The objective was to investigate the effect of nonprotein nitrogen source, dietary protein supply, and genetic yield index on methane emission, N metabolism, and ruminal fermentation in dairy cows. Forty-eight Danish Holstein dairy cows (24 primiparous cows and 24 multiparous cows) were used in a 6 × 4 incomplete Latin square design with 4 periods of 21-d duration. Cows were fed ad libitum with the following 6 experimental diets: diets with low, medium, or high rumen degradable protein (RDP):rumen undegradable protein (RUP) ratio (manipulated by changing the proportion of corn meal, corn gluten meal, and corn gluten feed) combined with either urea or nitrate (10 g NO₃^-/kg of dry matter) as nonprotein nitrogen source. Samples of ruminal fluid and feces were collected from multiparous cows, and total-tract nutrient digestibility was estimated using TiO₂ as flow marker. Milk samples were collected from all 48 cows. Gas emission (CH₄, CO₂, and H₂) was measured by 4 GreenFeed units. We observed no significant interaction between dietary RDP:RUP ratio and nitrate supplementation, and between nitrate supplementation and genetic yield index on CH₄ emission (production, yield, intensity). As dietary RDP:RUP ratio increased, intake of crude protein, RDP, and neutral detergent fiber and total-tract digestibility of crude protein linearly increased, and RUP intake linearly decreased. Yield of milk, energy-corrected milk, and milk protein and lactose linearly decreased, whereas milk fat and milk urea nitrogen concentrations linearly increased as dietary RDP:RUP ratio increased. The increase in dietary RDP:RUP ratio resulted in a linear increase in the excretion of total purine derivatives and N in urine, but a linear decrease in N efficiency (milk N in % of N intake). Nitrate supplementation reduced dry matter intake (DMI) and increased total-tract organic matter digestibility compared with urea supplementation. Nitrate supplementation resulted in a greater reduction in DMI and daily CH₄ production and a greater increase in daily H₂ production in multiparous cows compared with primiparous cows. Nitrate supplementation also showed a greater reduction in milk protein and lactose yield in multiparous cows than in primiparous cows. Milk protein and lactose concentrations were lower for cows receiving nitrate diets compared with cows receiving urea diets. Nitrate supplementation reduced urinary purine derivatives excretion from the rumen, whereas N efficiency tended to increase. Nitrate supplementation reduced proportion of acetate and propionate in ruminal volatile fatty acids. In conclusion, no interaction was observed between dietary RDP:RUP ratio and nitrate supplementation, and no interaction between nitrate supplementation and genetic yield index on CH₄ emission (production, yield, intensity) was noted. Nitrate supplementation resulted in a greater reduction in DMI and CH₄ production, and a greater increase in H₂ production in multiparous cows than in primiparous cows. As the dietary RDP:RUP ratio increased, CH₄ emission was unaffected and RDP intake increased, but RUP intake and milk yield decreased. Genetic yield index did not affect CH₄ production, yield, or intensity.

The objective of this study was to investigate the effects of heat treatment (toasting) and particle size alterations (grinding; rolling) on nutrient utilization, ruminal fermentation, and supply of metabolizable protein (MP), and to study the interaction between processing conditions of fava beans and forage type. Six Danish Holstein dairy cows fitted with ruminal, duode-nal, and ileal cannulas were used in a 6 x 4 incomplete Latin square design with 4 periods of 21-d duration. Cows were fed ad libitum with 6 experimental diets: diets high in either grass-clover silage or corn silage were combined with ground untoasted, ground toasted, or rolled untoasted fava beans. Samples of ruminal fluid, digesta from duodenum and ileum, and feces were collected, and nutrient digestibility was estimated using Cr2O3 and TiO2 as flow markers. Diets high in corn silage resulted in higher ruminal pH and higher proportion of propionate in ruminal volatile fatty acids compared with diets high in grass-clover silage. Diets high in corn silage resulted in higher apparent total -tract digestibility of crude protein and starch but lower apparent ruminal and total-tract digestibility of neutral detergent fiber compared with diets high in grass-clover silage. Rolling of fava beans decreased the in situ small intestinal disappearance of rumen-undegradable protein corrected for particle losses. Compared with grinding, rolling of fava beans reduced apparent ruminal digest-ibility of starch, true ruminal digestibility of organic matter, crude protein, and AA, and small intestinal digestibility of AA and starch. Grinding of fava beans increased apparent ruminal digestibility of neutral de-tergent fiber and reduced the proportion of propionate in ruminal volatile fatty acids compared with rolling of fava beans. In addition, rolling of fava beans had no ef- fect on MP supply. Toasting of fava beans had no effect on in vivo nutrient digestibility except for an interac-tion with forage source on apparent ruminal dry matter and organic matter digestibility. Toasting of fava beans did not affect small intestinal digestion of individual and total AA, and therefore failed to increase MP sup-ply. In conclusion, neither replacing grass-clover silage with corn silage, nor toasting nor rolling of fava beans had an effect on of MP.

