196 EFFECTS OF DIFFERENT IN VITRO MATURATION SYSTEMS ON BOVINE EMBRYO DEVELOPMENT

Abstract

Bovine oocytes and embryos have been established as a valuable model for studying human early development, specifically after assisted reproductive technologies (ART). Efforts for the improvement of ART in the last years have focused on culture media and conditions. Recently, Albuz et al. (2010) reported that the culture of bovine cumulus–oocyte complexes (COC) with cyclic adenosine 3′, 5′-monophosphate (cAMP) modulators, before and during extended in vitro maturation (IVM), improved blastocyst quality and yields in mice and cattle. In this study, we investigated the influence of an extended IVM phase in combination with cAMP modulators on blastocyst yields and quality, the effects on mRNA expression profiles and epigenetic marks. We compared these results to the standard protocol (Wrenzycki et al., 2001) used in our laboratory with oocytes from different retrieval methods. Oocytes were retrieved from slaughterhouse ovaries either by slicing or follicular aspiration. The COC were either subjected directly to IVM using the standard TCM-based protocol for 24 h (TCM24-slicing and TCM24-aspiration, respectively) or oocytes that were retrieved by aspiration were treated with forskolin and IBMX for a 2-h pre-IVM period, followed by an extended IVM phase of 30 h in TCM, supplemented with cilostamide (cAMP30-aspiration). Statistical analyses were performed using 1-way ANOVA followed by the nonparametrical Kruskal–Wallis test. Maturation rates were 79.3 ± 2.6% in TCM24-aspiration, 74.2 ± 8.8% in cAMP30-aspiration and 70.4 ± 5.1% in TCM24-slicing oocytes. Matured oocytes were fertilized in vitro with semen from a bull previously proven to be suitable for IVF. Blastocyst rates from presumptive zygotes were significantly higher (P = 0.003) in the TCM24-aspiration group (32 ± 7%) compared to TCM24-slicing (23 ± 7%) and cAMP30-aspiration (22 ± 5%). Analysis revealed that cell numbers were rather similar in the 3 experimental groups (125 ± 19, 128 ± 15 and 129 ± 9), while in vivo-produced blastocysts possessed slightly more cells (134 ± 17; P ≥ 0.05). RT-qPCR analysis of mRNA expression for a panel of genes indicative of embryo quality including DNMT3a, SLC2A8, COX2 and PCK2, showed that blastocysts derived from both aspiration protocols were similar to in vivo embryos, but were different from blastocysts resulting from the ovary-slicing protocol. Specifically, the expression profile of COX2, which is involved in pregnancy outcome and in the response to growth factors, indicates an enhanced developmental competence of aspirated oocytes. However, the transcript level of EGR1 (early growth response) was significantly higher (P = 0.009) in in vivo-derived blastocysts in comparison to all in vitro treatments. The investigation of the epigenetic status of the in vitro-derived blastocysts based on bisulfite sequencing of 2 satellite repeat sequences is currently underway. Results so far indicate that the method of obtaining the oocytes (slicing vs aspiration) for in vitro production of bovine embryos is of greater influence on blastocyst quality than IVM conditions.