Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.

Long noncoding RNAs (LncRNAs) have been identified as important regulators of testis development; however, their expression patterns and roles in sheep are not yet clear. Thus, we used stranded specific RNA-seq to profile the testis transcriptome (lncRNAs and mRNAs) in premature and mature sheep. Hormone levels and the testis index were examined, and histological analyses were performed at five stages of testis development, 5-day-old (D5), 3-month-old (3M), 6-month-old (6M), 9-month-old (9M), and 2-year-old (2Y), the results of which indicate a significant difference in hormone levels and testis morphometries between the 3M and 9M stages (P < 0.05). Based on the comparison between 3M and 9M samples, we found 1,118 differentially expressed (DE) lncRNAs and 7,253 DE mRNAs in the testes, and qRT-PCR analysis showed that the results correlated well with the transcriptome data. Furthermore, we constructed lncRNA-protein-coding gene interaction networks. Forty-seven DE lncRNA-targeted genes enriched for male reproduction were obtained by cis- and trans-acting; 51 DE lncRNAs and 45 cis-targets, 2 DE lncRNAs and 2 trans-targets were involved in this network. Of these, 5 lncRNAs and their targets, PRKCD, NANOS3, SERPINA5, and CYP19A1, were enriched for spermatogenesis and male gonad development signaling pathways. We further examined the expression levels of 5 candidate lncRNAs and their target genes during testis development. Lastly, the interaction of lncRNA TCONS__00863147 and its target gene PRKCD was validated in vitro in sheep Leydig cells. This study provides a valuable resource for further study of lncRNA function in sheep testis development and spermatogenesis.

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Goat’s milk, considered a substitute for cow’s milk, has a high nutritional value. However, goat’s milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%–9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

Simple SummaryHeat stress can induce oxidative stress and has an adverse effect on the growth and reproductive performance in animals. Curcumin, a plant-derived substance, with the effect of scavenging oxidative free radicals, improving immune response and anti-apoptosis, has been widely used as a dietary supplement in the livestock industry. The present study aims to investigate the effect of a curcumin dietary supplement on the blood metabolites, antioxidant status, immune response, and testicular gene expression in Hu Sheep in summer. The results show that dietary curcumin supplementation (450 and 900 mg/per sheep daily) can promote lipid metabolism, antioxidant capacity, and immune response as well as testicular development in Hu sheep, which provides evidence for the protective role of curcumin against heat stress in sheep.AbstractIn summer, the high temperature affects animal growth and reproductive performance. Curcumin is a flavonoid with anti-oxidant and anti-inflammatory effects. To evaluate the effects of dietary curcumin supplement on the blood biochemical parameters and testicular gene expressions in Hu sheep in summer, a total of 144 male Hu sheep aged four months were randomly divided into three groups (Con, Cur1, and Cur2, n = 48). Sheep in Con, Cur1, and Cur2 groups were fed a basal diet supplement with 0, 450, and 900 mg (per sheep) curcumin daily, respectively. Sheep were fed for 35 days, including a pre-feed for seven days. The results showed that the supplement with 450 mg and 900 mg curcumin increased serum free fatty acid (NEFA) and glutathione peroxidase (GPX), as well as IgA and IgM. The supplement with 450 mg curcumin increased the IgG level, while the supplement with 900 mg curcumin had a lower IgG level than the supplement with 450 mg curcumin (p < 0.05). Dietary curcumin supplement increased testicular organ index, serum testosterone level, and testicular star mRNA expression (p < 0.05). Furthermore, dietary curcumin supplement linearly inhibited testicular apoptosis with increased testicular bcl-2 mRNA expression and decreased caspase-3 mRNA expression (p < 0.05). In conclusion, dietary curcumin supplement can promote lipid metabolism, antioxidant capacity, and immune response, as well as testicular development, in Hu sheep, which provides evidence of application of curcumin in sheep production.

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In mammals, their proper development during the early cleavage stages strongly relies on the gene products newly transcribed by zygotic genome activation (ZGA). Long noncoding RNAs (lncRNAs) have been characterized as key regulators of the ZGA process in mice and human. However, the ZGA stage has not yet been identified and epigenetic regulations of the ZGA process remain largely unknown in goats. Here, we show that ZGA occurred at the 8-cell stage in goats. During ZGA, trimethylation of H3K9 was dynamically changed but maintained strong staining in development arrested embryos. Using single-cell RNA sequencing, we identified 800 mRNAs and 250 lncRNAs as candidates of key molecules in goat preimplantation embryos. These mRNAs and lncRNAs were differentially expressed from 4- to 8-cell stage embryos and were strongly enriched in terms of retinoic acid receptor signaling pathway as well as signaling pathway regulating pluripotency of stem cells. In particular, we found that microinjection of siRNA against lnc_137 caused development arrest. Our results are consistent with the notion that lncRNAs play vital roles during ZGA, and the data presented here provide an excellent source for further ZGA lncRNA studies.