FREEZABILITY OF PORCINE BLASTOCYSTS AT DIFFERENT PERI-HATCHING STAGES

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196-degrees-C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P < 0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300-mu-m, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (> 300-mu-m) survived the cryopreservation. High post-thaw survival (32/39, 82.1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatched blastocysts (diameter = 150 to 300-mu-m) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P < 0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196-degrees-C; however, a difference in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.