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Detection of early damage of sperm cell membrane in Gilthead seabream ( Sparus aurata ) with the nuclear stain YO‐PRO 1
Author(s) -
Beirão J.,
PérezCerezales S.,
MartínezPáramo S.,
Herráez M. P.
Publication year - 2010
Publication title -
journal of applied ichthyology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 62
eISSN - 1439-0426
pISSN - 0175-8659
DOI - 10.1111/j.1439-0426.2010.01560.x
Subject(s) - sperm , cryopreservation , biology , cryoprotectant , flow cytometry , extender , andrology , membrane permeability , staining , stain , membrane , microbiology and biotechnology , biochemistry , embryo , chemistry , botany , genetics , medicine , organic chemistry , polyurethane
Summary For an accurate evaluation of spermatozoa membrane state, both in fresh and cryopreserved sperm, it is important to have an assay capable of identifying small changes in membrane permeability. In this study we examined for the first time in fishes the feasibility of the YO‐PRO 1 nucleic acid stain to detect early membrane changes in samples frozen using different sperm cryopreservation protocols. Gilthead seabream ( Sparus aurata ) sperm were cryopreserved using 1% NaCl as extender and three different cryoprotectant mixtures: 5% DMSO, 5% DMSO plus 10 mg ml −1 BSA and 5% DMSO plus 10% egg yolk. Fresh and frozen/thawed sperm motility were evaluated with CASA software and sperm membrane integrity was evaluated by flow cytometry with two assays: PI/SYBR 14 and PI/YO‐PRO 1. The PI/YO‐PRO 1 assay allowed the identification of a subpopulation of cells, comprised of disturbed cell membrane, which was not noticed when using the common PI/SYBR 14 assay. This subpopulation increased in abundance after sperm cryopreservation and, as was perceived by flow cytometry, these changes in membrane permeability and/or integrity were also accompanied by morphometric modifications.