S.P. Leibo

Cryobiology of in vitro-derived bovine embryos

Abstract The capability to produce bovine embryos from ovarian oocytes by in vitro methods of maturation, fertilization and culture has progressed very rapidly during the past few years. Efficient experimental and practical use of these in vitro-derived embryos requires that they be cryopreserved. Until recently, in vitro embryos were considered to be much more sensitive to freezing than in vivo embryos. Recent observations are beginning to reveal that high survival of cryopreserved in vitro embryos requires improvements in the techniques of maturation and culture, rather than simply changes in cryopreservation methods. The merging of these two aspects of mammalian embryology is yielding success; pregnancies have been produced and calves have been born as a result of the transfer of cryopreserved in vitro-derived embryos.

Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos.

The effect of cooling and warming rates during cryopreservation on subsequent embryo survival was studied in 607 bovine morulae and 595 blastocysts produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC). Morulae and blastocysts were prepared by co-culturing presumptive zygotes with bovine oviductal epithelial cells (BOEC) in serum-free TCM199 medium for 6 and 7 d, respectively. The embryos in 1.5 M ethylene glycol in plastic straws were seeded at -7 degrees C, cooled to -35 degrees C at each of 5 rates (0.3 degrees, 0.6 degrees , 0.9 degrees, 1.2 degrees, or 1.5 degrees C/min) and then immediately plunged into liquid nitrogen. The frozen embryos were warmed either rapidly in a 35 degrees C water bath (warming rate > 1,000 degrees C/min) or slowly in 25 degrees to 28 degrees C air (< 250 degrees C/mm). With rapid warming, 42.1% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts. The proportions of rapidly wanned morulae that hatched decreased with increasing cooling rates (30.4, 19.0, 15.8 and 8.9% at 0.6 degrees , 0.9 degrees, 1.2 degrees and 1.5 degrees C/min, respectively). With slow warming 25.9% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts, while <10% of the morulae that had been cooled faster developed. The hatching rate of blastocysts cooled at 0.3 degrees C/min and warmed rapidly (96.3%) was higher than those cooled at 06 degrees and 0.9 degrees C/min (82.7 and 84.6%, respectively), and was also significantly higher than those warmed slowly after cooling at 0.3 degrees, 0.6 degrees or 0.9 degrees C/min (69.1, 56.6 and 51.8%, respectively). Cooling blastocysts at 1.2 degrees or 1.5 degrees C/min resulted in lowered hatching rates either with rapid (71.2 or 66 0%) or slow warming (38.2 or 38.9%). These results indicate that the survival of in vitro-produced bovine morulae and blastocysts is improved by very slow cooling during 2-step freezing, nevertheless, slow warming appears to cause injuries to morulae and blastocysts even after very slow cooling.

EFFECT OF AMBIENT TEMPERATURES DURING OOCYTE RECOVERY ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.