Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a...

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Autores principales:	Catarina Anjos, Ana Luísa Santos, Daniel Duarte, Domitília Matias, Elsa Cabrita
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	cryopreservation oyster sperm trehalose sucrose cryodamage Physiology QP1-981
Acceso en línea:	https://doaj.org/article/4b64148674a94eb787884fdb44d76610
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spelling	oai:doaj.org-article:4b64148674a94eb787884fdb44d766102021-12-01T01:40:03ZEffect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm1664-042X10.3389/fphys.2021.749735https://doaj.org/article/4b64148674a94eb787884fdb44d766102021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fphys.2021.749735/fullhttps://doaj.org/toc/1664-042XSperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.Catarina AnjosCatarina AnjosAna Luísa SantosDaniel DuarteDomitília MatiasElsa CabritaFrontiers Media S.A.articlecryopreservationoysterspermtrehalosesucrosecryodamagePhysiologyQP1-981ENFrontiers in Physiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cryopreservation oyster sperm trehalose sucrose cryodamage Physiology QP1-981
spellingShingle	cryopreservation oyster sperm trehalose sucrose cryodamage Physiology QP1-981 Catarina Anjos Catarina Anjos Ana Luísa Santos Daniel Duarte Domitília Matias Elsa Cabrita Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
description	Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.
format	article
author	Catarina Anjos Catarina Anjos Ana Luísa Santos Daniel Duarte Domitília Matias Elsa Cabrita
author_facet	Catarina Anjos Catarina Anjos Ana Luísa Santos Daniel Duarte Domitília Matias Elsa Cabrita
author_sort	Catarina Anjos
title	Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
title_short	Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
title_full	Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
title_fullStr	Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
title_full_unstemmed	Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
title_sort	effect of trehalose and sucrose in post-thaw quality of crassostrea angulata sperm
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/4b64148674a94eb787884fdb44d76610
work_keys_str_mv	AT catarinaanjos effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm AT catarinaanjos effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm AT analuisasantos effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm AT danielduarte effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm AT domitiliamatias effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm AT elsacabrita effectoftrehaloseandsucroseinpostthawqualityofcrassostreaangulatasperm
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Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

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