COMPARISON OF 2 COCULTURE SYSTEMS TO ASSESS THE SURVIVAL OF IN-VITRO PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION

The ability of bovine blastocysts to recover after cryopreservation an d thawing procedures is often assessed by evaluating their re-expansio n during in vitro co-culture. However, the influence of factors such a s feeder cell type and gas atmosphere on blastocyst survival and evolu tion have never been considered. This study therefore compared two cel l co-culture systems and two different gas atmospheres to assess survi val of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n = 181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured ei ther on Buffalo rat liver cells (BRL CC cell line) or on granulosa cel ls (GR CC primary culture) in TCM 199 supplemented with 10% FCS and un der an atmosphere of 5% or 20% O-2. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, survivin g blastocysts were treated for differential counting of inner cell mas s (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O-2: 62% vs. 5% O-2: 25%, P < 0.0001). Howe ver, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O-2 than under 5% O-2 as indicated by lo wer total and trophectoderm cell numbers (respectively 79 +/- 6 and 56 +/- 6 at 20% O-2 vs. 100 +/- 10 and 74 +/- 10 at 5% O-2, P < 0.05), b y an altered ICM/trophectoderm ratio (20% O-2: 28% vs. 5% O-2: 23%, P < 0.05), by a higher total nuclear fragmentation (20% O-2: 13.7% vs. 5 % O-2: 1.5%. P < 0.05) and a trend to decreased hatching (20% O-2: 32% vs. 5% O-2: 81%, P = 0.07). Whereas, for BRL co-culture, 20% O-2 yiel ded higher quality blastocysts than 5% O-2 as evaluated by higher ICM and trophectoderm cell numbers (19 +/- 1 and 71 +/- 5 at 20% O-2 vs. 1 5 +/- 2 and 48 +/- 9 at 5% O-2, respectively, P < 0.05), by lower nucl ear fragmentation in the ICM (20% O-2: 2.2% vs. 5% O-2: 6.7%, P < 0.05 ). In conclusion, co-culture conditions may influence blastocysts surv ival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O-2 seems to be the best combination to eval uate blastocyst survival and quality after vitrification. (C) 1998 Els evier Science B.V. All rights reserved.