While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Timed artificial insemination (TAI) is a very important tool in production systems, as it has a direct impact on cost-efficiency by reducing labour resulting from oestrus detection. However, to make TAI commercially feasible, hormonal treatments need to assure acceptable pregnancy rates and be economically viable. Recent advances in the knowledge of ovarian physiology (e.g. determination of follicular waves, sensitivity of the early corpus luteum) in small ruminants allowed the development of new treatments focused on an efficient estimation and synchronisation of the time of ovulation of the females of a treated flock/herd. In this review we summarise new information and concepts in the hormonal control of the life span of the corpus luteum, as well as methods to manipulate follicular growth in small ruminants. Additionally, we elaborate on recent studies concerning the use of TAI associated with short progestogen treatment in goats and the newly developed Synchrovine™ protocol (two doses of prostaglandin F2a given seven days apart) in sheep.

Management of the ovine oestrous cycle is mainly based on the use of exogenous hormones to mimic or enhance (progesterone and its analogues) or manipulate (prostaglandin F2α and its analogues) the activity of the corpus luteum, combined with the application of other hormones mimicking the pituitary secretion of gonadotrophins (e.g. equine chorionic gonadotrophin). These protocols have been applied without major change for decades but, now, there are two reasons to reconsider them: (1) our greatly improved knowledge of the dynamics of ovarian physiology, following the application of transrectal ultrasonography, indicates that modification of the protocols may improve fertility yields and (2) increasing concerns about animal health and welfare, food safety and the environmental impact of the treatments, as evidenced by public opinion and therefore market forces. Here, we offer an overview of these issues, introduce an updated protocol and suggest ways for future improvements to the protocols.