title: Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential.
abstract: We investigated the effects of two trout sperm activation solutions on sperm physiology and membrane organization prior to and following cryopreservation using flow cytometry and investigated their impact on in vitro fertility. Overall, frozen-thawed samples had greater phospholipid disorder when compared with fresh samples (high plasma membrane fluidity; P < 0.0001) and sperm activated with water also had high plasma membrane fluidity when compared to sperm activated with Lahnsteiner solution (LAS; P < 0.0001). Following cryopreservation water activated samples had membranes with greater membrane protein disorganization compared with LAS but the membrane protein organization of LAS samples was similar to samples prior to freezing (P < 0.0001). Post-thaw water activation resulted in significant increases in intracellular calcium compared to LAS (P < 0.002). In vitro fertility trials with frozen-thawed milt and LAS activation resulted in greater fertility (45%) compared to water activated samples (10%; P < 0.0001). Higher fertility rates correlated with lower intracellular calcium with water (R(2) = -0.9; P = 0.01) and LAS (R(2) = -0.85; P = 0.03) activation. Greater plasma membrane phospholipid (R(2) = -0.89; P = 0.02) and protein (R(2) = -0.84; P = 0.04) disorder correlated with lower water activation fertility rates. These membrane organization characteristics only approached significance with LAS activation in vitro fertility (P = 0.09, P = 0.06, respectively). Potentially the understanding of sperm membrane reorganizations and the physiology associated with activation following cryopreservation may enable users in a repository or hatchery setting to estimate the fertilizing potential of a sample and determine its value.
authors.affiliation: USDA, ARS, NLGRP, National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO 80521-4500, USA. Electronic address: phil.purdy@ars.usda.gov.
authors.forename: P H
authors.initials: PH
authors.lastname: Purdy
authors.affiliation: University of Brasilia, Brasilia, 70910-900, Brazil; Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB s/n°., Brasília, DF, Brasil, CEP 70770-901, Brazil. Electronic address: eleonora_vet@hotmail.com.
authors.forename: E A
authors.initials: EA
authors.lastname: Barbosa
authors.affiliation: Colorado Parks and Wildlife, Fish Research Hatchery, 5500 WCR 50C, Bellvue, CO 80512, USA. Electronic address: chris.praamsma@state.co.us.
authors.forename: C J
authors.initials: CJ
authors.lastname: Praamsma
authors.affiliation: Colorado Parks and Wildlife, 317 West Prospect Street, Fort Collins, CO 80526, USA. Electronic address: george.schisler@state.co.us.
authors.forename: G J
authors.initials: GJ
authors.lastname: Schisler
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D005298:Fertility; D005615:Freezing; D008297:Male; D008560:Membrane Fluidity / Q000187:drug effects; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects*; D014337:Trout
keywords: Cryopreservation; Fertility; Flow cytometry; Merocyanine 540; Oncorhynchus clarki; Phospholipid; Quanti-iT protein assay; Trout sperm