Supplementation of culture medium with foetal calf serum or insulin – transferrin – selenium affects the integrity of equine oviduct explants

Equine oviduct explants provide an excellent tool to unravel embryo-maternal interactions. They can be cultured in vitro for several days in DMEM F12 and serum whilst remaining functionally intact and highly differentiated. However, dark cell degeneration (DCD) has been observed inside the explants (Nelis et al. 2014 RFD 26 954-966). Since serum has been reported to negatively affect cell and embryo culture (Fernandez-Gonzalez 2004 PNAS 101 5880-5885), we aimed to assess the effect of serum and the serum replacer insulin-transferrin-selenium on the prevalence of DCD, ciliary activity, membrane integrity and ultrastructure of equine oviduct explants. Oviducts ipsilateral to the ovulation side were gathered from mares in the early postovulatory stage. Oviduct explants were harvested by scraping and cultured for 6d in 50 µl drops under oil in 5% CO2 in air in DMEM/F12 (control; Invitrogen, Merelbeke, Belgium), in DMEM/F12 with 10% foetal calf serum (FCS; Greiner Bio-one, Wemmel, Belgium) or in DMEM/F12 supplemented with 5 µg/ml insulin, 5 µg/ml transferring and 5 ng/ml selenium selenite (ITS; Sigma, Schnelldorf, Germany). Three replicates of 60 droplets per condition were performed. With an inverted microscope, every 24h, the percentage of explants with dark zones and the percentage of explants showing ciliary activity were determined. In addition, the percentage of membrane-damaged cells was determined using Trypan blue (Sigma-Aldrich, Diegem, Belgium). At d0, 3 and 6, ultrastructure was assessed by TEM. To compare DCD prevalence, ciliary activity and membrane integrity, binary logistic regression was implemented (SPSS 21 for windows; SPSS IBM, Brussels, Belgium). During the first two days, the prevalence of DCD was significantly lower in the FCS group (36%), when compared to ITS (68%, P 98% membrane intact cells (P=0.9). No qualitative differences in the development of DCD was detected by TEM. The outer surface of explants in all groups was highly differentiated and intact. In conclusion, without affecting morphology, components of FCS, which may be depleted after 2 days of culture, turn out to partly protect while ITS enhances the development of DCD. Furthermore, FCS and ITS seem to preserve ciliary activity. Since the toxic margin of insulin and transferrin, but not of selenium, is far above the applied levels in our culture system, amongst others, selenium may play a role in the development of DCD. Further research is needed to unravel the exact cause in the development in DCD in oviduct explants.