Enteric CH₄ produced from dairy cows contributes to the emission of greenhouse gases from anthropogenic sources. Recent studies have shown that the selection of lower CH₄-emitting cows is possible, but doing so would be simpler if performance measures already recorded on farm could be used, instead of measuring gas emissions from individual cows. These performance measures could be used for selection of low emitting cows. The aim of this analysis was to quantify how much of the between-cow variation in CH₄ production can be explained by variation in performance measures. A dataset with 3 experiments and a total of 149 lactating dairy cows with repeated measures was used to estimate the between-cow variation (the variation between cow estimates) for performance and gas measures from GreenFeed (C-Lock, Rapid City, SD). The cow estimates were obtained with a linear mixed model with the diet within period effect as a fixed effect and the cow within experiment as a random effect. The cow estimates for CH₄ production were first regressed on the performance and gas measures individually, and then performance and CO₂ production measures were grouped in 3 subsets for principal component analysis and principal component regression. The variables that explained most of the between-cow variation in CH₄ production were DMI (R² = 0.44), among the performance measures, and CO₂ production (R² = 0.61), among gas measures. Grouping the measures increased the R² to 0.53 when only performance measures were used, and to 0.66 when CO₂ production was added to the significant performance measures. We found the marginal improvement to be insufficient to justify the use of grouped measures rather than an individual measure because the latter simplifies the model and avoids over-fitting. Investigation of other measures that can be explored to increase explanatory power of between-cow variation in CH₄ production is briefly discussed. Finally, the use of residual CH₄ as a measure for CH₄ efficiency could be considered by using either DMI or CO₂ production as the sole predicting variables.

Summary: Nitrate has been extensively studied and is confirmed to have a significant methane mitigation effect in ruminants. However, it is proven that nitrate supplementation can stimulate the emission of nitrous oxide. In this study, we investigated the effect of nitrate supplementation on ruminal fermentation and related emissions of CH₄, CO₂, and H₂ on cow level, CH₄, CO₂, and N₂O on rumen level, and prediction of N₂O on cow level from rumen volume proportion. The results suggested that nitrate supplementation reduced CH₄ production and yield, with the largest effect on production just after feeding, and increased H₂ production and yield compared with urea supplementation. However, nitrate supplementation also increased N₂O production compared with urea supplementation.

The objective of this study was to evaluate the effect of fecal bacterial community and metabolomics in goats when consuming a lower-protein diet with different energy levels. Eight healthy Leizhou goats, with 11 ± 0.78 kg of body weight, were selected and housed individually in cages. The animals were randomly allocated to a lower-protein diet that varied with four metabolites energy levels (7.01, 8.33, 9.66, and 10.98 MJ/kg DM) in a replicated 4 × 4 Latin square design. Notably, energy-dependent microbial restructuring was observed at both phylum and genus levels. At the phylum level, the relative abundances of Firmicutes and Spirochaetote increased linearly, whereas the Bacteroidota and Patescibacteria decreased linearly with increasing dietary energy levels (p < 0.05). The relative abundances of Verrucomicrobiota increased quadratically, whereas others decreased quadratically with increasing dietary energy levels (p < 0.05). At the genus level, a total of 316 bacteria were identified in the 32 fecal samples. The relative abundances of Christensenellaceae_R-7_group, unclassified_f__Lachnospiraceae, Ruminococcus, norank_o__Clostridia_UCG-014, Treponema, [Eubacterium]_siraeum_group, and [Eubacterium]_ruminantium_group increased linearly, whereas the Oscillospiraceae_UCG-005, norank_f__[Eubacterium]_coprostanoligenes_group, Prevotellaceae_UCG-004, unclassified_c__Clostridia, norank_f__Ruminococcaceae, unclassified_f__ Oscillospiraceae, and others decreased linearly with an increasing dietary energy levels (p < 0.05). In addition, the metabolomic analysis of feces showed that there are many differential metabolites in goats when consuming a lower-protein diet with different energy levels; for example, lipid metabolism and amino acid metabolic pathways were increased in MLE, MHE, and HE groups compared to the LE group. In conclusion, this study provides further information regarding the effects on fecal bacterial community composition and metabolites in goats when consuming a lower-protein diet with different energy levels.

This study investigated the effects of Bacillus subtilis (B. subtilis) supplementation on microbiota and metabolites in the feces of Leizhou goats. Eight Leizhou goats were used in a replicated 4 × 4 Latin square design according to their gender (nanny goats and billy goats) with a 4 × 2 factorial arrangement of treatments that included four B. subtilis additive doses (control [0 g/d; NC, BC], low [2.5 g/d, NL, BL], medium [5 g/d, NM, BM], and high [7.5 g/d, NH, BH]) and 28 d periods (n = 4 per group), each consisting of 27 d adaption and 1 d sample collection. After collecting 32 fecal samples, 16S rRNA gene sequencing and LC-MS were performed to analyze microbial composition and metabolites, respectively. At the genus level, the relative abundance of Rikenellaceae_RC9_gut_group was significantly higher (p < 0.05) in the NM group than in the NC group. The relative abundance of Treponema sp. was significantly lower (p < 0.05) in the NM group than in the NC group. In billy goats, the relative abundances of UCG-005 and Rikenellaceae_RC9_gut_group were significantly higher (p < 0.05) in the BH group than in the BC group. The relative abundance of Treponema sp. was significantly lower (p < 0.05) in the BL, BM, and BH groups than in the BC group. Furthermore, metabolomic analysis revealed that B. subtilis significantly altered the concentrations of glucose metabolism modulators (1-deoxynojirimycin, 1-DNJ) and certain bioactive peptides. Many amino acid metabolic pathways were also enriched. Correlation analysis demonstrated close connections between differential metabolites and the top 10 bacterial genera in fecal samples. These results provide new insights into the impact of B. subtilis on the microbial community and metabolic profile of the feces of Leizhou goats. In this experiment, the appropriate doses of B. subtilis for nanny goats and billy goats were 5 g/d and 7.5 g/d, respectively, but the optimal doses still need to be verified based on performance-based feeding tests in the next study.