Background: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. Conclusions: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.

Low-temperature environments can strongly affect the normal growth and health of livestock. In winter, cold weather can be accompanied by strong winds that aggravate the effects of cold on livestock. In this study, two experiments were conducted to investigate the effect of low temperature and/or wind speed on physiological indices, rumen microbiota, immune responses and oxidative stress in sheep. When sheep were exposed to cold temperature and/or stronger wind speeds, the average daily gain (ADG) decreased (p < 0.05), and the abundance of Lachnospiraceae was significantly higher (p < 0.05). The acetate and propionate contents and the proportion of propionate in the rumen also significantly reduced (p < 0.05). The immunoglobulin G (IgG) and TH1-related cytokines in the blood were significantly lower (p < 0.05). However, antioxidant enzyme contents were significantly increased and the concentration of malondialdehyde (MDA) was reduced (p < 0.05). In a cold environment, the abundance of Lachnospiraceae in the rumen of sheep was highly enriched, and the decreasing of propionate might be one of the factors affecting the immunity of the animals, the sheep did not suffer from oxidative damage during the experiment.

Mulberry leaves, which have high nutritional value, have not been fully utilized. Few research systems have indicated whether mulberry leaves can replace traditional feed ingredients in goats. In this study, we investigated the effects of feeding white cashmere goats ensiled (Group E) or sun-dried mulberry leaves (Group S) on changes in ruminal microbial communities, rumen fermentation parameters and serum biochemical indices. The control group (Group C) received a typical total mixed ration (TMR). 16S rRNA gene sequencing revealed 209 genera belonging to 19 bacterial phyla dominated by Firmicutes and Bacteroidetes. Only the relative abundances of Erysipelotrichaceae_UCG-009 were significantly different among the three groups (p < 0.05). Physiological and biochemical findings revealed that only the serum leptin concentrations were significantly decreased when mulberry leaves were added to the diets (p < 0.05). Correlation analysis revealed that Ruminococcus_2 were significantly positively correlated with the butyrate concentration. These findings suggested that supplementation with mulberry leaves only induced minor changes in the abovementioned indicators, implying that the rumen fermentation status was still stable after adding mulberry leaves to the diets.

This study was conducted to investigate the impacts of dietary energy and protein on rumen bacterial composition and ruminal metabolites. A total of 12 ruminal samples were collected from Shaanbei white cashmere goats which were divided into two groups, including high-energy and high-protein (Group H; crude protein, CP: 9.37% in dry matter; metabolic energy, ME: 9.24 MJ/kg) and control (Group C; CP: 8.73%; ME: 8.60 MJ/kg) groups. Thereby, 16S rRNA gene sequencing and a quantitative polymerase chain reaction were performed to identify the rumen bacterial community. Metabolomics analysis was done to investigate the rumen metabolites and the related metabolic pathways in Groups C and H. The high-energy and high-protein diets increased the relative abundance of phylum Bacteroidetes and genera Prevotella_1 and Succiniclasticum, while decreasing the number of Proteobacteria (p < 0.05). The dominant differential metabolites were amino acids, peptides, and analogs. Tyrosine metabolism played an important role among the nine main metabolic pathways. Correlation analysis revealed that both Prevotella_1 (r = 0.608, p < 0.05) and Ruminococcus_2 (r = 0.613, p < 0.05) showed a positive correlation with catechol. Our findings revealed that the diets with high energy and protein levels in Group H significantly altered the composition of ruminal bacteria and metabolites, which can help to improve the dietary energy and protein use efficiency in goats.