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.

The present paper reviews the current state of knowledge in multiple ovulation and embryo transfer (MOET) technology in small ruminants, focusing on recently reported information. Major new findings are related to follicular wave patterns in small ruminants, the elucidation of follicular dominance and the integration of this information into ovarian superstimulatory treatment protocols. Follicular dynamics determine steroid and gonadotrophin secretion, follicular responses to FSH, ovulatory responses and embryo yield. Protocols that control follicular dominance have been designed to allow the initiation of superstimulation at the beginning of a follicular wave. New approaches consist of (1) synchronisation of ovulation for superstimulation during Wave 1 (i.e. Day 0 protocol), (2) pretreatment with a gonadotrophin-releasing hormone (GnRH) antagonist from 10 days prior to FSH treatment to avoid follicular dominance and (3) progesterone-oestradiol cotreatment to synchronise follicle wave emergence. These protocols provide a homogeneous pool of small follicles that are gonadotrophin responsive, enhancing the superovulatory response and embryo yield with a reduction in the incidence of unovulated follicles and early regression of corpora lutea. In addition, the rate of fertilisation failure has been reduced by using an inducer of ovulation (i.e. GnRH) associated with intrauterine insemination. In summary, the application of recently acquired knowledge has resulted in relevant improvements in MOET programmes in small ruminants.

Aims: Monothiol glutaredoxins (1-C-Grxs) are small proteins linked to the cellular iron and redox metabolism. Trypanosoma brucei brucei, model organism for human African trypanosomiasis, expresses three 1-C-Grxs. 1-CGrx1 is a highly abundant mitochondrial protein capable to bind an iron-sulfur cluster (ISC) in vitro using glutathione (GSH) as cofactor. We here report on the functional and structural analysis of 1-C-Grx1 in relation to its ISC-binding properties. Results: An N-terminal extension unique to 1-C-Grx1 from trypanosomatids affects the oligomeric structure and the ISC-binding capacity of the protein. The active-site Cys104 is essential for ISC binding, and the parasite-specific glutathionylspermidine and trypanothione can replace GSH as the ligands of the ISC. Interestingly, trypanothione forms stable protein-free ISC species that in vitro are incorporated into the dithiol T. brucei 2-C-Grx1, but not 1-C-Grx1. Overexpression of the C104S mutant of 1-C-Grx1 impairs disease progression in a mouse model. The structure of the Grx-domain of 1-C-Grx1 was solved by nuclear magnetic resonance spectroscopy. Despite the fact that several residues-which in other 1-C-Grxs are involved in the noncovalent binding of GSH-are conserved, different physicochemical approaches did not reveal any specific interaction between 1-C-Grx1 and free thiol ligands. Innovation: Parasite Grxs are able to coordinate an ISC formed with trypanothione, suggesting a new mechanism of ISC binding and a novel function for the parasite-specific dithiol. The first 3D structure and in vivo relevance of a 1-C-Grx from a pathogenic protozoan are reported. Conclusion: T. brucei 1-C-Grx1 is indispensable for mammalian parasitism and utilizes a new mechanism for ISC binding. Antioxid. Redox Signal. 19,[665][666][667][668][669][670][671][672][673][674][675][676][677][678][679][680][681][682]

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a b s t r a c tPrecise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time-and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.

SummaryLipoamide dehydrogenase (LipDH) is a component of four mitochondrial multienzyme complexes. RNA interference or the deletion of both alleles in bloodstream Trypanosoma brucei resulted in an absolute requirement for exogenous thymidine. In the absence of thymidine, lipdh-/-parasites showed a severely altered morphology and cell cycle distribution. Most probably, in bloodstream cells with their only rudimentary mitochondrion, LipDH is required as component of the glycine cleavage complex which generates methylene-tetrahydrofolate for dTMP and thus DNA synthesis. The essential role of LipDH in bloodstream parasites was confirmed by an in vivo model. Lipdh-/-cells were unable to infect mice. Our data further revealed that degradation of branched-chain amino acids takes place but is dispensable. In cultured bloodstream -but not procyclic -African trypanosomes, the total cellular concentration of LipDH increases with increasing cell densities. In procyclic parasites, LipDH mRNA depletion caused an even stronger proliferation defect that was not reversed by thymidine suggesting that in the fully elaborated mitochondrion of these cells the primary effect is not on the glycine cleavage complex. Since the medium used for the cultivation of procyclic cells was not supplemented with glucose, impairment of the 2-ketoglutarate dehydrogenase complex is probably the main effect of LipDH depletion.

The objective of this study was to test the effectiveness of ivermectin for the treatment of mouse hepatitis virus (MHV), a type 2 family RNA coronavirus similar to SARS-CoV-2. Female BALB/cJ mice were infected with 6,000 PFU of MHV-A59 (group infected, n = 20) or infected and then immediately treated with a single dose of 500 µg/kg ivermectin (group infected + IVM, n = 20) or were not infected and treated with PBS (control group, n = 16). Five days after infection/treatment, the mice were euthanized and the tissues were sampled to assess their general health status and infection levels. Overall, the results demonstrated that viral infection induced typical MHV-caused disease, with the livers showing severe hepatocellular necrosis surrounded by a severe lymphoplasmacytic inflammatory infiltration associated with a high hepatic viral load (52,158 AU), while mice treated with ivermectin showed a better health status with a lower viral load (23,192 AU; p < 0.05), with only a few having histopathological liver damage (p < 0.05). No significant differences were found between the group infected + IVM and control group mice (P = NS). Furthermore, serum transaminase levels (aspartate aminotransferase and alanine aminotransferase) were significantly lower in the treated mice than in the infected animals. In conclusion, ivermectin diminished the MHV viral load and disease in the mice, being a useful model for further understanding this therapy against coronavirus diseases.

This review summarizes the latest advances and main limitations for the implementation of in vitro embryo production programs in sheep and goats. We describe the laparoscopic assisted technique for oocyte retrieval and propose new insights for follicular manipulation to improve oocyte quality. Further description of the routine conducted in our laboratory for the in vitro process of oocyte maturation, fertilization and embryo culture is presented, with emphasis in the main issues for the success of the technique. Protocols for fixed time embryo transfer (FTET) are proposed and the optimal number of in vitro produced (IVP) embryos to be transferred per female is discussed. In addition, we present pregnancy outcomes and birth rates recently obtained with FTET with IVP embryos cryopreserved by vitrification with new minimum volume methods. In summary, due to important refinements for in vitro embryo production in sheep and goats achieved in the recent years, this technology is now available for its implementation in commercial programs for genetic improvement, for the production of genetically engineered sheep and goats, and for basic research in reproduction.