Embryotransplantatie bij het paard: onmisbaar in de moderne fokkerij

Nowadays, a valuable competition mare can produce offspring without interrupting its sport career which is made possible by a technique called embryo transfer. The valuable mare is inseminated and an embryo is flushed seven days later. The early embryo is then transferred to the uterus of a recipient mare that carries the pregnancy to term. In 50% of the cases, flushing of the donor mare results in an embryo. After transfer, an average of 70% of the recipient mares become pregnant. These percentages are influenced by several factors related to both the donor and recipient mares

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

It has been previously demonstrated that equine oviduct explants are an excellent tool to unravel embryo-maternal interactions. The system is easy to set-up and the equine oviduct explants remain in vitro functionally intact and highly differentiated. Although the outer surface of the explants does not undergo ultrastructural or functional changes, dark cell degeneration (DCD), as exteriorized by dark central zones in the explants, is observed inside the explants. Since serum has been reported to negatively affect cell and embryo culture the effect of serum and the serum replacer insulin-transferrin-selenium on the prevalence of DCD, the percentage of explants showing ciliary activity, membrane integrity and ultrastructure was assessed. Therefore, ipsilateral oviducts from mares in the early postovulatory cycle stage were gathered. Oviduct explants were harvested by scraping and cultured for 6 days in 50 µl drops under oil in 5 % CO2 in air in DMEM/F12 (unsupplemented medium) or DMEM/F12 with 10 % foetal bovine serum (FBS) or in DMEM/F12 supplemented with 5 µg/ml insulin and transferrin and 5 ng/ml selenium selenite (ITS). One droplet contained one µl (20-30 explants) of explant suspension. Three replicates of 60 droplets per treatment group were cultured. Using an inverted microscope, every 24 h, the percentage of explants with dark zones and the percentage of explants showing ciliary activity were determined and compared between the groups. In addition, membrane integrity, as evidenced by Trypan blue staining, was compared by recording the percentage of membrane-damaged cells. At day 0, 3 and 6, ultrastructure was assessed by TEM. Compared to ITS (68 %, P < 0.0005) and the unsupplemented medium (67 %, P < 0.0005), FBS seems to protect (36 %) against the development of DCD during the first 2 days of culture while it fails to do so from day 3 on. From then on, the prevalence of DCD was the lowest in the unsupplemented group (81 %) compared to ITS and FBS (87-92 %, P < 0.0005). FBS and in lesser extent ITS seem to sustain ciliary activity (respectively 97 and 94 %, P < 0.0005) compared with the unsupplemented medium (87 %, P < 0.0005). In all groups, as shown by Trypan blue staining, the explants consisted of more than 98 % membrane intact cells (P = 0.9). TEM revealed that there was no qualitative difference in the development of DCD. The outer surface of all explants in all groups was, similar to the in vivo situation, highly differentiated and intact. In conclusion, FBS and ITS supplementation sustains ciliary activity. while ITS seems to enhance the development of DCD, components of FBS, which may be depleted after 2 days of culture, turn out to protect partly against DCD. Since the toxic margin of insulin and transferrin is known to be far above the applied levels in our culture system, next to other factors present in the culture system, selenium may play a role in the development of DCD. Further research is needed to unravel the exact cause and/or trigger in the development in DCD in oviduct explants