In this experiment, B. subtilis was added to pelletized straw-based total mixed ration, and the rumen microbial diversity of house-fed Duhan hybrid sheep was compared. Ten 3-month-old weaned Duhan hybrid lambs were separated into two groups and fattened for 80 days using a single-factor trial design. During the fattening period, the control and the experiment groups were fed with the same ration, except that the experiment group was supplemented with B. subtilis. The results showed that the addition of B. subtilis could significantly increase the daily weight gain, total weight gain, rumen microbial abundance, and rumen microbial diversity of the Duhan lamb. Among them, the proportion of microbial flora such as Bacteroidetes was significantly increased, producing more acetate, iso-butyrate, and butyrate, obtaining higher energy efficiency.

The glucose metabolism homeostasis in the follicular fluid microenvironment plays an important role in follicular maturation and ovulation, and excessively high or low glucose concentrations have adverse effects on the differentiation of follicular granulosa cells (GCs). However, a limited number of microRNAs (miRNA) have been reported to be involved in glucose-stimulated GCs differentiation. In this study, we characterized the miRNA expression profiles of sheep ovarian GCs cultured in high-glucose and optimal glucose concentrations and focused on a differentially expressed miRNA: miR-17-5p, which may be involved in regulating high-glucose-induced GC apoptosis by targeting KPNA2. We found that overexpression of miR-17-5p significantly promoted GCs proliferation and inhibited cell apoptosis, while downregulated the mRNA and protein expression of apoptosis-related makers (Bax, Caspase-3, Caspase-9, and Bcl-2). In contrast to the classical mechanism of miRNA silencing target gene expression, miR-17-5p overexpression significantly upregulated the expression of target gene KPNA2. A dual luciferase reporter gene assay verified the targeted binding relationship between miR-17-5p and KPNA2 promoter. Meanwhile, overexpression of KPNA2 further promoted the downregulation of apoptosis-related genes driven by miR-17-5p mimics. Knockdown of KPNA2 blocked the inhibitory effect of miR-17-5p mimics on the expression of apoptosis-related genes. Our results demonstrated that miR-17-5p activated the KPNA2 promoter region and upregulated KPNA2 expression, thereby inhibiting GCs apoptosis under high glucose.

Cold stimulation can impair the immune and antioxidant functions of animals, which further leads to a decline in animal production performance. This study aimed to investigate the effect of the aqueous extract of Caragana korshinskii (CK) on the antioxidant and immunological properties of sheep under oxidative stress induced by cold stimulation. In this study, a total of 54 healthy 2-month-old sheep were randomly divided into six groups, namely the control group and experimental groups I–V. The control group sheep were fed the basal diets, while the sheep in experimental groups I–V had their diets supplemented with 4, 6, 8, 10, and 12 g kg⁻¹ of CK aqueous extract, respectively; the sheep from all groups were housed in semi-closed enclosures for 65 d (the pre-trial period was 15 d, and the experimental period was 50 d) in a cold environment (−20 to −10 °C). The results showed that the final body weight and average daily gain (ADG) of sheep in groups I, II, and III were significantly increased compared with the control group (p<0.05), but there was no significant difference in average daily feed intake (ADFI) (p>0.05). Total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were decreased (p<0.05); malondialdehyde (MDA) was increased (p<0.05); and the tumour necrosis factor-α (TNF-α), interleukin-2 (IL-2), and interleukin-6 (IL-6) were increased (p<0.05), whereas interleukin-10 (IL-10), immunoglobulin G (IgG), and immunoglobulin M (IgM) were decreased (p<0.05) in the blood of the control sheep after 50 d of cold stimulation. In contrast, T-AOC, GSH-Px, and SOD increased (p<0.05); MDA decreased (p<0.05); and TNF-α, IL-2, and IL-6 decreased (p<0.05), whereas IL-10, IgM, and IgG increased (p<0.05) in the group with the addition of CK aqueous extract. Moreover, the CK aqueous extract decreased MDA (p<0.05) and increased T-AOC, GSH-Px, and SOD in sheep liver (p<0.05) and decreased TNF-α, IL-2, and IL-6 (p<0.05) and increased IL-10, IgM, and IgG in the thymus and spleen (p<0.05). These results suggest that the addition of CK aqueous extract can promote sheep growth and improve oxidative stress, inflammation, and immune response to chronic cold stimulation in sheep.

