Can We Save Valuable Stallion Semen by Refreezing?

Approximately 100 million sperm are contained in a standard 0.5-mL straw of frozen equine semen, yet intracytoplasmic sperm injection (ICSI) requires only a single sperm to fertilize an egg. This means that millions of sperm are going to waste during each ICIS procedure, resulting in a significant loss of genetic material, especially  if straws are from popular or genetically sought-after stallions with limited semen supply.

To curb these losses, veterinary researchers looked at refreezing thawed semen in lower concentrations to produce multiple straws for future use.^* Because stallion semen responds variably to freezing, some semen samples perform better after thawing when using different types of extenders. Because of this, three different extenders were evaluated in this study.

Ejaculates were obtained from six stallions and frozen using extender 1 (a low egg yolk/glyceride/amide extender). All semen samples were then thawed, extended again using three different extenders: extender 1 (described above), extender 2 (high egg yolk/glycerol), and extender 3 (low egg yolk/milk/glycerol). All samples were then refrozen. In each of these extenders, the egg yolk/milk serves as a lipid and protein source and the glycerol and amide act as cryoprotectants.

After thawing the refrozen sperm, semen analysis (e.g., various motility parameters) and ICSI were performed.

Results showed that extender affected ICSI outcomes.

 “Semen refrozen in extender 1 performed less favorably for ICSI than the once-frozen sperm. When extenders 2 and 3 were used, however, no differences in key parameters such as cleavage rate and blastocyst development were noted compared to once-frozen sperm,” explained Ashley Fowler, Ph.D., a Kentucky Equine Research nutritionist.

Extender 1 contained amides as a cryoprotectant, and the research team therefore advised using extenders without amides when refreezing semen samples.

Interestingly, the semen refrozen in extender 1  had the lowest ICSI outcomes but the highest sperm motility and viability.

“Traditional measures of sperm quality were inadequate to assess the suitability of refrozen sperm for ICSI,” wrote the researchers.

“Another way to improve sperm quality during freezing cycles is supplementing the stallion with long-chain polyunsaturated omega-3 fatty acids, such as those found in EO-3. Studies show that feeding long-chain polyunsaturated omega-3 fatty acids can improve sperm motility and maintain better motility after freezing due to increased sperm membrane elasticity. Supplementing with EO-3 has the greatest effects on stallions with semen that has a low tolerance for cryopreservation,” said Fowler.

 ^*Brito, L.F.C., M.R. Felix, R.L. Linardi, E.V. Martinez de Andino, N.S. Balamurugan, C. Hernández-Avilés, and K. Hinrichs. 2024. Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection. Theriogenology 231:29-35.