Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.

Garlic, an important economic crop, provides nutrient-rich straw. When appropriately balanced with silage corn stalks, it is a high-quality forage resource. However, studies on the impact of garlic straw with silage corn stalks on Hu sheep’s digestive metabolism and rumen microbiota are scarce. In this study, different addition ratios of garlic straw and silage corn stalks were utilized for in vitro experiments. We designed six experimental groups (CON, G0, G20, G40, G60, G80, and G100) based on varying ratios of garlic straw to silage corn stalks. Rumen microbiota was analyzed through 16S rRNA sequencing. Nutrient composition analysis indicated that garlic straw’s relative feeding value (RFV) closely resembled that of silage corn stalks. After 24 h of fermentation, dry matter digestibility and in vitro gas production significantly increased, reaching peak values at a 60% addition ratio. Furthermore, volatile fatty acids (VFAs) such as acetic, propionic, and butyric acid exhibited elevated contents, with the highest yields observed at 60% inclusion. At the genus level, Prevotella, Rikenellaceae RC9 gut group, and Succiniclasticum were identified as the dominant bacterial groups. The gas production test showed a significant decrease in the G80 group compared to others. Microbial analysis revealed a higher abundance of Prevotella in G80 compared to G20, offering valuable insights for reducing greenhouse gas emissions from ruminant animals. Finally, this study predicted the impact of garlic straw with silage corn stalks’ addition on Hu sheep’s metabolic pathways and biological functions of the rumen microbiota. This research highlights the potential for effectively utilizing garlic straw as a feed resource for Hu sheep and proposes a rational proportion for combining garlic straw with silage corn stalks.

Sheep birth and weaning weights indicate their growth and survival. Thus, identifying molecular genetic markers for early body weight is important in sheep breeding. Pleomorphic adenoma gene 1 (PLAG1) is important for regulating birth weight and body length in mammals; however, its relationship with sheep body weight remains unknown. Here, the 3′-untranslated region (3′-UTR) of the Hu sheep PLAG1 gene was cloned, single nucleotide polymorphisms (SNPs) were screened, genotype–early body weight relationships were analyzed, and the possible molecular mechanism was explored. PLAG1 3′-UTR sequences with five forms of base sequences plus poly(A) tails were detected in Hu sheep and the g.8795C>T mutation was identified. Luciferase reporter assay indicated that the g.8795C>T mutation influenced PLAG1 post-transcriptional activity. miRBase prediction showed that the g.8795C>T mutation was located in the miR-139 seed sequence binding region, and miR-139 overexpression significantly decreased both PLAG1-CC and PLAG1-TT activities. Moreover, the luciferase activity of PLAG1-CC was significantly lower than that of the PLAG1-TT, but miR-139 inhibition substantially increased both PLAG1-CC and PLAG1-TT luciferase activities, suggesting that PLAG1 is the target gene of miR-139. Thus, the g.8795C>T mutation upregulates PLAG1 expression by weakening its binding with miR-139, promoting PLAG1 expression, and increasing Hu sheep birth and weaning weights.

Pretreating feed via fermentation to enhance its nutritional value is effective for young ruminants. This study optimized fermented feed using a microbial consortium of Saccharomyces cerevisiae , Bacillus licheniformis , and Enterococcus faecalis , and explored its mechanisms on growth performance and ruminal function in goat kids. The Bacillus licheniformis is the most critical determinant of the nutritional value of FF. Optimized FF had higher levels of crude protein, calcium, phosphorus, and limiting amino acids (AA). The FF contained viable microbiota with lower diversity but greater evenness. The microbiota was enriched in AA and carbohydrate metabolism by enhancing the abundance of Leuconostoc lactis and Lactococcus lactis . A 40-day feeding trial was then conducted with 20-day-old goat kids. Compared to conventional feed, FF increased feed digestibility, body weight and carcass weight. Mechanistically, FF enhanced the interactions among rumen microbes and increased the gene abundance related to carbohydrate and AA metabolism. The FF then upregulated expression of mitochondrial respiratory chain-related genes in the rumen epithelium, thereby improving epithelial energy metabolism. Overall, this study shows that fermented feed optimized by microbial consortium improves goat kid growth and carcass quality through the fermented feed–rumen microbiota axis, offering a sustainable approach for ruminant production.