Effects of Delipidation with Forskolin During In Vitro Culture of Bovine Embryos and Recipient Synchronization on Pregnancy Rates. (2010)

Cryotolerance of in vitro produced embryos can be improved by reduction of lipid droplet accumulation by microsurgery or chemical delipidation. We previously determined that forskolin caused delipidation during culture of bovine embryos; however, pregnancy rates were not evaluated; also, we hypothesized that thawing embryos and culturing them in vitro for 10 h in order to transfer only the expanded blastocysts could result in improved pregnancy rates. Recent research has shown that synchronization with a 5 day CO-synch plus progestagen protocol results in higher pregnancy rates after artificial insemination than a 7 day protocol; however, these protocols have not yet been compared for embryo transfer. The aim of this experiment was to evaluate pregnancy outcomes of delipidated, frozen-thawed-cultured in vitro produced embryos vs. in vitro and in vivo controls, as well as the two estrus synchronization protocols mentioned above. Oocytes were collected from slaughterhouse ovaries, matured, fertilized, and the resulting embryos cultured in vitro by standard procedures in a chemically defined medium. Day 0 of culture was ~18 h after the onset of IVF. At 6.0 days of culture, half of morulae were exposed to 10 μM forskolin, and the rest served as controls. At 7.5 days after fertilization, embryos were conventionally frozen with ethylene glycol. Also, in vivo produced embryos were collected nonsurgically from superovulated cows, and cryopreserved conventionally. Recipients were assigned to two synchronization protocols: 5 (n=59) or 7 day (n=56) CO-synch plus progestagen plus 400 IU equine chorionic gonadotrophin on the day of CIDR removal; embryos were transferred nonsurgically 7 days after the last GnRH injection. Recipients were assigned to 4 embryo cryopreservation treatments: 1) in vivo produced control (n=29), 2) in vitro produced control (n=21), 3) in vitro produced plus forskolin (n=36), 4) in vitro produced plus forskolin, plus thawed in the laboratory and cultured for 10 h, and only transferring expanded blastocysts (n=29; ~70% expanded). Pregnancies were evaluated at 41 days post GnRH by ultrasonography by detecting an amniotic vesicle or live fetus. Data was analyzed by Proc GenMod, using 1 for pregnant and 0 for nonpregnant. Pregnancy rate with the 5 day synchronization protocol was not different (P > 0.05) from that obtained with the 7 day protocol (27.1 vs. 23.2%, P > 0.1). There was an effect (P < 0.05) of cryopreservation treatment on pregnancy rates; in vivo control 41.4%a, in vitro plus forskolin plus 10 h culture 31.0a,b in vitro plus forskolin 16.7%b,c, and in vitro control 9.3%c (means without common superscripts differ, P < 0.05). There was no interaction (P > 0.1) among treatments. In conclusion, 5 day CO-Synch + progestagen treatment was similar to 7 day, and in vitro treatment of embryos to delipidate them with forskolin plus 10 h of culture post-thaw was similar to the in vivo control, and could be an alternative to improve pregnancy rates. Research supported by PROMEP, SEP, Grant UACH-PTC-087. (poster)