181 Equine androgenic embryos: ability of the equine sperm to develop in a heterospecific ooplasm

Abstract

Androgenic haploid embryos were originally produced for the study of certain aspects of early embryo development. The generation of androgenic haploid embryos allows us to better understand the complementary parental contribution to embryonic development, and to examine the effects of haploid development on gene expression. Because mare oocytes for research are scarce, the generation of heterospecific androgenic embryos could be useful to study aspects of the biology of early embryo development, or to identify genes and their variations or mutations that are responsible for reproduction-related problems in mares and stallions, which is of interest for the breeding industry. Therefore, the aim of this work was to study the capability of equine sperm to induce embryonic development after injection into an enucleated oocyte from a different species. Porcine cumulus-oocyte complexes (COC) were obtained from abattoir ovaries and placed in 100-µL drops in vitro maturation (IVM) medium for 42 h. Cumulus cells were removed with hyaluronidase and vortexing. Then, mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) with stallion frozen-thawed semen (according to Rodriguez et al. 2015). Immediately after the last injection, the zona pellucida of injected oocytes was removed with protease treatment, the oocytes were treated with cytochalasin B, and the metaphase II enucleated with a 20-µm micropipette. Finally, embryos were placed in culture medium (SOF) in plates with the well-of-the-well (WOW) system. As control treatment, non-enucleated pig oocytes were injected with stallion (CE) and boar (CC) semen. At Day 4, embryos were evaluated for cleavage and number of blastomeres, and stained with Hoechst 33342 to verify the presence of DNA in each blastomere under the UV light. Embryos were stored for future PCR studies to validate the presence of equine DNA. Data were analysed by chi-squared test to compare the cleavage of both controls with the androgenic embryos. From a total of 53 androgenic haploid embryos, the cleavage rate was 62% (33/53). Embryos were cleaved in 2 to 4 cells in 72.7%, 5 to 8 cells in 18.2%, and 9+ cells in 9.1% at Day 4. Presence of DNA in all blastomeres was observed in 60.6% (20/33) of the androgenic haploid embryos, while 21.2% (7/33) of the embryos had 10 to 50% of blastomeres with DNA, and 18.6% (6/33) of the embryos did not have DNA in their blastomeres. The ICSI control embryos cleaved in 45.3% (34/75) and 64.9% (98/151) for groups CC and CE, respectively. Cleavage rates in control CE were significantly higher than those in control CC (P < 0.004). No statistical difference was observed in the control groups versus androgenic embryos. This preliminary results showed that a heterospecific ooplasm can be successfully used to allow an equine sperm DNA to decondense and to develop, even in absence of the female counterpart. Using this method, copies of a single sperm DNA can be produced to potentially evaluate individual aspects of early embryo development concerning the male contribution. This is the first report of successful androgenic embryos using a heterospecific oocyte to create copies of a horse sperm DNA.