Isolation of high-quality stallion spermatozoa: a comparison of methods and their effects on sperm quality and yield, DNA damage, and functional competence by ICSI.

Overview

• This study compared four different techniques for isolating stallion sperm to determine their effects on sperm quality, DNA damage, and developmental success when used in intracytoplasmic sperm injection (ICSI) for equine embryo production.
• Results indicated that while all methods produced sperm capable of supporting embryo development, they differed in sperm recovery efficiency and impact on sperm quality parameters.

Background and Objective

• ICSI is the main approach for producing horse embryos in vitro, where a single sperm is injected directly into an oocyte.
• The success of ICSI can be influenced by how sperm are selected and isolated from the semen sample.
• The study aimed to compare four sperm isolation methods—EquiPure™, Vetmotl™, Felix™, and Samson™—to evaluate their effects on:
• Sperm motility and viability
• Integrity of sperm DNA
• The yield of high-quality sperm cells relative to the original sample
• The ability of isolated sperm to support embryonic development to cleavage and blastocyst stages following ICSI

Methods

• Semen collection was conducted on four fertile stallions to provide representative samples.
• Each semen sample was processed using the four isolation methods plus a control procedure involving simple centrifugation without isolation (non-isolated).
• Sperm parameters evaluated after isolation included motility (ability to move progressively), viability (live vs dead cells), DNA integrity (checking for breaks or damage), and overall yield of viable sperm in relation to input quantities.
• Isolated spermatozoa were used for ICSI with oocytes matured in vitro.
• Embryo development metrics recorded were cleavage rates (early cell division) and blastocyst rates (later-stage embryo formation).

Results

• Sperm quality improvements:
  • Vetmotl, Felix, and Samson isolation protocols improved sperm motility and viability compared to non-isolated centrifuged samples.
  • Vetmotl particularly improved DNA integrity, indicating better selection against damaged sperm.
• Cell recovery efficiency:
  • When accounting for concentration and total numbers, Samson, EquiPure, and Felix retrieved significantly higher numbers of motile, viable, and DNA-intact sperm than Vetmotl.
  • Vetmotl showed the lowest overall cell recovery despite high quality.
  • Samson isolation achieved the greatest recovery of progressively motile sperm, which are most important for fertilization.
• Embryo development outcomes:
  • All four isolation methods yielded sperm capable of supporting embryo development in vitro.
  • Cleavage rates ranged from 51.2% to 69.4%, and blastocyst rates ranged from 20.5% to 24.5% across all methods.
  • No statistically significant differences in cleavage or blastocyst rates were observed between the different isolation techniques.

Conclusions

• Established and newer sperm isolation methods effectively isolate high-quality stallion sperm suitable for ICSI and subsequent embryo production.
• Vetmotl, Felix, and Samson performed similarly to conventional methods in supporting embryo development.
• There are trade-offs between isolation methods in terms of:
  • Efficiency of sperm recovery (number of cells obtained)
  • Labor intensity (effort and time required)
  • Stringency of sperm selection (quality vs quantity)
• These differences have implications for:
  • Storage of sperm for later use
  • The ability to perform low-dose insemination, which requires fewer sperm cells
  • In vitro fertilization and embryo production success rates
• Further research is needed involving more stallions and in vivo embryo transfer outcomes to establish the clinical reproductive relevance and optimize choice of isolation methods.