Effect of Pre-IVM Duration with cAMP Modulators on the Production of Cloned Equine Embryos and Foals

Abstract

The asynchrony of cytoplasmic and nuclear maturation in cumulus-oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20-22 °C. Within the cryovials, the COCs were either untreated (Control) for 18 h or treated with 50 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine for the first 4 h (Pre-IVM 4 h) or the entire 18 h (Pre-IVM 18 h). Oocytes were then transferred to maturation medium and incubated for a further 22-24 h at 38.5 °C in 5% CO₂ in air. Somatic cell nuclear transfer embryos were then produced using the meiotically mature oocytes and donor cells from six different fibroblast cell lines. The rates of maturation and embryo development did not differ significantly between the groups, though blastocyst formation tended to be inferior in the Pre-IVM 4 h group compared with the Control group (p = 0.06). Of 67 blastocysts produced, 23 were transferred to recipient mares on Day 4 or 5 post-ovulation. Regarding the pregnancy outcomes, no significant differences were found between the groups, and four viable foals were born, each derived from a different donor cell line. The findings expand on those from previous evaluations of this biphasic IVM system, and indicate that the cAMP-modulating treatments exert limited effects under the pre-IVM conditions used here.

Keywords: biphasic IVM; blastocyst; embryo transfer; horse; in vitro maturation (IVM); simulated physiological oocyte maturation (SPOM); somatic cell nuclear transfer (SCNT).

Figure 1

Effect of the cAMP-modulating pre-IVM treatments on SCNT embryo production (Control, Pre-IVM 4 h, and Pre-IVM 18 h groups). (A) The percentage of oocytes that matured after an additional 22–24 h of IVM, as determined by the presence of a first polar body. (B) The percentage of constructed couplets that fused. (C) The percentage of fused couplets that cleaved after 48 h of embryo culture. (D) The percentage of fused couplets that formed blastocysts. (E) The percentage of cleaved embryos that formed blastocysts. (F) The overall cloning efficiency rate, expressed as the percentage of cumulus–oocyte complexes (COCs) that resulted in blastocysts. Values are presented as the mean ± s.e.m. No significant differences were detected between the groups (p > 0.05). Values labeled with an asterisk tend to differ (p < 0.1).

Figure 2

Effect of the donor cell lines (denoted as #01 to #06) on SCNT embryo production. (A) The percentage of constructed couplets that fused. (B) The percentage of fused couplets that cleaved. (C) The percentage of cleaved embryos that formed blastocysts. Values are presented as the mean ± s.e.m. No significant differences were detected between the donor cell lines (p > 0.05).