PIGLETS PRODUCED BY TRANSFER OF VITRIFIED PORCINE EMBRYOS AFTER STEPWISE DILUTION OF CRYOPROTECTANTS

A total of 498 porcine embryos at various stages of development collec ted from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blas tocysts (HB) were used to determine the effect of exposure to concentr ated solutions of ethylene glycol as cryoprotective additives (CPAs) o n embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethyl ene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solu tion of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. T o vitrify the embryos, they were exposed to 2.0 M EG for 5 min and the n were pipetted directly into shea columns of 8.0 M EG-PVP contained w ithin 0.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, th e embryos were immediately mixed with galactose within the straws by s haking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method I: Embryo s in the EG-PVP-galactose mixture were expelled from the straws and ri nsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cul tured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. Af ter 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method I I, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB v itrified embryos diluted by Method III and transferred into a recipien t, four developed into live piglets; two other recipients failed to li tter although one had been pregnant for 65 days. These results demonst rate that porcine embryos can be successfully cryopreserved by rapid c ooling in EG-PVP and by careful dilution of the CPA after warming. (C) 1998 Academic Press.