Studies on Optimal Conditions of Two Freezing Methods for Preservation of Rabbit Embryos

Since the first report on the successful deep cryopreservation of mam-malian embryos in 1972,slow progressing cooling rate has been employ-ed in conventional embryos-freezing techniques;while more recent studieson freezing preimplantation embryos have focussed on the simplificationof cooling and thawing procedures and improvement of viability of embr-yos.Dimethylsulfoxide (DMSO)has been used as an effective cryoprotec-tant for freezing human and other mammalian embryos.It has been foundthat glycerol and other alcohols are effective to protect embryos fromcryoinjury

Since the first report on the successful deep cryopreservation of mam- malian embryos in 1972,slow progressing cooling rate has been employ- ed in conventional embryos-freezing techniques;while more recent studies on freezing preimplantation embryos have focussed on the simplification of cooling and thawing procedures and improvement of viability of embr- yos.Dimethylsulfoxide （DMSO）has been used as an effective cryoprotec- tant for freezing human and other mammalian embryos.It has been found that glycerol and other alcohols are effective to protect embryos from cryoinjury during rapid freezing procedure,particularly,when glycerol and non-permeating sucrose are used together as the freezing medium,the protective effect is good for free zing morulae and early blastocysts.Us- ing this combined medium,murine and bovine embryos have been success- fully preserved by rapid freezing in liquid nitrogen vapour.The present theses compares and analyzes the various conditions of two rapid freezing and warming methods for rabbit morulae,and try to establish an easy and effective technique for freezing embryos.Viability of frozen-thawed embryos were assessed by their ability to develop to the blastocyst stage in vitro and liviborn rate after transfer to recipient foster-mothers, and by using FDA and DAPI fluorescence tests to assess viability of thawed ones,in which living cells ought to be FDA positive,and dead cells DAPI positive.The ultrastructure of thawed embryos was studied concerning their morphological changes associated with freezing and tha- wing. 1.Temperature control freezing method:Embryos were exposed to 1.5 MDMSO.The samples were then cooled from room temperature to -5℃ atl 1℃/min,in this temperature,the samples were seeded,then being rapid- ly cooled to -20℃ at 8-10℃/min.After 10 min,they were further rapid- ly cooled to -100℃ at same cooling rate.10 min later,they were placed into liquid nitrogen （LN2）.Thawing was accomplished by taking out sa- mples from LN2 and immersing into a 37℃ water bath,when ice melted, DMSO were removed by stepwise dilutions with inactive rabbit serum in PBS at room temperature.After thawing,22.65％ of 181 thawed embryos appeared with ruptured mucin coat ard/or zona pellucida;70.27％ of 37 embryos developed into blastocysts in vitro,18.18％ of 44 thawed embryos succeeded in implantation after transfer to synchronized recipients and liviborn rate was 13.64％. 2.LN2 vapour freezing method:Embryos were exposed to glycerol （2.0M,3.0M or 4.0M）for 10 min at 20℃,then aspirated into the freezing medium containing glycerol （2.0M,3.0M or 4.0M） and sucrose （0.25M） another 10 min.The freezing tube used was a 0.5 mlforplastic straw fill- ed sequentially with 440 μl sucrose diluent（0.5 or 1.0M）,20 μl air and- embryos in 40 μl of freezing medium.The straws wereheld horizontally 1- 1.5cm above the surface of LN2 in a container for 5min and then trans- ferred into LN.Thawing was performed by puttingthe straw directly into a 35-36℃ water bath for 20 s.The freezing medium was immediately mix ed with sucrose diluent after thawing.The glycerol was removed by the sucrose dilution in the straw at 20℃.After th-awing,5.07％ of embryosa ppeared with ruptured mucin coat and/or zona pellucida.The freezing me- dium containing 2.0M glycerol and 0.25 M sucrose showed almost no pre- servation effect.When.sucrose diluent was 1.0M high survival rate were obtained when the freezing medium was of 3.0/4.0M glycerol with addit- ion of 0.25M sucrose and their developmcntrates were 73.68％ and 80.56％, respectively.When sucrose diluent was0.5M,the survival rates of embr- yos in the above freezing media lowered to 40％ and 65.79％,respectively; the survival rate in freezing medium containing 3.0M glycerol and 0.25M sucrose decreased obviously.After embryos transfer,the imp lantation rate was 26.67％,and livibornrate 13.33％. Based upon and using the above LN vapour technique,rabbit morulae were also tried to be frozen in a medium of 3.0M DMSO and 0.25M suc- rose and diluted with 1.0M sucrose diluent,only 30％ of embryosbecame viable after thawing,obviously lower than that of the glycerolsucrose fr- eezing medium of the same molality.（P<0.005）. Statistically there is no significant difference between methods no. 1 and 2 in the development rate of thawed embryos in vitro and liviborn rate after thawed embryos transfer.Difference between the respective percentages of damaged mucin coat and/or zona pellucida in these two methods is very significant （P<0.005）. 83.33％ of the FDA-positive embryos could continue normal develop- ment in vitro.The partially or completely FDA-negative embryos hadlost ability of developmeat.In the DAPI fluorescence test,if the number of dead cells per embryo was less than two,the development rate in vitro was 94.1％,if the number reached 9-12,the development rate dropped to 21.43％,and,when the number exceeded 15,the embryos lost entirely their developmental ability. Results of ultrastructural study showed that,in the optimal condi- tions of the freezing and thawing procedure,the apparently uninjured fro- zen and thawed embryos revealed no obvious morphological damage.Whire in the injured embryos,the damage were predominantly located in the cell membrane and the intracellular membrane systems. The above results indicate that the rapid freezing method by LN2vap- our can offer effective preservation to rabbit morulae,it is rapid,ensy and practicable.In its freezing medium,higher glycerol conacentration is necessary.The FDA and DAPI fluorescence tests are rapid andprecise met- hods for evaluation of the viability of the frozen and thawed embryos. （81 references）