Canine IVM With SOF Medium, Insulin-Transferrin-Selenium, and Low O2 Tension Improves Oocyte Meiotic Competence and Decreases Reactive Oxygen Species Levels.

Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48-72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA-2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 µl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

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 species 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. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) 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 Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async 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 Async4n 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 Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.