title: A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio.
abstract: The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.
authors: 1 Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center , Baton Rouge, Louisiana.|2 School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences, University of Florida , Gainesville, Florida.
authors: Huiping
authors: H
authors: Yang
authors: 1 Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center , Baton Rouge, Louisiana.
authors: Jonathan
authors: J
authors: Daly
authors: 3 Zebrafish International Resource Center, University of Oregon , Eugene, Oregon.
authors: Carrie
authors: C
authors: Carmichael
authors: 3 Zebrafish International Resource Center, University of Oregon , Eugene, Oregon.
authors: Jen
authors: J
authors: Matthews
authors: 3 Zebrafish International Resource Center, University of Oregon , Eugene, Oregon.
authors: Zoltan M
authors: ZM
authors: Varga
authors: 1 Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center , Baton Rouge, Louisiana.
authors: Terrence
authors: T
authors: Tiersch
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D008297:Male; D012662:Semen Preservation; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D015027:Zebrafish / Q000502:physiology*
chemical_list: D003451:Cryoprotective Agents