VITRIFICATION OF MATURE MOUSE OOCYTES IN A 6 M ME2SO SOLUTION SUPPLEMENTED WITH ANTIFREEZE GLYCOPROTEINS - THE EFFECT OF TEMPERATURE

Oocytes have been successfully cryopreserved using rapid and slow free zing procedures. However, variability in the success of replicates has limited its practical application. In the present study, mature mouse oocytes were vitrified in 6 M dimethyl sulfoxide supplemented with 1 mg/ml antifreeze glycoproteins (AFGP) (solution known as VSD + AFGP) f rom the blood of Antarctic notothenioid fish. Such AFGPs have been use d to protect mammalian cells during hypothermia and cryopreservation. However, the degree of protection afforded is a contentious issue. Ste pwise addition of cryoprotectant was performed either at room temperat ure (19-21 degrees C) or on ice (2-4 degrees C), at the final stage of which oocytes were pipetted into 0.25 mi plastic insemination straws and held in liquid nitrogen vapor at -140 degrees C for 3 min before b eing plunged into liquid nitrogen. Thawing involved holding the straw in the air for 10 s and then in water at 20 degrees C for 10 s before dilution of the VSD solution with 1 M sucrose. Viability was assessed by in vitro fertilization; results have been quoted as median (range). Statistical analyses were performed using Kruskall-Wallis and Mann-Wh itney U tests (P < 0.05). Of the oocytes cryopreserved following expos ure to VSD + AFGP at room temperature (n = 518, 15 experimental runs), 78% (0-94%) retained normal morphology and, of these, 53% (0-100%) cl eaved to two cells. Of these two-cell embryos, 56% (0-100%) went on to develop to blastocyst. The overall percentage development to blastocy st, i.e., number of blastocysts/total number of oocytes treated x 100, was 20% (0-76%). Exposure of oocytes to the VSD + AFGP on ice prior t o cryopreservation yielded significantly improved rates of fertilizati on (94%, 82-100%) and overall development to blastocyst (66%, 24-89%) when compared with oocytes cryopreserved following exposure to the VSD + AFGP at room temperature. Rates of normality (86%, 35-95%) and deve lopment to blastocyst (89%, 64-100%) were also improved. Cryopreservat ion in 6 M dimethyl sulfoxide supplemented with 1 mg/ml AFGP resulted in poor rates of survival which were highly variable when exposure to cryoprotective agent (CPA) was performed at room temperature. Lowering the temperature of exposure to CPA prior to cryopreservation resulted in improved viability, (C) 1998 Academic Press.