|Barbosa, E - University Of Brazil|
|Praamsma, C - Colorado Parks And Wildlife, Fish Research Hatchery|
|Schisler, G - Colorado Parks And Wildlife|
Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/23/2016
Publication Date: 6/1/2016
Citation: Purdy, P.H., Barbosa, E.A., Praamsma, C.J., Schisler, G.J. 2016. Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential. Cryobiology. 73:73-79.
Interpretive Summary: Activation and freezing induce membrane lipid and protein reorganization and alter cellular processes in trout sperm. Excessive reorganization may be detrimental to sperm function and inhibit fertilization. Therefore, we investigated the effects of two activation solutions on trout sperm to determine if we could develop a method to effectively evaluate the quality and fertilizing potential of a trout sperm sample. Our analyses revealed that we can identify inferior sperm samples based on the levels of intracellular calcium, membrane lipid organization and membrane protein organization following freezing and thawing. Use of these methods could enable producers to identify sperm samples of inferior quality and avoid their use thereby increasing the productivity of the hatchery, laboratory or gene bank.
Technical Abstract: 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. Cryopreservation caused greater phospholipid disorder (high plasma membrane fluidity; P < 0.0001) compared with fresh samples but water activation resulted in more fluid plasma membranes compared to Lahnsteiner solution activated samples (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 (R2 = -0.9; P = 0.01) and LAS (R2 = -0.85; P = 0.03) activation. Greater plasma membrane phospholipid (R2 = -0.89; P = 0.02) and protein (R2 = -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.