No abstract

UBACyT from the University of Buenos Aires. http://cyt.rec. uba.ar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the specie to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. To do this purpose we compared in vitro development of synchronic (S) or asynchronic (AS) and asynchronic tetraploid (AST) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA-fragmentation. Additionally, the developmental rates of AST aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. AS aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by AST aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by AST aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of 2cell fused embryos to generate tetraploid blastomeres and demonstrate that AST aggregation generates good quality embryos.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

The transfer of nuclear genomic DNA from a cell to a previously enucleated oocyte or zygote constitutes one of the main tools for studying epigenetic reprogramming, nucleus-cytoplasm compatibility, pluripotency state, and for genetic preservation or edition in animals. More than 50 years ago, the first experiences in nuclear transfer began to reveal that factors stored in the cytoplasm of oocytes could reprogram the nucleus of another cell and support the development of an embryo with new genetic information. Furthermore, when the nuclear donor cell is an oocyte, egg, or a zygote, the implementation of these technologies acquires clinical relevance for patients with repeated failures in ART associated with poor oocyte quality or mitochondrial dysfunctions. This review describes the current state, scope, and future perspectives of nuclear transfer techniques currently available for assisting mammal reproduction.

Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before. To this purpose, it is necessary to introduce the system into early stage zygotes or to edit cells followed by somatic cell nuclear transfer. In this review, several strategies for pig knock-out gene editing, using the CRISPR-Cas9 system, will be summarized, as well as genotyping methods and different delivery techniques to introduce these tools into the embryos. Finally, the best approaches to produce homogeneous, biallelic edited animals will be discussed.

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Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24h) or late (28–30h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).