep receiving the oaten chaff diets with and without pot scrubbers (P < 0.01 and P < 0.05, respectively). In conclusion, pot scrubbers combined with grain alter the rumen fermentation, and introducing pot scrubbers into the rumens of livestock consuming low levels of grain may be a way to lower methane emissions.

Reducing methane emissions in sheep by immunization against rumen methanogens.

This work was conducted to determine if methane emissions from sheep immunized with an anti-methanogen vaccine were significantly lower than methane emissions from non-immunized sheep, to test the effectiveness of two different vaccine formulations (VF) on methane abatement, and to compare methane emissions measured using a closed-circuit respiration chamber and the sulphur-hexafluoride (SF6) tracer technique. Thirty mature wether sheep were randomly allocated to three treatment groups (n = 10). One group received an immunization of adjuvant only on days 0 and 153 (control), a second group received an immunization with a 3-methanogen mix on days 0 and 153 (VF3 + 3), and a third group received an immunization of a 7-methanogen mix on day 0 followed by a 3-methanogen mix on day 153 (VF7 + 3). Four weeks post-secondary immunization, there was a significant 7.7% reduction in methane production per kg dry matter intake in the VF7 + 3 group compared to the controls (P = 0.051). However, methane emissions from sheep immunized with VF7 + 3 were not significantly different when compared to the sheep in the control group (P = 0.883). The average IgG and IgA antibody titres in both plasma and saliva of the VF3 + 3 immunized sheep were four to nine times higher than those immunized with VF7 + 3 (P< 0.001) at both 3 and 6 weeks post-secondary immunization. Data also revealed that SF6 methane estimates were consistently higher than the respiration chamber estimates and that there was no significant correlation between the SF6 methane estimates and the respiration chamber methane estimates (R2 = 0.11).