Evaluation of cryopreservation methods for in vitro produced bovine embryos

English: The objective of this study was to evaluate four cryopreservation techniques for in vitro produced bovine embryos, and to select the best method for practical application. The cryopreservation methods investigated were three vitrification methods and a slow freezing method. This study was done at the ARC-Animal Improvernent Institute in conjunction with the University of the Orange Free State (Department of Animal Science). Embryos were obtained by the IVM, IVF and IVC of bovine follicular oocytes. A total of 136 early blastocysts, blastocysts and expanded blastocysts were randomly assigned to four different treatment groups. In the conventional slow freezing method, the IVP bovine embryos were first held in ViGro™Holdingplus medium before being transferred to 1.5M ViGro™EG Freezeplus medium (TMT 4). In this technique, the IVP embryos were loaded into 0.25ml straws. The straws containing the embryos were immediately placed into a programmable freezer (CL-863 cryo-chamber) at -6°C. Straws were seeded after a 5 minutes equilibration period. Embryos were initially cooled from -6 "C to -30°C at a rate of 0.3 °C/min. Thereafter, from -30°C to -33°C the rate was changed to 0.1 °C/min. After the target temperature was reached, straws were immediately transferred to liquid nitrogen. Vitrification of IVP bovine embryos was performed according to the following procedures: Embryos were initially placed in 10% EO in ViGro™Holdingplus medium for 5 minutes (Equilibration I), thereafter in 40% EO + 0.3M trehalose in ViOro ™Holdingplus medium for 5 minutes (Equilibration 11), both at room temperature. Embryos were then transferred to vitrification solutions, containing 40% EO (TMT 1); 40% EG + 0.3M trehalose (TMT 2); 40% EG + 0.3M trehalose + 20% PVP (TMT 3) in ViGro TM Holdingplus. Embryos were then loaded into 0.25ml straws, and plunged directly into liquid nitrogen (LN2). The straws were vertically stored in liquid nitrogen (- 196°C) until thawing and evaluation took place. Thawing of embryos within the straws was carried out in a water bath (32 DC). Each straw was placed in a water bath for 30 seconds. The straws were dried, cut and the contents transferred to ViGro ™Holdingplus medium. Recovered embryos were washed twice in fresh ViGro™I-Ioldingplus, and embryos were morphological examined for their viability under a stereo microscope. The viable embryos were cultured in IVC media. Embryo survival was recorded immediately after thawing, 24 hours and 48 hours post-thawing by monitoring the re-expansion of the blastocoel and expansion of the blastocyst. Statistically, there was a significant (P<0.05) difference in survival rate between embryos frozen in TMT 3 (77%), compared to those frozen in TMT 2 (41%), immediately after thawing. There was no significant difference in embryo survival rate for the other treatment groups. At 24 hours post-thawing, there was a significant (P<0.05) difference in survival rate between embryos frozen in TMT 3 (60%), compared to those frozen in TMT 1 (26%). There was also a significant (P<0.05) higher survival rate for embryos frozen in TMT 3 (60%), compared to those frozen in TMT 2 (21%). At 48 hours post-thawing, however, there was no significant difference in survival rate for embryos frozen in all the treatment groups. TMT 3 had the highest survival rates of embryos (37%). The generalized linear model (Bonferroni multiple comparison test) was used to test and predict the embryo survival rate between the treatment groups. The predicted (theoretical) embryo survival rate correlated highly and significantly (P<0.05) higher with the survival rate of embryos frozen in TMT 3. Embryos Frozen in TMT 3 were also predicted to be more likely to survive, compared to the other treatment groups. The results clearly indicate the beneficiary effect of this vitrification method (TMT 3). Vitrification is simple and more cost effective, compared to the slow freezing method (TMT 4), which is time consuming and expensive. Although there was no significant difference 48 hours ostthawing, TMT 3 could be recommended as the method for cryopreservation of IVP bovine embryos. The addition of 0.3M trehalose with 40% EO in the ViGro™Holdingplus medium decreased the survival rates of the IVP bovine embryos. Embryos frozen and thawed in 40% EO in ViGro™Holdingplus had higher survival rates, compared to those frozen/thawed in TMT 2, from immediately after thawing, to 48 hours post-thawing. Perhaps the addition of trehalose in the solution (ViGro™Holdingplus), already containing non-permeating agent (sucrose), increased the concentration of non-permeating agent in the freezing solution. High concentrations of non-permeating agent may be detrimental or toxic to the embryos. The presence of 20% PVP with 0.3M trehalose and 40% EO dramatically increased the survival rate of IVP bovine embryos. The PVP plays some kind of protective role during the freezing and thawing processes. Although the mechanism of protection is not clear, it may be that it prevents water from entering the cells during vitrification and thawing, which in turn prevents intracellular ice formation. Intracellular ice formation is lethal to embryos during thawing. It can be concluded that the combination of 40 % EO + 0.3M trehalose + 20% pyp (TMT 3), used as a vitrification solution, be recommended as suitable method for cryopreservation of IVP bovine embryos. It gave the highest embryo survival rate from immediately after thawing to 48 hours post-thawing. The advantage of this vitrification technique is that it is simple, quick and inexpensive. Additional research is needed to develop an effective cryopreservation method that will reduce the sensitivity problem of in vitro produced embryos. In vitro produced embryos contain lipids that cause them to be more sensitive to freezing, compared to those produced in vivo. The ability of vitrified in vitro produced bovine embryos still needs to be evaluated for their development in utero, in controlled embryo transfer programs.