The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

The objective of this study was to evaluate the effects of exogenous non-starch polysaccharidases (a mixture of cellulase, xylanase, β-glucanase and mannanase) on the growth performance and nutrient digestibility, rumen fermentation, and rumen microflora of sheep. The animal trial was conducted using 36 5-month-old female fattening hybrid sheep (Duolang♂ × Hu♀) who were randomly assigned into four groups comprising nine sheep per treatment: CON, T1, T2, and T3, with 0, 0.1, 0.3, and 0.5% NSPases/kg DM of TMR, respectively. This complex enzyme product was screened for optimal ratios based on previous in vitro tests and responded positively to the in vitro fermentation of the TMR. When treated with NSPases, there was a non-linear effect of average daily gain and feed conversion rate, with the greatest improvement observed in the T2 group. There were no significant differences (p > 0.05) in nutrient intake or apparent digestibility among the NSPase-supplemented groups. In addition, T2 group had a significantly higher acetate to propionate ratio and pH (p < 0.05) than the other groups, and NH₃-N and microbial protein concentrations showed a quadratic curve. The results revealed that both immunoglobulins and serum hormones increased linearly with addition (p < 0.05). As the T2 group showed the best growth performance, the CON and T2 groups were subjected to rumen metagenomic analysis. The results showed higher abundance of bacteria and lower abundance of Viruses in the rumen microbiota of the T2 group compared to the CON group. In addition, Uroviricota and Proteobacteria abundance was significantly lower in the T2 group than in the CON group at the phylum level (p < 0.05). These results suggest that the supplementation of high-concentrate rations with NSPases enhance immunity, reduces virus abundance in the rumen, improves rumen health, and promotes rumen fermentation. Our findings provide novel insights for improving growth performance and alleviating inflammatory responses arising from high concentrate feeding patterns in ruminants. However, the biological mechanisms cannot be elucidated by exploring the composition of rumen microbe alone, and further studies are required.

Weaning is one of the most stressful periods in yak growth. However, the impact of weaning on microbial diversity, structure, and potential function of yak feces is not clear. In this study, 12 Xinjiang yaks aged 3, 4, 5, and 6 months old were selected to collect fresh feces before and after weaning. Through 16S rRNA and ITS high‐throughput sequencing, the dynamic distribution and potential function of yak fecal, bacterial, and fungal communities in each month were revealed. The study found that the richness of fungi had a significant impact on weaning. At the phylum level, Firmicutes, Bacteroidetes, Ascomycota, and Basidiomycota, and at the genus level, 5-7N15, Oscillospira, Roseburia, Dorea, Preussia, Neoascochyta, Naganishia, and Sporormiella were enriched in yak feces of different months old. The abundance and proportion of bacteria Firmicutes, Bacteroidetes, 5-7N15, and fungi Mucoromyceta changed significantly before and after weaning. With the increase of months, Verrucomicrobia and Akkermansia have shown a downward trend. Through the prediction and analysis of fecal microbial function, it was found that at the level of primary pathways, weaning has a significant impact on cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. At the level of secondary metabolic pathways, weaning has a significant impact on cell motility, signal transduction, folding, sorting and degradation, translation, amino acid metabolism, glycan biosynthesis and metabolism, metabolism of terpenoids and polyketides, and xenobiotics biodegradation and metabolism. In addition, by analyzing the differences in functional pathways and microbial composition between sample groups of different months, it was found that the differences in functional pathways were related to the abundance differences of some microorganisms. In general, the changes in the composition and structure of yak fecal microflora may reflect the adaptability of the intestinal microbiota.

This study aimed to explore the protective effects of raffinose (Raf) against inflammatory bowel disease in mice with colitis. Mice were administered 100, 200, or 400 mg/kg Raf for 21 d, followed by drinking-water containing 3% dextran sulfate sodium salt (DSS) for 3 d. Thereafter, the phenotype, pathological lesions in the colon, cytokines levels, and gut microbiota were evaluated. Treatment with Raf reduced the severity of the pathological changes in the colon, mitigating the reduction in colon length. Following Raf intervention, serum levels of inflammatory cytokines (IL-2, IL-6, IL-1β, and TNF-α) tended to return to normal. These results suggest that the anti-inflammatory effects of Raf are associated with a reduction in TLR4–MyD88–NF-κB pathway expression in mouse colonic tissues. Analysis of gut microbiota abundance and its correlation with colitis parameters revealed that DSS-induced dysbiosis was partially mitigated by Raf. In conclusion, Raf exerts a protective effect in colitis by modulating the gut microbiota and TLR4-MyD88-NF-κB pathway.

We evaluated the effects of cottonseed meal protein hydrolysate (CPH) on the intestinal microbiota of yellow-feather broilers. We randomly divided 240 chicks into four groups with six replicates: basal diet with 0% (CON), 1% (LCPH), 3% (MCPH), or 5% (HCPH) CPH. The test lasted 63 days and included days 1–21, 22–42, and 43–63 phases. The ACE, Chao1, and Shannon indices in the MCPH and HCPH groups of 42-day-old broilers were higher than those in the CON group (p < 0.05), indicating that the cecum microbial diversity and richness were higher in these groups. Firmicutes and Bacteroidetes were the dominant phyla; however, the main genera varied during the different periods. The abundance of Lactobacillus in CPH treatment groups of 21-day-old broilers was high (p < 0.05); in the 42-day-old broilers, the abundances of Barnesiella, Clostridia_vadinBB60_group, and Parasutterella in the LCPH group, Desulfovibrio, Lactobacillus, Clostridia_vadinBB60_group, and Butyricicoccus in the MCPH group, and Megamonas and Streptococcus in the HCPH group increased; in the 63-day-old broilers, the abundance of Clostridia_UCG-014 and Synergistes in the LCPH and HCPH group, respectively, increased (p < 0.05), and that of Alistipes in the LCPH and MCPH groups decreased (p < 0.05). And changes in the abundance of probiotics were beneficial to improve the intestinal morphology and growth performance. In addition, the LCPH treatment increased the complexity of the microbial network, while the MCPH treatment had the same effect in 42-day-old broilers. Thus, CPH increased the relative abundance of beneficial intestinal microbiota and enhanced the richness and diversity of the bacterial microbiota in broilers aged <42 days; this effect was weakened after 42 days.

Microbial degradation techniques are often considered an environmentally friendly and cost-effective strategy for reducing gossypol toxicity. However, the mechanism by which Candida tropicalis degrades gossypol remains unclear. In the current study, we aimed to establish the mechanisms of biodegradation and adaptation mechanisms by C. tropicalis ZD-3. The toxicological evaluation results revealed that ZD-3 adapts to gossypol primarily by activating the antioxidant defense system to alleviate the oxidative stress response induced by gossypol. Transcriptomic analyses further suggested that ZD-3 protects against gossypol toxicity via cell wall remodeling. The intracellular enzyme CTRG_04744 gene was significantly up-regulated under gossypol stress, and then expressed in Pichia pastoris. The purified AKR_Z1 degraded 92 % of gossypol within 48 h. In addition, the aldehyde group of gossypol was effectively eliminated to achieve the desired detoxification. Collectively, these results provide theoretical guidance for the continued development of bio-efficient strategies capable of degrading gossypol.