Reproductive Technology

A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles

Abstract

Evaluating post-thaw viability and acrosome integrity of spermatozoa simultaneously by flow cytometry would provide a valuable testing tool in research and routine work as well. In this study, a new, triple stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR 14 and propidium iodide (PI) enabled to discriminate sperm cells from egg-yolk and debris particles; which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm because it implied that washing steps to remove egg-yolk were not anymore required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome damaged/reacted sperm cells from acrosome intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method agreement analysis between the SYBR 14/PE-PNA/PI and fluoresce in isothiocyanate-conjugated peanut agglutinin, FITC-PNA/PI staining was carried out on 14 frozen/thawed semen samples immediately after thawing and after a three-hour-long incubation at 37°C. The British Standards Institution repeatability index of the SYBR 14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple stain combination is highly repeatable and easy in routine application and it provides a more precise estimate of the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.