Follicular fluid secretome enhances the meiotic competence of in vitro matured equine oocytes, but not the developmental competence of zygotes after intracytoplasmic sperm injection (ICSI).

Overview

• This study investigated the impact of supplementing mare preovulatory follicular fluid (PFF) secretome during the in vitro maturation (IVM) of equine oocytes on their maturation and developmental competence.
• The addition of PFF secretome improved oocyte maturation rates but did not enhance embryo development or influence gene expression in embryos after fertilization by intracytoplasmic sperm injection (ICSI).

Background

• In vitro embryo production (IVP) is used to assist reproduction in horses but typically shows low efficiency, mostly because oocytes matured in vitro often have reduced developmental competence.
• The follicular fluid surrounding the oocyte contains molecules secreted by follicle cells (secretome) which may support oocyte maturation and developmental potential.
• This study focused on testing whether adding secretome isolated from mare preovulatory follicular fluid to the IVM medium could improve oocyte maturation and development.

Methods

• Oocytes were collected from mare ovaries after death (post-mortem).
• Secretome was isolated from preovulatory follicular fluid of mares and added to the oocyte maturation medium at two dosages: 20 µg/ml (S20) and 40 µg/ml (S40).
• After in vitro maturation, oocytes were fertilized by intracytoplasmic sperm injection (ICSI).
• Outcomes measured included:
  • Percentage of oocytes reaching meiosis (maturation rate).
  • Proportion of degenerated oocytes post-maturation.
  • Embryo cleavage rate (early cell divisions post-fertilization).
  • Blastocyst development rate (ability to reach the blastocyst stage by day 7).
  • Expression levels of seven candidate genes in day 7 embryos via real-time quantitative PCR: BEX2, FABP3, ODC, MOBKL3, HSP90AA1, BAX, and BCL2.

Results

• The higher concentration of secretome (S40, 40 µg/ml) significantly improved oocyte maturation rates compared to the control without secretome:
  • Maturation rates: 46.5% (S40) vs. 35.4% (Control).
  • Reduced degenerated oocytes: 44.0% (S40) vs. 58.6% (Control).
• The lower secretome concentration (S20) did not show significant improvements.
• No significant differences were observed between the groups in:
  • Cleavage rates (initial embryonic cell divisions).
  • Blastocyst formation rates at day 7 post-ICSI.
  • Expression of the seven candidate genes in the resulting day 7 embryos.

Interpretation

• Supplementation with PFF secretome at 40 µg/ml during IVM supports the meiotic competence of oocytes, meaning it helps the oocytes to mature properly.
• However, this improvement in maturation did not translate into better embryonic development or changes in the expression of genes linked to embryo quality or development.
• This suggests that while follicular fluid secretome contains factors that promote meiotic progression, additional factors or conditions may be needed to enhance the subsequent developmental competence of equine embryos produced in vitro.

Significance and Future Directions

• The findings provide a potential method to improve oocyte maturation rates in equine IVP by adding PFF secretome during IVM.
• However, since embryo development and gene expression were unaffected, this approach alone may not increase the overall success of embryo production.
• Further research could explore:
  • Identifying specific bioactive components within the secretome responsible for improved meiotic competence.
  • Combining secretome supplementation with other culture improvements to promote embryonic development.
  • Evaluating long-term outcomes post-embryo transfer and live foal rates to assess clinical usefulness.