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Effect of Slow Cooling End Point Temperature on Survival of Frozen Bovine Embryos¹

Colorado State University², Fort Collins 80523

Abstract

One hundred sixty-two embryos were collected from superovulated crossbred beef cattle 7 to 8 d after the onset of estrus. Embryos were frozen in modified Dulbecco's phosphate-buffered saline supplemented with 20% heat-inactivated fetal calf serum (PBS + FCS) and dimethylsulfoxide (DMSO), which was added in three steps to a final concentration of 1.5 M. Embryos were placed in .25 ml of 1.5 M DMSO in PBS + FCS in 1-ml glass ampules and cooled at 1.0 C/min from ambient temperature to –7 C, seeded and then cooled at .3 C/min to –19, –26, –33, –38, –43, –50 or –57 C before immersion (plunging) in liquid nitrogen. Ampules were thawed in 25 C water, and DMSO was removed in six steps at .25 M increments, 10 min/step. After removal of DMSO, embryos were cultured 24 h in PBS + FCS and then fixed and stained. Just after thawing, embryos for which slow cooling was terminated at –50 C were of lower (P<.05) morphological quality than other groups. After removal of cryoprotectant, embryos from both the –19 and –50 C treatments had deteriorated more (P<.05) than had embryos from other treatments. After 24-h culture, embryos slow-cooled to –19, –26 and –50 C had a lower rate of survival (P<.05) than did embryos from –33, –38, –43 and –57 C temperatures. Embryos slow-cooled to –33, –38 and –43 C showed a higher percentage of healthy nuclei than did embryos slow-cooled to –19, –26 and –50 C. The –33, –38 and –43 treatments also had better cytoplasmic scores and higher overall ratings after staining (P<.05) than those in the other treatments. Embryos that had formed a blastocele before freezing had higher survival rates than did morulae (P<.01), but survival rates of morulae slow-cooled to –43 C before plunging approached those of blastocysts. Under the conditions of the experiment, post-thaw survival of embryos was optimal with plunging temperatures between –33 and –43 C.

¹ This research was supported in part by Achilles Cattle Co., Lexington, KY and the Colorado State Univ. Exp. Sta. We thank H. R. Tervit for help in designing the experiment.

² Anim. Reprod. Lab.