Freeze-thawing impairs the motility, plasma membrane integrity and mitochondria function of boar spermatozoa through generating excessive ROS

Bin Zhang,Shulei Qiu,Xiaolan Chen,Huimei Xie,Bingyan Cai,Yan Wang,Caihong Wu

doi:10.1186/s12917-021-02804-1

Bin Zhang, Shulei Qiu + Show 5 more

Open Access

https://doi.org/10.1186/s12917-021-02804-1

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Abstract

BackgroundCryopreservation is an efficient way to store spermatozoa and is closely associated with the quality of sperm after the freeze-thaw process. During freeze-thaw cycling, excessive reactive oxygen species (ROS) are produced, and the effects of ROS on boar sperm during cryopreservation have not been identified.ResultsIn this study, we evaluated the quality of boar spermatozoa in different steps of cryopreservation (extension, cooling, and thawing for 30 min and 240 min) with or without boar-sperm antioxidant (N-acetylcysteine (NAC)). The ROS levels, sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, ATP content, and sperm apoptosis were assayed. After thawing, the ROS level and sperm apoptosis were significantly increased, and the sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, and ATP content were significantly impaired compared with those at the extension period and cooling period. Moreover, the addition of N-acetyl L-cysteine (NAC) reversed these changes.ConclusionThe freeze-thawing of boar spermatozoa impaired their motility, plasma membrane, mitochondrial activity, sperm chromatin structure and apoptosis by producing excessive ROS. Thus, the downregulation of ROS level by antioxidants, especially the NAC, is important for manufacturing frozen pig sperm to increase reproductive cells and livestock propagation, as well as to improve the application of frozen semen in pigs worldwide.

Highlights

Cryopreservation is an efficient way to store spermatozoa and is closely associated with the quality of sperm after the freeze-thaw process
Effects of cryopreservation on the boar spermatozoa reactive oxygen species (ROS) levels The ROS level of frozen-thawed spermatozoa was detected by fluorescence using the DCFH-DA assay
A significantly high ROS level was observed in thawed spermatozoa with an increase in the thawing time compared with the spermatozoa at the extension period and cooling period in the control group, and the ROS level of thawed spermatozoa was inhibited in the N-acetyl L-cysteine (NAC) group (Fig. 1a and b)

Summary

Introduction

Cryopreservation is an efficient way to store spermatozoa and is closely associated with the quality of sperm after the freeze-thaw process. During freeze-thaw cycling, excessive reactive oxygen species (ROS) are produced, and the effects of ROS on boar sperm during cryopreservation have not been identified. Physical changes during freezing and thawing can lead to changes in sperm morphology and function, affecting sperm survival and reducing fertilization ability, Reactive oxygen species (ROS) have physiologically relevant roles in improving intracellular cAMP levels, acrosome reactions, hyperactivation and spermatozoa. Boar sperm are more vulnerable to peroxidative damage during cryopreservation due to its high content of polyunsaturated fatty acids [18,19,20] that serve as preferred substrates for ROS generation in membranes [21], resulting in limitations of the use of frozen pig sperm [19]. Little is known about the influence of ROS on the cryopreservation of boar sperm

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