title: 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: 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 degrees 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.
authors.affiliation: Research Institute for Animal Breeding and Nutrition, Department of Cell Biology, Herceghalom, Hungary.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Nagy
authors.forename: Johannes
authors.initials: J
authors.lastname: Jansen
authors.forename: Einko K
authors.initials: EK
authors.lastname: Topper
authors.forename: Barend M
authors.initials: BM
authors.lastname: Gadella
mesh_terms: D000177:Acrosome / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D004396:Coloring Agents; D015925:Cryopreservation; D004530:Egg Yolk / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000502:physiology; D019887:Peanut Agglutinin; D015203:Reproducibility of Results; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D004396:Coloring Agents; D005456:Fluorescent Dyes; D019887:Peanut Agglutinin; D016650:Fluorescein-5-isothiocyanate