Bacillus subtilis and Macleaya cordata extract regulate the rumen microbiota associated with enteric methane emission in dairy cows.

BACKGROUND: Ruminant livestock production is a considerable source of enteric methane (CH4) emissions. In a previous study, we found that dietary inclusions of Bacillus subtilis (BS) and Macleaya cordata extract (MCE) increased dry matter intake and milk production, while reduced enteric CH4 emission in dairy cows. The objective of this study was to further elucidate the impact of feeding BS and MCE on rumen methanogenesis in dairy cows using rumen metagenomics techniques. RESULTS: Sixty dairy cows were blocked in 20 groups of 3 cows accordingly to their live weight, milk yield, and days in milk, and within each group, the 3 cows were randomly allocated to 1 of 3 treatments: control diet (CON), control diet plus BS (BS), and control diet plus MCE (MCE). After 75 days of feeding experimental diets, 12 cows were selected from each treatment for collection of rumen samples for the metagenomic sequencing. Results showed that BS decreased ruminal acetate and butyrate, while increased propionate concentrations, resulting in decreased acetate:propionate ratio. The metagenomics analysis revealed that MCE reduced relative abundances of Methanobrevibacter wolinii, Methanobrevibacter sp. AbM4, Candidatus Methanomassiliicoccus intestinalis, Methanobrevibacter cuticularis, Methanomicrobium mobile, Methanobacterium formicicum, and Methanobacterium congolense. Both BS and MCE reduced relative abundances of Methanosphaera sp. WGK6 and Methanosphaera stadtmanae. The co-occurrence network analysis of rumen bacteria and archaea revealed that dietary treatments influenced microbial interaction patterns, with BS and MCE cows having more and stronger associations than CON cows. The random forest and heatmaps analysis demonstrated that the Halopenitus persicus was positively correlated with fat- and protein-corrected milk yield; Clostridium sp. CAG 269, Clostridium sp. 27 14, Haloarcula rubripromontorii, and Methanobrevibacter curvatus were negatively correlated with rumen acetate and butyrate concentrations, and acetate:propionate ratio, whereas Selenomonas rumiantium was positively correlated with those variables. CONCLUSIONS: The present results provided new information for mitigation of enteric methane emissions of dairy cows by feeding BS and MCE to influence rumen microbial activities. This fundamental knowledge is essential for developing enteric CH4 reduction strategies to mitigate climate change and reduce dietary energy waste. Video Abstract.

Requirements of metabolizable protein by Dorper × thin-tailed Han crossbred ewe lambs.

The requirement of net protein (NP) and metabolizable protein (MP) by Dorper crossbred ewe lambs grown from 35 to 50 kg of body weight (BW) was assessed by comparative slaughter experiment. Thirty-five ewe lambs (33.5 ± 0.6 kg BW) of F1 crosses of Dorper × thin-tailed Han sheep were used: 7 lambs were slaughtered as reference animals at the start of the trial, and the remaining 28 lambs were randomly divided into 4 groups of 7 lambs each. Three of the 4 groups were fed a pelleted mixed diet (concentrate/roughage = 44:56, dry matter basis) for ad libitum intake or 65% or 45% of ad libitum intake, and they were all slaughtered when the lambs that were fed ad libitum reached 50 kg BW. The lambs from the fourth group were also fed ad libitum and slaughtered at 43 kg BW as the intermediate group. The intake of MP by the animals of these 4 groups was estimated, and their total body protein and protein retention were measured. The daily requirements of NP and MP for maintenance were 1.52 and 3.98 g/kg BW0.75 , respectively, with a partial efficiency of MP utilization for maintenance of 0.38. The MP requirement for growth ranged from 77.4 to 124.5 g/day for average daily gains from 100 to 250 g BW, and the partial efficiency of MP utilization for growth was 0.66. The Dorper crossbred ewe lambs required more MP for both maintenance and growth in comparison with the recommendations of the US nutritional system.

Early Feeding Regime of Waste Milk, Milk, and Milk Replacer for Calves Has Different Effects on Rumen Fermentation and the Bacterial Community.

