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.

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.

The objectives of this study were to determine how dietary supplementation of N-carbamylglutamate (NCG) and rumen-protected L-arginine (RP-Arg) in nutrient-restricted pregnant Hu sheep would affect (1) maternal endocrine status; (2) maternal, fetal, and placental antioxidation capability; and (3) placental development. From day 35 to day 110 of gestation, 32 Hu ewes carrying twin fetuses were allocated randomly into four groups: 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations, 50% of NRC recommendations supplemented with 20g/day RP-Arg, and 50% of NRC recommendations supplemented with 5g/day NCG product. The results showed that in maternal and fetal plasma and placentomes, the activities of total antioxidant capacity and superoxide dismutase were increased (P0.05) in both NCG- and RP-Arg-treated underfed ewes. A supplement of RP-Arg and NCG reduced (P<0.05) the concentrations of progesterone, cortisol, and estradiol-17β; had no effect on T₄/T₃; and improved (P<0.05) the concentrations of leptin, insulin-like growth factor 1, tri-iodothyronine (T₃), and thyroxine (T₄) in serum from underfed ewes. These results indicate that dietary supplementation of NCG and RP-Arg in underfed ewes could influence maternal endocrine status, improve the maternal–fetal–placental antioxidation capability, and promote fetal and placental development during early-to-late gestation.

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, tri-methylation 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.

This study was conducted with an ovine intrauterine growth restriction (IUGR) model to test the hypothesis that dietary N-carbamylglutamate (NCG) and rumen-protected L-Arg (RP-Arg) supplementation are effective in ameliorating fetal growth restriction in undernourished ewes. Beginning on d 35 of gestation, ewes were fed a diet providing 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations (50% NRC), 50% of NRC recommendations supplemented with 20 g/d RP-Arg (providing 10 g/d of Arg), and 50% of NRC recommendations supplemented with 5 g/d NCG product (providing 2.5 g/d of NCG). On d 110, maternal, fetal, and placental tissues and fluids were collected and weighed. Ewe weights were lower (P < 0.05) in nutrient-restricted ewes compared with adequately fed ewes. Maternal RP-Arg or NCG supplementation did not alter (P = 0.26) maternal BW in nutrient-restricted ewes. Weights of most fetal organs were increased (P < 0.05) in RP-Arg–treated and NCG-treated underfed ewes compared with 50% NRC-fed ewes. Supplementation of RP-Arg or NCG reduced (P < 0.05) concentrations of β-hydroxybutyrate, triglycerides, and ammonia in serum of underfed ewes but had no effect on concentrations of lactate and GH. Maternal RP-Arg or NCG supplementation markedly improved (P < 0.05) concentrations of AA (particularly arginine-family AA and branched-chain AA) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids within nutrient-restricted ewes. These novel results indicate that dietary NCG and RP-Arg supplementation to underfed ewes ameliorated fetal growth restriction, at least in part, by increasing the availability of AA in the conceptus and provide support for its clinical use to ameliorate IUGR in humans and sheep industry production.