# Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential

## TL;DR

Researchers developed a simplified vitrification method for preserving feline sperm without cryoprotectants, which maintains sperm quality and fertilization potential.

## Contribution

The study introduces a permeating cryoprotectant-free vitrification protocol using trehalose for feline epididymal spermatozoa.

## Key findings

- Optimized vitrification using 0.5 M trehalose preserved sperm motility and viability.
- Vitrified sperm supported fertilization and early embryo development, though blastocyst formation was reduced.
- DNA fragmentation remained minimal despite increased acrosomal damage in vitrified sperm.

## Abstract

Cryopreservation of feline sperm is an important tool for assisted reproduction and conservation programs in domestic and wild felids. This study investigated a simplified, permeating cryoprotectant-free vitrification method using disaccharides (sucrose and trehalose) and evaluated factors influencing post-warming sperm quality. The findings demonstrated that optimized vitrification using 0.5 M trehalose, combined with a rapid mixing protocol, preserved sperm motility and viability and supported fertilization and early embryo development. This approach offered a practical alternative for feline sperm cryopreservation in clinical and field settings.

The aim of this study was to optimize a permeating cryoprotectant-free vitrification protocol for feline epididymal spermatozoa and evaluate its effects on post-warming sperm quality and fertilizing potential. Four experiments were conducted to assess the impact of the disaccharide concentration (sucrose), type (sucrose vs. trehalose), and stepwise mixing volume with equilibration time and fertilization potential. Post-warming sperm motility, viability, DNA fragmentation, and acrosomal integrity were evaluated. Fertilizing ability was assessed by in vitro fertilization (IVF) and embryo development. The optimal condition—0.5 M trehalose with 20 µL stepwise mixing and no equilibration—resulted in the highest post-warming motility and viability. Vitrified sperm exhibited significantly higher acrosomal damage compared to fresh controls, but DNA fragmentation remained minimal. IVF using vitrified sperm achieved comparable cleavage rates, although blastocyst formation was reduced compared to fresh spermatozoa. These findings confirm the feasibility of vitrification without permeating cryoprotectants and provide a simplified, efficient protocol for feline sperm preservation.