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.

Sirtuin 4 (SIRT4), a member of the SIRT family, has been reported to be a key factor involved in antioxidant defense in mitochondria. This study aimed to explore the potential molecular mechanism via which SIRT4 regulates heat stress-induced oxidative stress and lactoprotein synthesis in bovine mammary epithelial cells (BMECs). Our results showed that SIRT4 was significantly decreased in heat stressed mammary tissue. Depletion of SIRT4 in BMECs induced the generation of ROS, which, as exhibited by the decreased activity of antioxidant enzymes, changed mitochondrial morphology through mediating protein and mRNA levels related to mitochondrial fission and fusion. Moreover, we found that depletion of SIRT4 or stress conditions inhibited the expression of milk proteins, as well as lipid and glucose synthesis-related genes, and activated the AMPK/mTOR signaling pathway. Increased SIRT4 expression was found to have the opposite effect. However, blocking the AMPK/mTOR signaling pathway could inhibit the regulatory function of SIRT4 in milk synthesis-related gene expression. In summary, our results suggest that SIRT4 may play critical roles in maintaining mammary gland function by regulating the AMPK/mTOR signaling pathway in dairy cows, indicating that SIRT4 may be a potential molecular target for curing heat stress-induced BMEC injury and low milk production in dairy cows.

Extracellular vesicles (EVs), which exist in the follicular fluid of ruminant ovaries, are considered as cargo carriers for the transfer of biomolecules to recipient cells. However, the functions and changes in EVs in antral follicles remain ambiguous. In the present study, we isolated and characterized EVs from goat follicular fluid by means of differential ultracentrifugation and Western blotting of marker proteins. Bioinformatics tools were used to detect miRNA expression levels in EVs. Different miRNA expression patterns of EVs exist in small to large follicles. Thirteen differentially expressed miRNAs (seven upregulated and six downregulated) were identified and used for analysis. A total of 1948 predicted target genes of 13 miRNAs were mapped to signaling pathways, and three significantly enriched pathways (FoxO, MAPK, and PI3K-AKT signaling pathways) were involved in follicular development, as revealed by KEGG enrichment analysis. Our findings suggest that EVs in follicular fluid play biofunctional roles during follicular development in goats.

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.

With global warming, heat stress has become a primary factor that compromises the health and milk quality of dairy cows. Here, we investigated the function and underlying regulatory mechanism of miR-27a-3p in bovine mammary epithelial cells (BMECs) under heat-stress conditions. The current study showed that miR-27a-3p could prevent heat stress-induced BMEC oxidative stress and mitochondrial damage by regulating the balance between mitochondrial fission and fusion processes. Importantly, we found that miR-27a-3p could increase cell proliferation under heat stress conditions by regulating the MEK/ERK pathway and cyclin D1/E1. Interestingly, miR-27a-3p is also involved in the regulation of milk protein synthesis-related protein expression, such as CSN2 and ELF5. Inhibition of the MEK/ERK signaling pathway by AZD6244 blocked the regulatory function of miR-27a-3p in cell proliferation and milk protein synthesis in BMECs under heat stress conditions. Our findings demonstrated that miR-27a-3p protects BMECs from heat stress-induced oxidative stress and mitochondrial damage through the MEK/ERK pathway, thereby promoting BMECs proliferation and lactation in dairy cows. Graphical Abstract The potential regulatory mechanism of miR-27a-3p in attenuating heat stress-induced apoptosis and lactation defect in BMECs.

MicroRNAs (miRNAs) play key roles in sperm as the regulatory factors involved in fertility and subsequent early embryonic development. Bta-miR-6531 is specifically a highly enriched miRNA in low-motility sperms in previous study. To investigate the mechanism of bta-miR-6531, 508 shared target genes of bta-miR-6531 were predicted using two miRNA target databases (TargetScan7 and miRWalk). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the calcium and cAMP signaling pathways were the most enriched of the target genes. A dual-luciferase assay indicated that bta-miR-6531 targeted ATP2A2 mRNA by binding to the coding sequence region. In bovine Leydig cells, bta-miR-6531 overexpression affected the intracellular calcium concentration by restraining ATP2A2 expression. Moreover, we observed high calcium concentrations and high ATP2A2 protein levels in high-motility sperm compared with those in low-motility sperms. Furthermore, high-linkage single-nucleotide polymorphisms (SNPs) of the pre-bta-miR-6531 sequence were identified that related to sperm traits. Genotype TCTC of bta-miR-6531 showed high sperm motility and density and low deformity rate in Holstein bulls. However, the mutation in pre-miR-6531 did not significantly affect mature bta-miR-6531 expression in the sperm or cell models. Our results demonstrate that bta-miR-6531 might involve in sperm motility regulation by targeting ATP2A2 of the calcium signaling pathway in bovine spermatozoa.