Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.

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