29 Co-Incubation of Equine Cloned Embryos with Sialic Acid: Effect on Pregnancy Rate

Abstract

In vitro-produced equine embryos have certain morphological characteristics that differ from embryos produced in vivo. One of them is the absence or inadequate formation of the embryo capsule. This capsule is composed of mucin-like glycoproteins produced by the trophectoderm with high proportion of sialic acid, which confers anti-adhesive properties. This characteristic is necessary for the intrauterine embryo migration process to occur, which is vital and fundamental for pregnancy recognition. For this reason, inadequate formation of the glycoprotein capsule could result in a lower pregnancy rate. In this study, we aimed to evaluate the effect of cloned equine embryo co-incubation with sialic acid on blastocyst and pregnancy rates. To achieve this, equine oocytes obtained from slaughterhouse ovaries were matured in TCM-199 HEPES medium with 2% fetal bovine serum, 2% antibiotics, and 1 μg mL⁻¹ FSH, incubated at 39°C for 24 h. Matured oocytes were denuded with pronase, enucleated, and fused to donor bone marrow mesenchymal cells according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Chemical activation was induced using 8.7 μM ionomycin for 4 min and embryos were incubated with 1 mM 6-DMAP and 5 mg mL⁻¹ cycloheximide for 4 h. Afterwards embryos were cultured in microwells for 8 days in DMEM-F12 medium. On Day 6, cloned equine embryos were exposed to 5 μM sialic acid for 48 h (SA group). On Day 8, blastocysts were transferred to recipient mares 5 days post-ovulation and pregnancy was confirmed 15 days post-transfer by transrectal ultrasound. Embryo clones generated without sialic acid exposure were used as a control (C) group. Fisher test was used to analyse both blastocyst and pregnancy rates. Blastocyst rates were 14% (46/328) and 15% (62/413) and pregnancy rates were 30.4% (7/23) and 19.4% (6/31) for SA and C groups, respectively. No statistical differences were observed between groups for the analysed parameters, even though pregnancy rates tended to be higher in the SA group. This effect could be a consequence of higher concentrations of the glycoprotein involved in the formation of the embryo capsule.