The low production efficiency of tannase and the insufficient utilization of high-tannin feed resources form the research background and research significance of this study. In this experiment, the tannase sequence TanLpl from Lactiplantibacillus plantarum ATCC14917T (obtained from a microbial culture collection) was selected. These sequences were respectively integrated into the expression systems of Bacillus subtilis 168 (BS168) and Bacillus subtilis WB600 (WB600) through plasmids TanLpl-p43NMK and TanLpl-pHT43. This successfully constructed three tannase-producing strains: TanLpl-p43NMK-Bacillus subtilis 168 (BS168(p43NMK)), TanLpl-pHT43-Bacillus subtilis 168 (BS168(pHT43)), and TanLpl-pHT43-Bacillus subtilis WB600 (WB600(pHT43)). An evaluation of the recombinant strains’ growth characteristics, expression stability, and enzymatic properties revealed that all three strains reached the stationary phase after 18 h of growth, with no significant differences in growth rate compared to the parental strains. At the 10th generation of subculture, the plasmid loss rate of BS168(p43NMK) was significantly higher than that of BS168(pHT43) or WB600(pHT43) (p < 0.05). The optimal temperature for tannase activity in all three recombinant strains was 30 °C, with an optimal pH value of 5.0. Under these conditions, the tannase activities were 68.81 U/mL, 397.36 U/mL, and 461.12 U/mL, respectively. The recombinant strain WB600(pHT43) exhibited superior expression stability and enzyme production capability compared to the other two strains. The research on the heterologous expression of tannase and its application in feed utilization has important theoretical and practical significance: it enriches the technical system for the heterologous expression of functional enzymes in Bacillus subtilis, provides new ideas for the efficient production of industrial enzymes, and promotes the development of bio-manufacturing technology.

Caragana korshinskii is an underutilized non-grain feed resource. This study investigated the optimal dietary inclusion level of fermented Caragana korshinskii for Mongolian lambs by assessing growth performance, antioxidant status, meat quality, fatty acid and amino acid profiles, and rumen microbiota. Thirty-six lambs were fed diets containing 0%, 10%, 15%, or 20% fermented Caragana korshinskii replacing Leymus chinensis hay for 105 days after a 10 d adaptation period. The 10% group exhibited higher final body weight, average daily gain, feed intake, and carcass weight (p < 0.05). All supplemented groups showed elevated serum T-AOC and reduced MDA (p < 0.05); furthermore, 10% supplementation increased CAT, and 10% and 15% supplementation increased SOD (p < 0.05). Meat quality improved in the 10% and 15% groups, with lower water loss, shear force, drip loss, L*, and b* (p < 0.05; p < 0.01), and higher a* (p < 0.05). Muscle nutrients remained unchanged. Amino acid alterations included increased Glu (all groups), Thr, Ile (15%, 20%), Leu (20%), and variable changes in Asp. Fatty acid analysis revealed higher C18:0, PUFA, C18:2n6c, C18:3n3 in 10% (p < 0.05; p < 0.01), but lower PUFA and C18:3n3 in the 15% and 20% groups (p < 0.05). Rumen 16S sequencing revealed higher abundances of Thermodesulfobacteriota, Methanobacteriota, Methanobrevibacter (10%, 20%), Xylanibacter, Succiniclasticum (15%, 20%), and lower Rikenellaceae_RC9_gut_group (20%) (p < 0.05; p < 0.01). Correlations linked rumen microbes to meat-related fatty acids and amino acids. In conclusion, supplementation with 10% fermented Caragana korshinskii optimally enhanced lamb growth, health, and meat quality through modulation of the rumen microbiota.