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

## TL;DR

This study investigates how pre-IVM treatments with cAMP modulators affect the development of cloned equine embryos and foals, finding limited impact on outcomes.

## Contribution

The study evaluates the effect of varying durations of pre-IVM cAMP modulator treatments on equine oocyte developmental competence for cloning.

## Key findings

- Pre-IVM treatment with cAMP modulators did not significantly improve in vitro embryo development or pregnancy outcomes in horses.
- Blastocyst formation tended to be lower with short pre-IVM treatment, but not significantly.
- Four viable foals were born from transferred blastocysts, with no significant differences between treatment groups.

## Abstract

The efficiency of equine embryo in vitro production is mainly limited by the capacity of current in vitro maturation (IVM) systems to support the acquisition of oocyte developmental competence. Oocyte quality may be improved by better coordinating nuclear and cytoplasmic maturation using pre-IVM treatments that modulate cAMP levels. The aim of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for short and long durations on equine oocyte quality. Following maturation with or without the pre-IVM treatments, the oocytes were used to produce cloned embryos and early development was assessed. Additionally, cohorts of blastocysts were transferred to recipient mares and the resulting pregnancies were monitored. The in vitro development of embryos did not differ significantly between the groups, though blastocyst formation tended to be inferior when the oocytes were subjected to the short pre-IVM. The in vivo development of transferred blastocysts was not adversely impacted by the pre-IVM treatments, with pregnancies established and foals born in all groups. While the use of cAMP modulators in this biphasic IVM system supported successful outcomes, it did not enhance the production of cloned equine embryos and foals.

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.