82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD

Abstract

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5°C in an humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean ± SEM; 109 ± 6 and 112 ± 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving embryos.