Marked blastomere herniation is the only post-thaw morphological characteristic associated with a reduced pregnancy rate for in vitro produced equine blastocysts.

Overview

• This study investigated whether the appearance of in vitro produced (IVP) equine embryos after thawing and biopsy before freezing affects pregnancy success rates after embryo transfer.
• The main finding was that significant herniation of blastomeres (cells) post-thawing is linked to lower pregnancy rates, while other morphological features and biopsy did not negatively impact outcomes.

Background and Purpose

• Equine IVP embryos can be vitrified (frozen) and later thawed for transfer to recipient mares, but success rates vary.
• Some embryos undergo biopsy before freezing to harvest cells, a process involving sampling through a hole created during the ICSI (intracytoplasmic sperm injection) procedure.
• The study aimed to determine if specific post-thaw morphological traits or the biopsy procedure itself affected pregnancy outcomes after transfer of frozen-thawed IVP blastocysts.

Methods

• Embryos were produced via ICSI and either biopsied or left intact before slow freezing.
• Biopsy involved collecting cells that herniated through the ICSI-derived hole in the zona pellucida (ZP).
• Post-thaw, the embryos were assessed for morphological characteristics including:
  • Shape
  • Extent of presumptive trophectoderm layer
  • Percentage of extruded blastomeres
  • Degree of expansion
  • Percentage of herniated cells
• A total of 480 embryos were studied: 36 biopsied and 444 non-biopsied.
• Embryos were transferred to recipient mares, and pregnancy outcomes were recorded and analyzed statistically using a multivariate generalized linear model to identify factors affecting pregnancy rates.

Key Findings

• The overall pregnancy rate after embryo transfer was 73.3%.
• Pregnancy rates were lower when blastocyst formation was delayed after ICSI, indicating quicker developing embryos had better pregnancy outcomes (p < 0.01).
• The stallion used for ICSI and the biological mother mare influenced pregnancy outcomes (stallion p < 0.01, mother p = 0.02), though no individual animal was singled out as significantly better or worse.
• Performing a biopsy prior to freezing did not significantly reduce pregnancy rates (p = 0.59), indicating the biopsy procedure itself is safe for embryo viability.
• Blastocysts with more than 10% herniated cells post-thaw had significantly reduced pregnancy rates (39%) compared to those with fewer than 10% (69%) or no herniated cells (77%) (p = 0.02).
• No other morphological post-thaw characteristics (shape, trophectoderm extent, expansion, extruded blastomeres) were linked to pregnancy success.

Conclusions and Implications

• The presence of marked blastomere herniation—cells extruding through the hole in the ZP after thawing—is the only morphology factor associated with reduced pregnancy success in frozen-thawed IVP equine blastocysts.
• The biopsy technique, involving sampling from herniated cells, does not compromise embryo viability or reduce pregnancy rates.
• Embryos that herniate extensively through the ICSI hole upon thawing may be less viable and less likely to establish pregnancy after transfer.
• Monitoring post-thaw herniation could be used as a criterion for selecting embryos with higher implantation potential.
• This research enhances understanding of factors influencing success in equine assisted reproduction and supports refinement of protocols for embryo freezing and transfer.