Comparative evaluation of three culture media for domestic cat in vitro embryo production and their effects on blastocyst development

In vitro embryo production (IVP) in felid species faces difficulties derived from their conservation status, challenging reproductive traits and low success rates during in vitro maturation (IVM), fertilization (IVF) and culture (IVC). This study aimed to evaluate different IVC strategies for domestic cat IVP, as a model to improve assisted reproduction techniques (ARTs) in wild felids. Three IVC media were compared: (i) synthetic oviductal fluid (SOF) supplemented with fetal bovine serum ("FBS"); (ii): SOF supplemented first with bovine serum albumin (BSA), followed by FBS addition ("BSA-FBS"); and (iii): a commercial IVC human medium (IVC-CULT®) ("COM"). A total of 1064 in vitro matured cat oocytes were fertilized with frozen-thawed epididymal spermatozoa, and 813 presumptive zygotes were cultured in the three experimental groups. Expanded blastocysts (n = 108) were snap-frozen or fixed for differential cell count and quantitative analysis of pluripotency-related gene expression. One-way ANOVA was used to assess differences in IVP rates and blastocyst developmental parameters. No significant differences in cleavage or blastocyst rates were found among groups, nor in total blastomere count (P > 0.05). However, the FBS group showed higher SOX2 pluripotency marker expression compared to the other groups, while BSA-FBS exhibited a more balanced SOX2/OCT4 ratio, linked to blastocyst competence in other species. Furthermore, a tendency was found towards a lower proportion of cells in the inner cell mass (ICM) in the FBS group. Our results suggest that elevated SOX2 expression alone may not reflect improved developmental competence in the domestic cat blastocysts. Instead, early BSA supplementation followed by FBS might enhance ICM differentiation, benefiting first from BSA's fatty acids and later from FBS's antioxidants and growth factors. Lastly, the commercial medium yielded comparable embryonic developmental outcomes to BSA-FBS, offering a viable alternative. Thus, this research represents a contribution to the refinement of IVP protocols in the domestic cat, promoting standardization that could be adapted for wild feline conservation.