Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa.

The research article aims to investigate the effects of follicular fluid and varying levels of progesterone on horse oocytes during in vitro maturation prior to insemination with both non-sorted and sex-sorted sperm.

Experimental Design

• The study is divided into two experiments. In Experiment 1, the maturation of compact cumulus oocyte complexes (COCs) was performed under three different conditions: a control medium, a medium with 20% follicular fluid, and a control medium containing 250 ng of progesterone per ml. For comparative analysis, mature oocytes were also collected through a process known as transvaginal aspiration.
• In Experiment 2, oocytes with expanded COCs were matured in a medium with varying concentrations of progesterone (0, 50, 250, and 1250 ng per ml). The chosen matured oocytes were then inseminated and the embryos cultured in a chemically defined medium.

In Vitro Fertilization and Embryo Culture

• A technique known as intracytoplasmic sperm injection (ICSI) was used to fertilize the oocytes. Following the ICSI process, the fertilized oocytes were cultured in Menezo’s B2 with an added 5% of fetal calf serum.
• The embryos cultured in medium containing 1250 ng of progesterone per ml presented fewer oocytes with a visible first polar body after maturation.
• Interestingly, it was noted that those cultured in the media containing 0 and 50 ng progesterone per ml had higher development rates, leading to the production of seven to eight-cell embryos compared to those cultivated in media with 250 or 1250 ng progesterone per ml.

Transfers and Outcome

• Certain resulting embryos were transferred to recipient mares. However, no pregnancies were established following the transfers.
• In a separate investigation, a portion of oocytes from Experiment 2 were injected with sex-sorted sperm, which had been separated into X- and Y-bearing spermatozoa using a Cytomation MoFlo flow cytometer/cell sorter. Two embryos resulted from ICSI with X-bearing spermatozoa and were transferred to a mare, again resulting in no established pregnancies.

Conclusions

• The research revealed no significant differences among in vitro maturation treatments in Experiment 1.
• In Experiment 2, lower levels of progesterone in the media appeared to result in higher development of the embryos.
• However, none of the embryo transfers in the experiments resulted in established pregnancies, prompting further investigation to determine the possible causes and to optimize the in vitro maturation and fertilization processes.