Oocyte pre-IVM 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 IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before 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 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before 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%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance

<i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.

<p><i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.</p

Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.

<p>Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.</p

Gene expression profiles in expanded blastocysts produced from adult and prepubertal oocytes treated with or without cAMP modulators prior to and during IVM.

<p>Data are shown as the mean ± SEM (n = 12). Data were analyzed using two-way ANOVA followed by a Tukey's range test. The asterisk represents statistical significance among treatments for the same transcript; <i>P</i> < 0.05. <i>In vivo</i> produced expanded blastocysts were used for comparison. The mRNA relative abundance of the EGR1 gene was lower in all <i>in vitro</i> derived blastocysts compared to <i>in vivo</i> produced counterparts. No differences among treatments were found for <i>DNMT3b</i>, <i>BCL2L1</i>, <i>PRDX1</i>, <i>SLC2A8</i>.</p