Evaluation of cryopreservation methods for in vitro produced bovine embryos

[摘要] English: The objective of this study was to evaluate four cryopreservation techniques for in vitroproduced bovine embryos, and to select the best method for practical application. Thecryopreservation methods investigated were three vitrification methods and a slowfreezing method. This study was done at the ARC-Animal Improvernent Institute inconjunction with the University of the Orange Free State (Department of AnimalScience).Embryos were obtained by the IVM, IVF and IVC of bovine follicular oocytes. A total of136 early blastocysts, blastocysts and expanded blastocysts were randomly assigned tofour different treatment groups. In the conventional slow freezing method, the IVPbovine embryos were first held in ViGro™Holdingplus medium before being transferredto 1.5M ViGro™EG Freezeplus medium (TMT 4). In this technique, the IVP embryoswere loaded into 0.25ml straws. The straws containing the embryos were immediatelyplaced into a programmable freezer (CL-863 cryo-chamber) at -6°C. Straws were seededafter a 5 minutes equilibration period. Embryos were initially cooled from -6 C to -30°Cat a rate of 0.3 °C/min. Thereafter, from -30°C to -33°Cthe rate was changed to 0.1°C/min. After the target temperature was reached, straws were immediately transferred toliquid nitrogen.Vitrification of IVP bovine embryos was performed according to the followingprocedures: Embryos were initially placed in 10% EO in ViGro™Holdingplus mediumfor 5 minutes (Equilibration I), thereafter in 40% EO + 0.3M trehalose inViOro ™Holdingplus medium for 5 minutes (Equilibration11), 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) inViGro TM Holdingplus. Embryos were then loaded into 0.25ml straws, and plungeddirectly 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). Eachstraw was placed in a water bath for 30 seconds. The straws were dried, cut and thecontents transferred to ViGro ™Holdingplus medium. Recovered embryos were washedtwice in fresh ViGro™I-Ioldingplus, and embryos were morphological examined for theirviability 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-thawingby 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 embryosfrozen in TMT 3 (77%), compared to those frozen in TMT 2 (41%), immediately afterthawing. There was no significant difference in embryo survival rate for the othertreatment groups. At 24 hours post-thawing, there was a significant (P<0.05) differencein survival rate between embryos frozen in TMT 3 (60%), compared to those frozen inTMT 1 (26%). There was also a significant (P<0.05) higher survival rate for embryosfrozen 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 frozenin all the treatment groups. TMT 3 had the highest survival rates of embryos (37%). Thegeneralized linear model (Bonferroni multiple comparison test) was used to test andpredict the embryo survival rate between the treatment groups. The predicted (theoretical)embryo survival rate correlated highly and significantly (P<0.05) higher with the survivalrate of embryos frozen in TMT 3. Embryos Frozen in TMT 3 were also predicted to bemore likely to survive, compared to the other treatment groups. The results clearlyindicate the beneficiary effect of this vitrification method (TMT 3). Vitrification is simpleand more cost effective, compared to the slow freezing method (TMT 4), which is timeconsuming and expensive. Although there was no significant difference 48 hours ostthawing,TMT 3 could be recommended as the method for cryopreservation of IVPbovine embryos.The addition of 0.3M trehalose with 40% EO in the ViGro™Holdingplus mediumdecreased the survival rates of the IVP bovine embryos. Embryos frozen and thawed in40% EO in ViGro™Holdingplus had higher survival rates, compared to thosefrozen/thawed in TMT 2, from immediately after thawing, to 48 hours post-thawing.Perhaps the addition of trehalose in the solution (ViGro™Holdingplus), alreadycontaining non-permeating agent (sucrose), increased the concentration of non-permeatingagent in the freezing solution. High concentrations of non-permeating agentmay be detrimental or toxic to the embryos.The presence of 20% PVP with 0.3M trehalose and 40% EO dramatically increased thesurvival rate of IVP bovine embryos. The PVP plays some kind of protective role duringthe freezing and thawing processes. Although the mechanism of protection is not clear, itmay 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 toembryos 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 forcryopreservation of IVP bovine embryos. It gave the highest embryo survival rate from immediately after thawing to 48 hours post-thawing. The advantage of this vitrificationtechnique is that it is simple, quick and inexpensive.Additional research is needed to develop an effective cryopreservation method that willreduce the sensitivity problem of in vitro produced embryos. In vitro produced embryoscontain lipids that cause them to be more sensitive to freezing, compared to thoseproduced in vivo. The ability of vitrified in vitro produced bovine embryos still needs tobe evaluated for their development in utero, in controlled embryo transfer programs.

[发布机构] University of the Free State