Effect on equine sperm of post-thaw glycerol dilution using two different semen extenders.

Overview

• This study investigated how post-thaw dilution with two different extenders affects the quality of frozen-thawed equine sperm, focusing on minimizing the toxic and osmotic effects of glycerol, a common cryoprotectant.
• The researchers compared semen diluted with Tyrodes solution and a commercial equine extender after thawing, assessing various sperm quality parameters.

Background

• Glycerol is widely used as a penetrating cryoprotectant in deep freezing of spermatozoa to protect sperm cells during freezing and thawing processes.
• Despite its protective role, glycerol can cause toxic, chemical, and osmotic stress, which may alter the lipid structure of the sperm membrane.
• Previous evidence suggests rapid glycerol addition is less harmful than its removal, so optimization of glycerol handling is important for maintaining sperm quality.
• Post-thaw dilution is of interest because it might reduce glycerol’s osmotic and toxic effects after thawing.

Objective

• The main goal was to evaluate and minimize the toxic and osmotic impact of glycerol on equine sperm after thawing by using two different dilution extenders: Tyrodes (a basic buffered salt solution) and a commercial equine extender (CE) designed for semen preservation.

Methods

• Equine semen from nine stallions was cryopreserved using 5% glycerol as the cryoprotectant.
• After thawing, semen samples were diluted 1:2 with either Tyrodes solution or the commercial equine extender.
• The diluted samples were incubated at 22°C for 30 minutes to simulate post-thaw handling conditions.
• Several sperm quality parameters were measured, including:
  • Kinematic parameters like total motility (TM) and progressive motility (PM), using Computer-Assisted Semen Analysis (CASA).
  • Plasma membrane integrity and acrosome status using fluorescein isothiocyanate-peanut agglutinin-propidium iodide (FITC-PNA-PI) staining.
  • Lipid peroxidation using BODIPY581/591 staining to measure oxidative damage.
  • DNA fragmentation assessed by the Halo test to identify sperm DNA damage.

Results

• No significant differences were found between the control (no dilution after thawing) and the two extenders (Tyrodes and commercial extender) for:
  • Total motility (TM): Control 43.8%, Tyrodes 41%, CE 40.4% (P > 0.05)
  • Progressive motility (PM): Control 30.6%, Tyrodes 27.5%, CE 29.7% (P > 0.05)
  • DNA fragmentation and lipid peroxidation levels also showed no significant differences between groups (P > 0.05).
• However, there were statistically significant improvements in plasma membrane and acrosome integrity for samples diluted with the commercial extender:
  • Sperm with intact plasma membranes and acrosomes were higher in the CE group (39.4%) compared to control (33.9%) and Tyrodes (33.5%) (P < 0.05).

Conclusions

• Post-thaw dilution with either Tyrodes or the commercial extender did not improve motility, DNA integrity, or oxidative stress markers compared to the control (no dilution).
• Dilution with the commercial equine extender showed benefits in maintaining membrane and acrosomal integrity, indicating it may help reduce the early acrosome reaction, which is premature sperm activation leading to reduced fertility.
• Overall, while post-thaw dilution is not broadly beneficial for all sperm quality parameters, the choice of extender matters, and using a commercial extender may enhance certain aspects of sperm viability after thawing.
• These insights can guide better semen handling protocols to optimize fertility outcomes in equine breeding programs utilizing cryopreserved sperm.