86 CULTURE OF PIG EMBRYOS BEFORE CRYOPRESERVATION

Abstract

Our previous experiment on cryoconservation of in vitro-cultured porcine embryos (Gajda and Smorag 2000 CryoLetters 21, 231–236) revealed that, unlike for other species, sensitivity to vitrification of such embryos is higher than for those obtained in vivo. Considering that, selection of an optimal in vitro culture medium for pig embryos before cryoconservation becomes more important. Two experiments have been done on 3/4-cell pig embryos. Embryos were obtained from superovulated gilts after flushing the oviduct with PBS medium supplemented with 20% fetal calf serum at 38°C. In experiment 1, the embryos were cultured in three chemically defined media: NCSU-23, NCSU-37, CZB. The culture was performed at 39°C, 5% CO₂ in air for 96 to 120 h. The main evaluation criterion was development into blastocysts. Additionally, embryos that developed into blastocysts were stained with Hoechst 33342 and the cells were counted under a fluorescence microscope. Data were analyzed using chi-square and Fisher tests. The highest percentage of embryos developing into blastocysts was observed for those cultured in NCSU-23 (a): 89.2%; lower in NCSU-37 (b): 78.9%; and lowest for those in CZB medium (c): 66.6% (a,c: P < 0.05; a,b and b,c: NS). There were significantly (P < 0.001) more total cells in blastocysts obtained after culture in NCSU-23 (139.5 ± 32.8) than after culture in NCSU-37 (71.9 ± 36.6) or in CZB medium (58.3 ± 8.6). In experiment 2, embryos were cultured in vitro in NCSU-23 medium to the blastocyst stage and then vitrified (Gajda and Smorag 2002 CryoLetters 23, 385–388). Embryos were vitrified in 0.25-mL plastic straws in a mixture of 40% v/v ethylene glycol, 18% w/v Ficoll, and 0.3 M sucrose. Straws with embryos were stored in liquid nitrogen for 3 to 6 months. Dilution after rapid thawing (water bath at 20°C) was done in one step in 0.5 M sucrose solution. Eighty-five thawed blastocysts were surgically transferred into the oviducts of four synchronized recipient gilts. Two recipients became pregnant and farrowed 11 live healthy piglets. These results indicated that NCSU-23 medium provided the best conditions (of those media tested) for in vitro culture of pig embryos before vitrification. Transfer into oviducts of blastocysts that developed from in vitro-cultured and vitrified embryos resulted in full development in vivo.

Research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26).