We investigated the effects of different types of early feeding on rumen fermentation parameters and the bacterial community in calves. Fifty-four Holstein calves were assigned to three treatments and fed whole milk (M), pasteurized waste milk (WM), or milk replacer (MR). Male calves were slaughtered at the age of two months to measure the stomach masses. The female calves were followed for six months to determine the body weight, blood indices, rumen fermentation, and ruminal bacterial community. At the age of two months, the average daily gain was lower, but the concentration of total volatile fatty acids was greater in the MR group. Starter intake and stomach mass were lower, but the isovalerate molar proportion was greater in the WM group. The blood indices and ruminal bacterial community of the WM group differed from those of the other groups. At the age of six months, the ruminal propionate molar proportion was lower, but the ruminal pH and acetate/propionate ratio were greater in the MR group. In conclusion, calves fed WM had different rumen fermentation and bacterial community during the weaning period, whereas feeding MR produced a long-lasting effect on the rumen environment.

Saponin-Induced Shifts in the Rumen Microbiome and Metabolome of Young Cattle.

The aim of this study was to explore the effects of saponins on the rumen microbiota and the ruminal metabolome. Alfalfa hay (AH) and soybean hulls (SH) were used as fiber sources for the control diets. The AH and SH diets were supplemented with tea saponins resulting in two additional diets named AHS and SHS, respectively. These 4 diets were fed to 24 young male Holstein cattle (n = 6 per diet). After 28 days of feeding, the rumen fluid from these cattle was collected using an oral stomach tube. Illumina MiSeq sequencing and ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) were used to investigate the changes in the ruminal microbes and their metabolites. The relative abundance of Prevotellaceae_YAB2003 increased, while Ruminococcaceae_NK4A214 and Lachnospiraceae_NK3A20 decreased in SHS and AHS compared to SH and AHS, respectively. Feeding SHS resulted in higher ruminal concentrations of squalene, lanosterol, 3-phenylpropanoic acid, and citrulline compared to SH. The different microbial genes predicted by Tax4Fun were involved in amino sugar and nucleotide sugar metabolism. The pathways of arginine and proline metabolism, purine metabolism, and pyrimidine metabolism were enriched by different metabolites. Moreover, in the SH group, a positive correlation was observed between Prevotella_1 (Bacteroidetes), Prevotellaceae_YAB2003 (Bacteroidetes), and Christensenellaceae_R.7 (Firmicutes), and the metabolites, including citrulline, lanosterol, and squalene. The increased abundances of Prevotella_1, Ruminococcaceae_UCG.002, and Prevotellaceae_YAB2003 might result in increased fiber digestion and nutrient utilization but nutrient digestion was not measured in the current study. In summary, saponins have the ability to modulate the ruminal microbial community and ruminal metabolites and thus affect the rumen environment. However, the response seems to be dependent on the composition of the basal diet. This study provides a comprehensive overview of the microbial and biochemical changes in the rumen of cattle fed saponins.

Effect of weaning age on growth performance, feed efficiency, nutrient digestibility and blood-biochemical parameters in Droughtmaster crossbred beef calves.

OBJECTIVE: The objective of this study was to determine the effect of weaning age on intake, performance, nutrition metabolism and serum parameters of beef calves. METHODS: Sixty Droughtmaster crossbred calves were assigned to 5 groups with 12 calves in each group. The calves in control group remained with the dams till the 22-week age, while the calves weaned at 28 d (4 wk), 42 d (6 wk), 56 d (8 wk), and 70 d (10 wk) of age were sent to group 4 wk, group 6 wk, group 8 wk, and group 10 wk, respectively, and then were fed on milk replacer till the 22-week age. Feed intake and body weight and size were record and blood metabolites were measured. And 24 calves of them (6 in each group) were picked randomly for digestion and metabolism trail. Feed, feces and urine sample were taken and measured. RESULTS: Dry matter intake of calves in group 4 wk was significantly lower than those in the remaining groups from wk 17 to 22 (p<0.05). Feed efficiency of the calves was higher in groups 4 wk and 6 wk than those in groups 8 wk and 10 wk from 11 to 13 wk (p<0.05), and calves had higher feed efficiency in group 4 wk, group 6 wk, and group 8 wk than those in group 10 wk from wk 14 to wk 22. Calves in group 4 wk and 6 wk had lower body weight than group 8 wk and group 10 wk and control group at 10-week age (p<0.05) and 13-week age (p<0.05), and calves in group 6 wk had no significant difference in body weight with control group, group 8 wk and 10 wk (p>0.05) but was higher than that of group 4 wk (p<0.05). Calves in group 6 wk had higher final body weight and total gain than group 4 wk, but no difference of total gain with that of groups 8 wk, 10 wk, and control group. And weaning calves at 6-week age brought higher feed efficiency and average daily gain from wk 14 to wk 22, and higher dry matter and organic matter digestibility at 21 wk. CONCLUSION: It is concluded that the weaning of calves at 6 weeks of age gave positive results.

