152 IN VITRO AND IN VIVO DEVELOPMENT OF BOVINE IVP EMBRYOS FOLLOWING SINGLE-CELL BIOPSY ON DAY 4

Abstract

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO₂ in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 10⁶/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO₂, 7% O₂, 88% N₂. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.