Pre-maturation of oocytes for equine cloning using cAMP modulators

Abstract

The asynchrony of cytoplasmic and nuclear maturation in cumulus oocyte complexes (COCs) due to decreased concentrations of cyclic adenosine phosphate (cAMP) has been shown to result in decreased oocyte developmental competence (Rose et al., Theriogenology. 2013;79: 142–148). The aim of this study was to evaluate the effect of pre-maturation treatment with cAMP modulators, previously shown to delay spontaneous nuclear maturation, on the developmental competence of oocytes used for equine cloning. Equine ovaries were obtained from a slaughterhouse and processed within 1-2h. The COCs were washed three times with H-SOF medium and transferred to cryovials filled with a 1:1 mixture of DMEM-F12 and TCM-199 containing 10% FCS and kept at 20-22°C. Groups of COCs were either untreated (control) or treated with 50 mM forskolin (FSK) and 100 mM 3-isobutyl-1-methylxanthine (IBMX) for 4 or 18h. Immature oocytes from all three groups were washed 3 times with H-SOF medium, then transferred to maturation medium, and incubated for 18h at 38.5°C in a humidified atmosphere of 5% CO2 in air. A total of 715 oocytes were collected in 6 replicates (191 oocytes inthe 4h FSK-IBMX group, 239 in the 18h FSK-IBMX group, and 285 oocytes in the control). The methods used for in vitro maturation (IVM), SCNT, and embryo culture were identical for all three groups. Six different fibroblast cell lines were used to provide the donor nuclei. Meiotic maturation, fusion, cleavage, and embryonic development in vitro were all evaluated. Data were analyzed using the Genstat 18th Edition statistical software package. While the meiotic maturation and cleavage rates were similar for the three groups, the proportion of fused couplets that developed to the blastocyst stage was lower for the 4 FSK-IBMX group (15.4 ± 5.7%) compared to the control group (25.6 ± 6.4%; P<0.05). Interestingly, the fusion rates for oocytes of the 4 and 18h FSK-IBMX groups (90.3 ± 4.7% and 94.3 ± 2.6%, respectively) were greater than that for oocytes of the control group (79.1 ± 2.1%; P<0.05). As a result, the pre-maturation treatment did not reduce the blastocyst yield from couplets reconstructed. Further studies are needed to determine the impact of such cAMP modulating treatments on embryos’ in vivo viability following transfer to recipient mares.