Dietary supplementation with mulberry leaf flavonoids inhibits methanogenesis in sheep.

The effects of flavonoids on methanogenesis and microbial flora in Dorper × thin-tailed Han crossbred ewes were evaluated in two experiments. To investigate the effects of flavonoids on nutrient digestibility and nitrogen balance, 18 ewes (60.0 ± 1.73 kg body weight (BW)) were allotted to two dietary treatments in experiment one, a control diet and the control diet supplemented with flavonoids (2 g/head/day). In experiment two, the effects of supplementary flavonoids on ruminal fermentation and microbial flora were investigated using quantitative polymerase chain reaction with six ewes (67.2 ± 0.79 kg BW) with ruminal cannula assigned to the identical dietary treatments used in experiment one. Supplementary flavonoids improved the apparent digestibility of nitrogen (N, P < 0.001) and neutral detergent fibre (NDF, P = 0.024) and decreased daily CH4 output (P < 0.001). The ruminal pH (P = 0.638) and ammonia (P = 0.690) were not affected by supplementary flavonoids, whereas the total volatile fatty acid (VFA) content increased (P = 0.037). Supplementary flavonoids decreased ruminal populations of protozoans (P = 0.002) and methanogens (P < 0.001) and increased the populations of Fibrobacter succinogenes (P = 0.016). In conclusion, flavonoids improved the digestibility of organic matter and reduced CH4 output by inhibiting the populations of microbes involved in methanogenesis.

Effect of Dietary Concentrate:forage Ratios and Undegraded Dietary Protein on Nitrogen Balance and Urinary Excretion of Purine Derivatives in Dorper×thin-tailed Han Crossbred Lambs.

THIS STUDY AIMED TO INVESTIGATE DIETARY CONCENTRATE: forage ratios (C:F) and undegraded dietary protein (UDP) on nitrogen balance and urinary excretion of purine derivatives (PD) in lambs. Four Dorper×thin-tailed Han crossbred castrated lambs with 62.3±1.9 kg body weight at 10 months of age were randomly assigned to four dietary treatments in a 2×2 factorial arrangement of two levels of C:F (40:60 and 60:40) and two levels of UDP (35% and 50% of CP), according to a complete 4×4 Latin-square design. Each experimental period lasted for 19 d. After a 7-d adaptation period, lambs were moved into individual metabolism crates for 12 d including 7 d of adaption and 5 d of metabolism trial. During the metabolism trial, total urine was collected for 24 h and spot urine samples were also collected at different times. Urinary PD was measured using a colorimetric method and creatinine was measured using an automated analyzer. Intake of dry matter (DM) (p<0.01) and organic matter (OM) (p<0.01) increased as the level of UDP decreased. Fecal N was not affected by dietary treatment (p>0.05) while urinary N increased as the level of UDP decreased (p<0.05), but decreased as dietary C:F increased (p<0.05). Nitrogen retention increased as dietary C:F increased (p<0.05). As dietary C:F increased, urinary excretion of PD increased (p<0.05), but was not affected by dietary UDP (p>0.05) or interaction between dietary treatments (p>0.05). Daily excretion of creatinine was not affected by dietary treatments (p<0.05), with an average value of 0.334±0.005 mmol/kg BW(0.75). A linear correlation was found between total PD excretion and PDC index (R(2) = 0.93). Concentrations of creatinine and PDC index in spot urine were unaffected by sampling time (p>0.05) and a good correlation was found between the PDC index (average value of three times) of spot urine and daily excretion of PD (R(2) = 0.88). These results suggest that for animals fed ad libitum, the PDC index in spot urine is effective to predict daily excretion of PD. In order to improve the accuracy of the spot sampling technique, an appropriate lag phase between the time of feeding and sampling should be determined so that the sampling time can coincide with the peak concentration of PD in the urine.