Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another wa...

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Autores: Domínguez-Rebolledo, Álvaro Efrén, Fernández-Santos, M. Rocío, García-Álvarez, Olga, Maroto-Morales, Alejandro, Garde, José Julián, Martínez-Pastor, Felipe
Tipo de recurso: artículo
Estado:Versión enviada para evaluación y publicación
Fecha de publicación:2009
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/408121
Acceso en línea:http://hdl.handle.net/10261/408121
Access Level:acceso abierto
Palabra clave:Antioxidants
Centrifugation
Oxidative stress
Washing
Red deer
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spelling Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoaDomínguez-Rebolledo, Álvaro EfrénFernández-Santos, M. RocíoGarcía-Álvarez, OlgaMaroto-Morales, AlejandroGarde, José JuliánMartínez-Pastor, FelipeAntioxidantsCentrifugationOxidative stressWashingRed deerThe effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 × g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4 h was assessed using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30 ± 3.52% to 67.94 ± 5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.This work was supported by the Spanish Ministry of Science and Innovation (grant no. AGL2004-05904/GAN) and by the Education and Science Council of Junta de Comunidades de Castilla-La Mancha (grant no. PAC06-0047). The authors thank Enrique del Olmo, Alfonso Bisbal, and José Luis Ros for help with the experiments. A.E. Domínguez-Rebolledo was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico). F. Martìnez-Pastor was supported by the Juan de la Cierva program and by the Ramón y Cajal program (Ministry of Science and Innovation, Spain), and M.R. Fernández-Santos was supported by the Juan de la Cierva program (Ministry of Science and Innovation, Spain). Olga García-Álvarez and Alejandro Maroto-Morales were recipients of scholarships from INIA and JCCM (Spain), respectively.Peer reviewedElsevierMinisterio de Ciencia e Innovación (España)Junta de Comunidades de Castilla-La ManchaConsejo Nacional de Ciencia y Tecnología (México)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202520252009info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Preprintinfo:eu-repo/semantics/submittedVersionapplication/pdfhttp://hdl.handle.net/10261/408121reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttps://doi.org/10.1016/j.theriogenology.2009.06.027Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4081212026-05-22T06:33:51Z
dc.title.none.fl_str_mv Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
title Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
spellingShingle Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
Domínguez-Rebolledo, Álvaro Efrén
Antioxidants
Centrifugation
Oxidative stress
Washing
Red deer
title_short Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
title_full Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
title_fullStr Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
title_full_unstemmed Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
title_sort Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa
dc.creator.none.fl_str_mv Domínguez-Rebolledo, Álvaro Efrén
Fernández-Santos, M. Rocío
García-Álvarez, Olga
Maroto-Morales, Alejandro
Garde, José Julián
Martínez-Pastor, Felipe
author Domínguez-Rebolledo, Álvaro Efrén
author_facet Domínguez-Rebolledo, Álvaro Efrén
Fernández-Santos, M. Rocío
García-Álvarez, Olga
Maroto-Morales, Alejandro
Garde, José Julián
Martínez-Pastor, Felipe
author_role author
author2 Fernández-Santos, M. Rocío
García-Álvarez, Olga
Maroto-Morales, Alejandro
Garde, José Julián
Martínez-Pastor, Felipe
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Junta de Comunidades de Castilla-La Mancha
Consejo Nacional de Ciencia y Tecnología (México)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Antioxidants
Centrifugation
Oxidative stress
Washing
Red deer
topic Antioxidants
Centrifugation
Oxidative stress
Washing
Red deer
description The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 × g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4 h was assessed using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30 ± 3.52% to 67.94 ± 5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.
publishDate 2009
dc.date.none.fl_str_mv 2009
2025
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Preprint
info:eu-repo/semantics/submittedVersion
format article
status_str submittedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/408121
url http://hdl.handle.net/10261/408121
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://doi.org/10.1016/j.theriogenology.2009.06.027
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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