50 Effect of colony-stimulating factor 2 on competence of bovine blastocysts to survive vitrification

Abstract

Colony-stimulating factor 2 (CSF2) is an important maternal regulator of embryonic development. Treatment of in vitro-produced embryos with CSF2 from Days 5 to 7 after insemination increased competence of embryos to establish pregnancy after transfer. One action of CSF2 is to protect embryos from stress. Treatment with CSF2 reduced heat-shocked induced apoptosis in morulae and regulated expression of multiple genes involved in cellular stress responses in the blastocyst. Here we tested whether CSF2 would promote survival of embryos to another stress: cryodamage caused by vitrification. Ovaries were recovered from an abattoir and used to harvest cumulus-oocyte complexes (COCs). Groups of 10 COCs were matured for 20-22 h. After maturation, COCs were fertilised with 1 × 10⁶ mL⁻¹ isolate-purified spermatozoa from a pool of frozen-thawed semen from 3 bulls. Fertilization proceeded for 20-22 h. Cumulus cells were removed from putative zygotes using 1000 U mL⁻¹ hyaluronidase by agitation and putative zygotes were cultured in groups of 30 in 50-µL drops of synthetic oviducal fluid-bovine embryo 2 (SOF-BE2) medium until Day 7 after insemination. Drops of cultured embryos were randomly assigned to receive either 10 ng mL⁻¹ recombinant bovine CSF2 or vehicle [90% (vol/vol) SOF-BE2 and 10% (vol/vol) Dulbecco's phosphate-buffered saline containing 1 mg mL⁻¹ bovine serum albumin) on Day 5. Blastocyst-stage embryos were selected for vitrification at Day 7. Blastocysts were vitrified in open-pulled straws and rewarmed according to procedures of Vajta et al. (1996 Theriogenology 45, 683-689). After warming, embryos were cultured for 72 h in groups of 5-12 in SOF-BE2 containing 10% fetal bovine serum and 50 µM dithiothreitol. Blastocysts were assessed for re-expansion (defined as restoration of a reconstituted blastocoele) and hatching (defined as partial or completion exit from the zona pellucida). A total of 11 replicates were performed using 1745 zygotes treated with CSF2 and 1690 zygotes treated with vehicle. The number of blastocysts vitrified were 280 for CSF2 and 289 for vehicle. Data on percent presumptive zygotes becoming blastocysts and on blastocyst re-expansion and hatching after thawing were calculated for each replicate and analysed by analysis of variance using the GLIMMIX procedure of SAS (SAS Institute Inc.). The percentage of putative zygotes that became blastocysts was not affected by treatment (21.5 ± 1% for CSF2 vs. 20.9 ± 1% for vehicle). Overall, CSF2 increased (P = 0.021) the percentage of blastocysts that re-expanded (77.8 + 0.05% vs. 73.3 + 0.05%), but there was no effect of CSF2 on the percentage of blastocysts that underwent hatching (39.3 + 0.06% vs. 37.4 + 0.06%). In conclusion, CSF2 increased blastocyst survival following vitrification. Further work will be performed to evaluate whether CSF2 also increases survival of vitrified embryos after embryo transfer.

Support was provided by NIH R01 HD088352.