Effects of 5,6-dimethylbenzimidazole and cobalt supplementation in high-concentrate diets on rumen fermentation, ruminal microorganisms and the metabolome in sheep

Background In the actual production of high-yielding cattle and fattening sheep, the regular use of high-concentrate diets can improve the growth performance of the animals. Notably, high-concentrate diets can also change the rumen fermentation and microbial community structure of ruminants, leading to problems associated with acidosis in the rumen. The aim of this study was to investigate the effects of 5,6-dimethylbenzimidazole and cobalt supplemented with high-concentrate diets on rumen fermentation, ruminal microorganisms and the metabolome in sheep. Methods Twelve rumen-fistulated Kazakh rams [location: Changji, China; age: 8 months; average initial body weight [BW]: 39.23 ± 2.61 kg] were selected for this study. The sheep were randomly allocated to one of two groups: the control group (CON, n = 6) or the DMB group (n = 6). Sheep in the CON group were fed a basal diet (concentrate supplement to forage ratio 70:30), and sheep in the DMB group were fed a basal diet supplemented with 100 mg/d 5,6-dimethylbenzimidazole (Yuanye Biotechnology Co., Ltd., China; analytical purity ≥ 99%) and 0.5 mg/kg cobalt (Co) (cobalt chloride as supplementary Co, zeolite powder as carrier, Yuanda Zhongzheng Biotechnology Co., Ltd., Shijiazhuang, Hebei, China; Co content ≥ 1%). The experiment lasted for 32 days. Days 1–14 were the adaptation period, and days 15–32 were the test period. During the experiment, feed samples and residual feed were collected, weighed and recorded. Ruminal fluid samples were collected at 0 h before morning feeding and at 1, 3, 5 and 7 h after feeding on days 29–32 of the experiment to measure the ruminal pH. Results The results revealed that (1) the ruminal pH of the DMB group was significantly greater than that of the CON group at 3, 5, and 7 h after feeding ( P  < 0.01). Compared with those in the CON group, the concentrations of NH ₃ -N ( P  < 0.01), propionate ( P  < 0.01), acetate/propionate ( P  < 0.05), and total volatile fatty acids (VFAs) ( P  < 0.05), lactate ( P  < 0.05) and lactate dehydrogenase ( P  < 0.05) were significantly lower in the DMB group. (2) The dominant phyla between the two groups were Firmicutes (46.70%), Bacteroidetes (43.79%), and Proteobacteria (2.66%). The dominant genera were Prevotella (14.78%), Quinell a (7.47%), Succiniclasticum (7.40%), Rikenellaceae_RC9_gut_group (4.44%), NK4A214_group (3.89%), and Ruminococcus (3.67%). (3) Alpha diversity analysis revealed that the Simpson ( P  < 0.05) and Shannon ( P  < 0.05) indices of the DMB group increased significantly. The PCoA results revealed that the two groups of samples were significantly separated on the basis of Bray‒Curtis analysis, with R ² and P values of 0.83 and 0.001, respectively. NMDS analysis revealed that stress < 0.2, indicating that the two groups of samples were well separated. There were 78 OTUs in the two groups, and 52 and 45 OTUs were unique to the CON and DMB groups, respectively. (4) OPLS-DA results revealed that the two groups of samples were clearly separated, with R2X, R2Y and Q2Y values of 0.85, 0.628 and 0.248, respectively, in positive ion mode and 0.536, 0.913 and 0.778, respectively, in negative ion mode, indicating the stability of the model. (5) There were 20 significantly different metabolites between the two groups, including 12 positive ions and 8 negative ions. There were 16 metabolic pathways, including the fatty acid metabolism pathway and the tricarboxylic acid cycle pathway. (6) Correlation analysis revealed a correlation between ruminal microorganisms and metabolites (propionate, L-malic acid and arginine). Conclusions In summary, the supplementation of high-concentrate diets with 5,6-dimethylbenzimidazole and cobalt changed the rumen fermentation parameters and microbial community structure, thereby affecting ruminal metabolites and alleviating acidosis symptoms in the rumen.