Changes in plasma membrane and acrosome integrity of frozen-thawed bovine spermatozoa during a 4 h incubation as measured by multicolor flow cytometry.
Cryopreservation of bull semen is sub-optimal, causing cell death of a majority of spermatozoa. Even the surviving cells are affected post-thaw, either structurally or functionally. The aim of this study was to investigate the sequence of events that take place when sperm plasma membrane and acrosome deteriorate during a 4 h incubation period post-thaw, with special attention paid to the acrosome status of dying cells. Frozen-thawed semen of six AI dairy bulls was used. Three straws per batch were pooled and incubated at 37 degrees C. Sub-samples were taken at 30 min intervals and stained with SYBR 14, propidium iodide (PI) and phycoerythrin-conjugated peanut agglutinin (PE-PNA). Plasma membrane and acrosome integrity were measured by flow cytometry. The experiment was repeated three times. Immediately after thawing, only 3.45% of the dying cells showed acrosomal exocytosis. This number increased dramatically during incubation, reaching 67% after 4 h. Within the intact cell population, the overall decrease in viability and acrosome integrity was kept at five percentage points. Flow cytometry and the triple fluorochrome combination presented a detailed picture of the time course in plasma membrane and acrosome deterioration of frozen-thawed bull semen. The results are expected to be useful for monitoring new cryopreservation protocols.
Nagy
Szabolcs
S
Department of Anatomy and Physiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), PO Box 7011, S-750 07 Uppsala, Sweden. szabio70@hotmail.com
Hallap
Triin
T
Johannisson
Anders
A
Rodriguez-Martinez
Heriberto
H
D000177:Acrosome / Q000648:ultrastructure*; D000818:Animals; D002417:Cattle; D016923:Cell Death; D002462:Cell Membrane / Q000648:ultrastructure*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D005089:Exocytosis; D005434:Flow Cytometry; D006358:Hot Temperature; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology / Q000648:ultrastructure*; D013997:Time Factors