PRACTICAL EXPERIENCES ON FROZEN BOVINE EM BRYOS TRANSFERRED JUST AFTER THAWING

An experiment was designed to examine factors influencing the efficien cy of the direct transfer of frozen/thawed embryos without the need to remove the embryos from straws. The viability of embryos was evaluate d by in vitro culture and implantation (in vivo) under field condition s in this experiment. The effect of developmental stages and quality o f embryos on survival was also examined. Embryos were recovered from s uperovulated cows and heifers on days 6, 7 and 8 after oestrus and wer e grouped on the basis of developmental stages and quality. The only d ifferences between experimental and control embryos were in the method of filling the straw and of the extraction of cryoprotectant after th awing. Embryos were equilibrated for 20 minutes in PBS containing 10% of glycerol and 10% of fetal calf serum (FCS). After equilibration, em bryos were aspirated into 0.25 mi artificial insemination straws at ro om temperature then placed into the freezing machine precooled to -7 d egrees C. The embryos in PBS medium containing glycerol were aspirated into straws followed by a small bubble and then by 1.0 M sucrose solu tion. The ratio of the two solutions was 1:8-10. After 10 min. at -7 d egrees C ice crystals were induced and after another 10 min., straws w ere cooled at 0.5 degrees C/min. to -30 degrees C. At that temperature , the straws were placed into liquid nitrogen. Straws were thawed at r oom temperature on air for 2 min. Glycerol was removed from the contro l embryos outside the straws in a 1.0 M sucrose solution. Glycerol was extracted from experimental embryos inside the straws by mixing the g lycerol and sucrose solutions for a period of 10 min. Embryos were the n either transferred into day 6 to 8 recipients or were placed into PB S+25% of FCS solution at 38 to 39 degrees C for 24 hours in vitro cult ures. Significant differences were not found between in vitro cultures of experimental and control groups of embryos in the compacted morula e/early blastocyst stage of development (experimental 80/104, 76.9%; c ontrol 52/63, 82.5%). Survival rate of expanded blastocysts was 42% (5 /12) for experimental and 53% (17/32) for control embryos (Table 1). T he developmental rate for poorer quality of embryos at the compacted m orulae and early blastocyst stages were 26% (4/15) for experimental an d 23% (4/17) for control groups (Table 2). After transcervical transfe r of 42 experimental and 55 control embryos, the respective results of pregnancy rate were 38 (16/42) and 56% (31/55, Tables 3 and 4). The r esults have suggested that the direct transfer method using sucrose in the straw can be applied with good results in the practice. However, results have to be improved, especially for poorer quality embryos.