86 COMPARISON OF TWO VITRIFICATION PROTOCOLS FOR CROSSBRED BOS INDICUS × BOS TAURUS IN VITRO-PRODUCED EMBRYOS

Abstract

Dairy herds in tropical countries are often maintained as crossbred B. indicus × B. taurus hybrids to take advantage of heterosis, such as resistance to heat stress. Creating crossbred B. indicus × B. taurus embryos by in vitro methods may offer a means of rapidly improving tropical dairy herds, especially if the embryos can be cryopreserved. The aim of this study was to compare the viability of in vitro-produced crossbred B. indicus × B. taurus embryos (1/2, 3/4) using two vitrification solutions and equilibration/dilution temperatures. Cumulus-oocyte complexes were aspirated from purebred B. indicus and crossbred (B. indicus × B. taurus hybrid) ovaries, matured in vitro, and fertilized with spermatozoa collected from a Holstein bull. Presumptive zygotes were co-cultured in CR2aa medium with cumulus cells, in a humid atmosphere of 5% CO₂-air at 38.8°C. On day 7 of co-culture, embryos were assessed and classified as good or excellent, and those at the appropriate developmental stage were vitrified using one of two vitrification solutions, a mixture of either glycerol/ethylene glycol (GE) or dimethylsulphoxide/ethylene glycol (DE). Embryos (n = 34) assigned to GE vitrification were equilibrated in a medium of PBS + 20% FCS (HM1) containing 10% v/v G for 5 min, followed by 10% v/v G + 20% v/v E for 5 min., and then transferred to a vitrification solution of 25% v/v G + 25% v/v E in HM1 for 30 s. The embryos were immediately aspirated into an Open Pulled Straw (OPS) and plunged into liquid nitrogen. Embryos vitrified in GE were warmed by immersing the OPS in HM1 containing 1 M sucrose for 1 min (37°C), then stepwise diluted in fresh HM1 containing 1 M, 0.5 M, and 0.25 M sucrose for 5 min; and finally washed in HM1. Stepwise equilibration and dilution of GE embryos was at 20°C. Embryos (n = 43) assigned to DE vitrification were equilibrated in a medium of PBS + 5% FCS (HM2) containing 10% v/v D + 10% v/v E for 1 min, and then transferred to a vitrification solution of 20% v/v D + 20% v/v E in HM2 for 30 s. The embryos were immediately aspirated into an Open Pulled Straw (OPS) and plunged into liquid nitrogen. Embryos vitrified in DE were warmed by immersing the OPS in HM2 containing 0.25 M sucrose for 1 min (39°C), then stepwise diluted in fresh HM2 containing 0.25 M and 0.15 M sucrose for 5 min, and finally washed in HM2. Stepwise equilibration and dilution of DE embryos was at 39°C. Diluted embryos from both groups and untreated control embryos (n = 49) were cultured in TCM-199 with monolayer granulosa cells for 72 h in conditions described above. Blastocyst re-expansion and hatching was assessed and analyzed by chi-square test. Overall, 67% of the thawed embryos were expanded blastocysts (remainder were blastocysts) and 56% were excellent quality (remainder were good). No significant difference (P > 0.05) was found between the rates of blastocyst re-expansion and hatching for the GE and DE vitrification procedures (respectively, 59 and 79%, and 41 and 58%). However the hatching rate of control embryos (77%) was significantly higher than that of vitrified embryos (P < 0.05). These results indicate that both vitrification procedures are promising for the cryopreservation of crossbred B. indicus × B. taurus in vitro-produced embryos. Supported by CNPq.