Humidity resistant live-dead stain for spermatozoa

Area of Science:

• Veterinary Science
• Reproductive Biology
• Microscopy Techniques

Background:

• Live-dead staining of spermatozoa is crucial for assessing cell viability.
• Conventional stains like eosin are susceptible to environmental factors, particularly high relative humidity (RH).
• Dye migration in conventional slides leads to loss of contrast and unreliable viability assessment.

Purpose of the Study:

• To evaluate the stability of live-dead stained bovine spermatozoa slides under high relative humidity.
• To identify alternative staining methods that resist dye migration and maintain slide contrast.
• To determine the efficacy of erythrosin B and brilliant green for sperm viability assessment in humid environments.

Main Methods:

• Preparation of live-dead stained bovine spermatozoa slides using conventional and alternative dye combinations.
• Exposure of prepared slides to high relative humidity conditions (up to 98% RH).
• Assessment of slide contrast and cell viability over a seven-day period at controlled high RH.

Main Results:

• Conventional eosin-based slides exhibited complete loss of contrast within 60 minutes at 85% RH due to water-soluble dye migration.
• Slides stained with erythrosin B and brilliant green maintained satisfactory contrast between live and dead cells.
• The enhanced contrast stability was observed even at 98% RH throughout the seven-day trial.

Conclusions:

• Conventional live-dead staining methods are unreliable in high humidity environments.
• Erythrosin B and brilliant green offer a stable and reliable alternative for bovine spermatozoa viability assessment.
• These alternative stains are suitable for use in challenging environmental conditions, ensuring accurate diagnostic outcomes.