105 BOVINE DEMI-BLASTOCYSTS ARE ABLE TO EXPAND TO A SIZE SIMILAR TO INTACT BLASTOCYSTS UNTIL AT LEAST DAY 13 OF IN VITRO CULTURE

Embryo bisection has been used to produce identical twins, to increase the pregnancy rate per embryo, and for preimplantation diagnosis. However, the invasive nature of splitting might affect development decreasing embryo survival. In general, after bisection or biopsy, embryos are transferred to surrogate mothers and their competence evaluated in terms of implantation and pregnancy maintenance. However, this makes it difficult to evaluate the immediate response of each embryo to bisection. Our aim was to evaluate embryo growth during the 5 days following bisection by using an extended in vitro culture system. We postulated that bisected blastocysts are able to counteract the injury and expand in size until Day 13 of in vitro culture. Two experiments were performed. First, two different culture systems were evaluated to determine the best to support embryo development from Day 9 to 13. One system consisted of conventional culture in plastic (CCP), while the other one included co-culture with endometrial cells derived from a cycling cow (CC). Both used SOFaa supplemented with 3 mg mL–1 of fatty acid-free BSA and 2% FBS in 4-well dishes. Twenty-six nonbisected in vitro-derived blastocysts were cultured. Embryo size and survival were recorded daily. All living embryos were measured with Micrometrics™ SE Premium software and statistical analyses were performed using the Kruskal–Wallis test. From Day 9 to 11, blastocysts cultured in the CCP system had smaller diameters than those cultured in CC [Day 9: CC 358 µm, CCP 277 µm (P = 0.04); Day 10: CC 456 µm, CCP 340 µm (P = 0.005); and Day 11: CC 535 µm, CCP 408 µm (P = 0.02)]. However, on Day 12 and 13, no difference was observed in embryo diameters [(Day 12: CC 560 µm, CCP 411 µm (P = 0.1) and Day 13: CC 470 µm, CCP 474 µm (P = 0.9)]. Additionally, embryos with diameters less than 200 µm on Day 9 did not develop further independent of the culture system (P < 0.001). Thus, in the second experiment, to determine embryo size after bisection, only well-expanded grade 1 blastocysts >200 µm were used in the conventional CCP system. Twenty four Day 8 bisected (B) and nonbisected (C) blastocysts were cultured from Day 9 until 13. In the bisected group, one-half was kept for further gene expression analysis. Significant differences were observed in embryo diameter between both groups on Day 9 and 10 of culture [Day 9: B 321 µm, C 277 µm (P = 0.05); D10: B 436 µm, C 340 µm (P = 0.01)]. However, on Days 11, 12, and 13, no differences in diameter were observed (Day 11: B 411 µm, C 408 µm (P = 0.8); Day 12: B 394 µm, C 411 µm (P = 0.5); Day 13: B 316 µm, C 474 µm (P = 0.3)]. In conclusion, we show that bovine embryos are capable of developing in vitro until Day 13, and embryo diameter on Day 9 impacts on the subsequent in vitro survival of nonsplit embryos, regardless of culture system. Finally, on Day 11 of culture, the split embryos were able to overcome the injury caused by the bisection procedure and expanded in size similar to controls, until at least Day 13 of culture.