77 PREGNANCY RATES AFTER VITRIFICATION OF FRESH AND COOLED EQUINE EMBRYOS USING THE CRYOTOP METHOD

Abstract

Cryopreservation of equine embryos is still not a routine procedure. Pregnancies have been obtained after transfer of vitrified embryos of less than 300 μm (Eldridge-Panuska et al. 2005). The aim of this study was to use the cryotop method (Kuwayama, 2007) to obtain pregnancies after transfer of vitrified thawed cooled and fresh embryos collected in our clinical embryo transfer programme. Embryos were assigned either to be vitrified within 3 h of collection or to be cooled for 18–24 h before vitrification. All embryos were vitrified and thawed by Cryotop Vitrification Kit^® (Cryo Tech Laboratory^®). Briefly, they were equilibrated in a solution containing ethylene glycol (EG), dimethylsulphoxide (DMSO) in TCM-199 for 10 to 25 min. Then they were moved to vitrification solution containing EG, DMSO, and sucrose in TCM-199 and loaded with a glass capillary onto the top of the film strip. After loading, almost all the solution was removed to leave only a thin layer covering the embryo, and the sample was quickly immersed into liquid nitrogen and covered with a protective cap. The time between entry to vitrification solution and nitrogen was from 1 to 3 min. At warming, the strip was immersed directly for 1 min into a 37°C medium containing sucrose in TCM-199. The embryo was incubated 3 min in a diluent solution, washed twice 5 min each in washing solution, and further cultured in DMEM F-12 with 10% FBS at 38.5°C 5% CO₂ between 2 to 5 h. For transfer, the embryo was loaded in 0.5-mL straws. All recipient mares had ovulated 4 to 7 days before nonsurgical transfer. Pregnancies were detected 6 to 8 days later. A total of 15 embryos, grades 1 to 2, were obtained. Fresh embryos (n = 7) ranged between 250 and 800 μm, and refrigerated embryo (n = 8) diameter was between 130 and 550 μm. Pregnancy rates were 37.5% (3/8) for embryos cooled before vitrification and 28.6% (2/7) for embryos vitrified within 3 h. The overall pregnancy rate was 33.3% (5/15). Shipping cooled embryos allows maintaining a large number of recipients far away from donors, without decreasing pregnancy rate. It also makes it possible to send embryos to a specialised laboratory in order to be vitrified and preserved until recipients are available. Equine embryos collected 6 days after ovulation are generally smaller than 300 μm and have shown the highest survival rate after cryopreservation. However, the embryo recovery rate is higher when flushing is performed at Day 7 or 8. This cryopreservation protocol could provide a way to vitrify fresh and cooled embryos up to 550 μm, which would prevent the loss of valuable embryos collected in more advanced stages of development. In summary, pregnancies can be obtained after cooling for 18 to 24 h and vitrification of embryos collected 7 or 7.5 days after.