The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts

A total of 678 bovine blastocysts, which had been produced by in vitro matu ration, fertilization, and culture, were placed into plastic straws and wer e vitrified in various solutions of ethylene glycol (EC) + polyvinylpyrroli done (PVP]. Part of the straw was loaded with TCM 199 medium + 0.3 M trehal ose as a diluent; the diluent portions of the straw were prefrozen to eithe r -30 or -196 degreesC. Then, the embryos suspended in the vitrification so lution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degreesC and then agitated to mix th e diluent and cryoprotectant solution for 5 min followed by culture in TCM1 99 + 10% FCS + 5 + mug/ml insulin + 50 mug/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitri fication, the PVP concentration, and the temperature of exposure to EG + PV P prior to vitrification. Survival and hatching rates of the blastocysts ex posed to 40% EG in four steps at 4 degreesC were higher than those of embry os exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% a nd 71.5 +/- 4.7%, respectively; P < 0.05). The same indices were superior f ollowing vitrification-thawing oi the blastocysts in 40% EG + 20% FVP than it was in 40% EG + 10% PVP (76.1 +/- 5.5% vs 63.7 +/- 1.8%; P < 0.05; and 6 1.6 +/- 6.0% vs 70.5 +/- 4.7%; P < 0.01, respectively). Exposure to the vit rification solution (40% EG + 20% PVP) at higher temperatures (37.5 degrees C vs 4 degreesC) reduced both survival and hatching rates (45.8 +/- 6.9% vs 83.9 +/- 4.4% and 41.5 +/- 1.8% vs 64.0 +/- 4.7%, respectively; P < 0.001) . These results indicate that blastocysts vitrified after prefreezing the: diluent portions of the straws do favor developmental competence of in vitr o produced embryos. (C) 2001 Academic Press.