Oocyte pre- in vitro maturation with caffeine improves bovine embryo survival after vitrification

Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during in vitro maturation (IVM) has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-in vitro maturation (pre-IVM) with caffeine on oocyte maturation, intra-oocyte cAMP concentration, developmental competence after in vitro fertilization and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2h culture prior to IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until day 8. Expanded blastocysts derived from either standard control or the 10 mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9h IVM in a concentration-dependent manner. The cAMP levels were similar prior to and after IVM. Matured oocytes, cleavage and blastocyst rates were reduced in the 30 mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10 mM caffeine group (73.8%) compared with the standard control (59.7%). Re-expansion rates and total number of cells after 48h were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation prior to IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance.