Background: Gastrointestinal tract (GIT) microbiomes in ruminants play major roles in host health and thus animal production. However, we lack an integrated understanding of microbial community structure and function as prior studies are predominantly biased towards the rumen. In this study, we used shotgun metagenomics to profile the microbiota of 370 samples that represent 10 GIT regions of seven ruminant species. Results: Our analyses reconstructed a GIT microbial reference catalog with > 154 million nonredundant genes and identified 8745 uncultured candidate species from over 10,000 metagenome-assembled genomes. The integrated gene catalog across the GIT regions demonstrates spatial associations between the microbiome and physiological adaptations, and 8745 newly characterized genomes substantially expand the genomic landscape of ruminant microbiota, particularly those from the lower gut. This substantially expands the previously known set of endogenous microbial diversity and the taxonomic classification rate of the GIT microbiome. These candidate species encode hundreds of enzymes and novel biosynthetic gene clusters that improve our understanding concerning methane production and feed efficiency in ruminants. Overall, this study expands the characterization of the ruminant GIT microbiota at unprecedented spatial resolution and offers clues for improving ruminant livestock production in the future. Conclusions: Having access to a comprehensive gene catalog and collections of microbial genomes provides the ability to perform efficiently genome-based analysis to achieve a detailed classification of GIT microbial composition and function. Our study will bring unprecedented power in future association studies to investigate the impact of the GIT microbiota in ruminant health and production.

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p < 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p < 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p < 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants.

The effect of early feed restriction on metabolic programming and compensatory growth was studied in broiler chickens. A total of 480 female 1-d-old broiler birds (Aconred) were randomly allocated to ad libitum and feed-restricted groups, each of which was replicated 6 times with 40 birds per replicate. Broilers were provided commercial diets. Feed-restricted broilers were deprived of feed for 4 h per day from 1 to 21 d of age. Effects of treatments were determined at 21 and 63 d of age. In feed-restricted birds at 21 d of age, BW, average daily gain and average daily feed intake, breast muscle (P < 0.01), carcass yield (P < 0.05), and abdominal fat (P < 0.05) were decreased. Ether extract content in breast muscle was increased (P < 0.01), whereas CP content was slightly decreased. Triiodothyronine (P < 0.01) and thyroxine (P < 0.05) were decreased in serum. Free fatty acid and very low density lipoprotein were slightly increased in serum, whereas triglyceride and glucose were decreased (P < 0.01). Activities of NADPH-generating enzymes in liver including malic dehydrogenase, isocitrate dehydrogenase, and glucose-6-phosphate remained unchanged in ad libitum birds, whereas hormone-sensitive lipase activity was increased (P < 0.01). In feed-restricted birds at 63 d of age, BW, average daily gain, average daily feed intake, carcass yield, breast muscle yield, and serum triiodothyronine and thyroxine remained as ad libitum birds, whereas abdominal fat yield was increased (P < 0.05). Ether extract content in breast muscle was decreased (P < 0.01), whereas CP content was increased (P < 0.05). Activities of NADPH-generating enzymes were significantly increased, except abdominal malic dehydrogenase and hormone-sensitive lipase activity was decreased (P < 0.01) in liver and abdominal fat. Lipoprotein lipase activity was increased (P < 0.05) in abdominal fat. In summary, feed restriction severely affected growth performance and lipid metabolism in broilers in the early period. Because there was no statistical difference among the final BW, near full compensatory growth was achieved. In addition, early feed restriction might have induced prolonged metabolic programming in chicks and led to adult obesity.

Dissolved hydrogen (dH₂) influences the pathways of VFA production and is a precursor of methane formation in the rumen. Measurements of dH₂ in rumen fluid taken at the same time as measuring other rumen fermentation end products would improve our quantitative understanding of the role of dH₂ as a controller of rumen fermentation. Sample collections though a rumen cannula and using oral stomach tubing were compared for measurements of dissolved gases and fermentation end products in the rumen fluid of 4 ruminally cannulated dairy cows fed a total mixed ration of corn silage and concentrate. Rumen fluid was collected at 0, 2.5, and 6 h after morning feeding through the cannula from cranial dorsal rumen, cranial ventral rumen, central rumen, caudal dorsal rumen, and caudal ventral rumen and in parallel by oral stomach tubing at 2 insertion depths of 180 cm (sampling the central rumen) and 200 cm (sampling the caudal dorsal rumen). The cranial dorsal rumen had the greatest pH and smallest VFA concentration among 5 sites sampled. Samples collected by oral stomach tubing had greater (P < 0.001) rumen pH and less (P < 0.001) dissolved methane (dCH₄) and lower VFA concentration than that collected through rumen cannula. The dH₂ concentrations were positively correlated (r > 0.8) in rumen samples collected by the 2 sampling techniques, with a concordance correlation coefficient larger than 0.8 and scale shift being about 0.1 away from unity. The variations in the measurement of dH₂, dCH₄, pH, and VFA in samples collected by oral stomach tubing are most likely the result of saliva contamination. The time of sampling relative to feeding had significant influence (P < 0.01) on dissolved gases and fermentation end products, with the greatest concentrations of dH₂, dCH₄, and VFA measured 2.5 h after morning feeding. The dH₂ was correlated positively (r > 0.58) with dCH₄ and negatively (r < −0.65) with the estimated net H₂ production relative to the amount of VFA produced. This indicated that greater dH₂ enhanced rumen CH₄ production and also led to fermentation pathways that produce less H₂, such as enhanced propionate and butyrate production. In summary, oral stomach tubing could be a feasible method to measure ruminal dH₂ in intact animals, but caution should be taken to minimize saliva contamination. Measurements made using both techniques yield similar conclusions for the effects of dH₂ on fermentation pathways and CH₄ generation.

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO3-/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (-33.1%) and plasma (-30.6%) as well as methane emissions (-15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = -0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = -0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.