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

• Published in: Biology of reproduction Vol. 68; no. 5; pp. 1828 - 1835
• Main Authors: Nagy, S, Jansen, J, Topper, E.K, Gadella, B.M
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01.05.2003
• Subjects: acrosome; Acrosome - physiology; Animals; Biological and medical sciences; Birth control; bulls; Cattle; Cell Membrane - physiology; Cell Survival - physiology; Coloring Agents; Cryopreservation; differential staining; egg yolk; Egg Yolk - chemistry; flow cytometry; Flow Cytometry - methods; Fluorescein-5-isothiocyanate; fluorescence; Fluorescent Dyes; fluorometric assessments; Gynecology. Andrology. Obstetrics; In Vitro Techniques; jc-1; Male; Medical sciences; Membranes - physiology; mitochondrial-function; Peanut Agglutinin; phycoerythrin-conjugated peanut agglutinin; plasma membrane; propidium iodide; repeatability; Reproducibility of Results; semen extenders; sperm viability; spermatozoa; Spermatozoa - physiology; Spermatozoa - ultrastructure; Sterility. Assisted procreation; SYBR-14; thawing; viability; Egg yolk; Spermatozoa; Structure integrity; Staining; Ox; Thawing; Germinal cell; Freezing; Particle; Vertebrata; Mammalia; Cryopreservation; Artificial insemination; Plasma membrane; Artiodactyla; Technique; Acrosome; Ungulata
• DOI: 10.1095/biolreprod.102.011445
• ISSN: 0006-3363

Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present 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 the discrimination of 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 no longer 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 fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h 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 to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.