Cryopreservation of in vitro-fertilised bovine embryos by vitrification: In vitro and in vivo evaluation

In cattle embryo transfer commercial enterprises, a reliable preservation method for the short- and long-term storage of in vitro-derived bovine embryos is lacking. A series of in vitro experiments, therefore, was designed and conducted in order to establish an efficient method for storage of in vitro-derived bovine embryos. Moreover, an in vivo experiment was conducted to validate the efficacy of the cryopreservation method. In Experiment 1, bovine embryos at expanded or pronuclear stages were generated from abattoir-obtained oocytes. Embryos were assessed during in vitro development that included in vitro maturation, in vitro fertilization and in vitro culture. The purpose of this experiment was to compare efficiency of three cryopreservation methods (slow freezing, short equilibration vitrification and long equilibration vitrification of bovine embryos at different developmental stages. The main finding from this experiment was the greater quality of embryos when using vitrification as compared to slow freezing. The hatching rates of expanded blastocysts after being thawed and cultured in vitro for 48 h were significantly greater with use of short duration equilibration (93.9%) and long duration equilibration (87.5%) for vitrification as compared to slow freezing (73.8%). With pronuclear stage cryopreservation, there was a greater percentage of vitrified embryos that developed to the blastocyst stage (approximately 22%) as compared to 4.9% in the group when slow freezing was used for cryopreservation. Analysis of the results from comparing three methods of cryopreservation that included standard slow freezing and the two vitrification procedures led to the conclusion that vitrification is more efficacious than slow freezing for embryo cryopreservation. Moreover, a short equilibration vitrification procedure is preferable because of the relatively greater viability of the embryos stored using this method. Consequently, short equilibration vitrification was used as a method in the subsequent experiments for the research reported in this thesis. Experiment 2 examined the inclusion or not of sucrose in the warming media used during the warming process for embryos following their storage by cryopreservation. Expanded blastocysts that were developed using abattoir-obtained oocytes were used in this study. The results indicate that there was no difference in embryo viability when sucrose was or was not included as a constituent of the warming media (91.3% and 87.0%, respectively). In Experiment 3, the effect on blastocyst formation and cryotolerance of salubrinal supplementation to the oocyte maturation medium was assessed. The inclusion of salubrinal did not enhance the embryo production nor did it affect cryotolerance. There was an in vivo assessment of the efficacy of using the vitrification method for embryo cryopreservation in Experiment 4. Blastocysts were generated from oocytes retrieved from live donor cows by using the ovum pick-up technique. The purpose of Experiment 4 was to compare the pregnancy rate of recipients implanted with vitrified embryos to that when fresh embryos were transferred into recipient animals. There was no difference in the pregnancy rate in the two groups. It is important to note that the pregnancy rate when using vitrified embryos is very much in keeping with those that are generally achieved by commercial embryo transfer enterprises and that there was no losses of pregnancies during the period ranging from Day 35-90 of gestation. In conclusion, the results of the research described in this thesis indicate that for cryopreservation of in vitro-derived bovine embryos a short duration equilibration vitrification is efficacious. When this procedure is used, there are desirable embryo viability rates after cryopreservation and when embryos derived using this procedure are transferred into recipient animals the resulting pregnancy rate meets industry expectations. Furthermore, the research contributed for this thesis project advances knowledge in the field of cryobiology of cattle embryos. Results of this research indicate that there is no advantage to using sucrose in warming media for survival of blastocysts; or to using salubrinal for in vitro maturation of oocytes when assessing the development of in vitro-derived embryo development to the blastocyst stage. Moreover, the research suggests that an application may be found for the vitrification procedure in a commercial setting to enhance the efficacy of assisted reproductive technologies in the cattle industry.