FGF2, LIF, and IGF1 supplementation during in vitro maturation or culture improves equine ICSI embryo development in an age-dependent manner.

Overview

• This study investigated whether adding a combination of growth factors—fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), collectively called FLI—to the maturation or culture media improves embryo development in horses following intracytoplasmic sperm injection (ICSI).
• It also explored if the age of the donor mare affects the response to this supplementation during in vitro maturation (IVM) or in vitro culture (IVC).

Background

• Equine assisted reproduction technologies like ovum pickup (OPU) and ICSI help produce embryos from horses that have limited natural breeding opportunities.
• Despite these advances, embryo production efficiency remains limited due to suboptimal in vitro maturation and culture conditions.
• Previous research in other species has suggested that supplementing maturation or culture media with growth factors such as FGF2, LIF, and IGF1 combined (FLI) enhances oocyte quality and embryo development.
• This study aimed to test this supplementation strategy in horses and examine whether donor mare age modulates its effect.

Methods

• Oocytes were collected from donor mares and divided into groups to receive in vitro maturation (IVM) with or without FLI supplementation.
• After intracytoplasmic sperm injection (ICSI), embryos were cultured in vitro (IVC) either with or without FLI added to the culture medium.
• Developmental milestones were tracked, including:
• Oocyte maturation rates
• Cleavage rates (earliest division following fertilization)
• Blastocyst formation rates (an important stage of embryo development)
• Blastocyst diameter, as a metric of embryo growth and quality
• Pregnancy establishment after embryo transfer, monitored up to Day 45
• Donor mares were categorized by age (e.g., young, middle-aged, older) to analyze age-dependent effects of FLI.

Results

• FLI supplementation during IVM or IVC did not significantly change overall oocyte maturation rates or early cleavage percentages.
• Blastocyst formation rates increased significantly only in oocytes from middle-aged mares when FLI was added in either maturation or culture phases, suggesting an age-related benefit.
• Adding FLI during embryo culture (IVC) also increased blastocyst diameter, indicating improved embryo quality or growth.
• Pregnancy rates established by Day 45 after embryo transfer were marginally higher for embryos from FLI-treated oocytes, especially after freezing and thawing (vitrification), which implies improved embryo viability.

Conclusions and Implications

• The beneficial effects of FLI supplementation appear to be age-dependent, with middle-aged donor mares benefiting most from the growth factor combination.
• This suggests that aging-related changes in oocyte or embryo physiology might be mitigated by growth factor support during maturation or culture.
• The study supports the inclusion of FLI supplementation in equine assisted reproduction protocols to enhance embryo production, especially for donor mares in the middle age range.
• These findings lay groundwork for future molecular studies to understand how exactly these growth factors improve embryo development at the cellular and genetic levels.
• Practically, implementing FLI supplementation could improve clinical outcomes in equine reproduction by increasing embryo yield and quality, particularly benefiting middle-aged mares whose reproductive efficiency may naturally decline.