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

Abstract

Background 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. Results In 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. Conclusion The 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.

Evaluation of supplementation of cryopreservation medium with gallic acid as an antioxidant in quality of post‐thaw human spermatozoa

“…ROS can interact with multiple cellular targets including membrane polyunsaturated fatty acids and DNA, respectively, resulting in lipid peroxidation and DNA damage (Dinga et al, 2021;Emokpae & Moronkeji, 2022). As a result, during cryopreservation, a high degree of ROS generation and weakened antioxidant defence might have negative effects on sperm structure, function, and viability, which can affect sperm fertilizing potential (Agarwal et al, 2014;Santonastaso et al, 2021;Zhang et al, 2021). Therefore, it is clear that adding antioxidants to the freezing solution during sperm cryopreservation should be a suitable approach (Bahmyari et al, 2020).…”

Ameliorative effect of silymarin on the quality of frozen–thawed boar spermatozoa

“…Based on these data, we suggested that the protective effects of the electron transport chain of mitochondria (Zhang et al, 2021).…”

“…Mitochondrial damage can cause an imbalance between ROS production and removal, resulting in the accumulation of ROS. Extensive ROS attacks polyunsaturated fatty acids on the sperm plasma membrane, forms lipid peroxide and in turn destroys the electron transport chain of mitochondria (Zhang et al, 2021). In our study, we found that the addition of SMN significantly upregulated ATP content and enhanced the mitochondrial activity of frozen–thawed sperm compared with the CTR (Table 3), which was in agreement with the sperm quality parameters results (Figure 1).…”

“…Besides, ROS accumulation will attack mitochondrial DNA (mtDNA), and in return mitochondrial oxidative damage will lead to abnormal mitochondrial electron transport, electron leakage, the ATP synthesis decline, stimulating ROS to continue to generate, resulting in a vicious circle (Aitken, 2017). At the same time, a large amount of ROS affects intracellular calcium ion balance, changes mitochondrial fusion protein‐2 and tyrosine phosphorylation patterns in sperm and affects mitochondrial activity (Hezavehei et al, 2018; Yadav et al, 2017; Zhang et al, 2021). The damage caused during the cryopreservation process eventually compromises the sperm motility, capacitation and fertilization capacity.…”

Sperm Selection Procedures for Optimizing the Outcome of ICSI in Patients with NOA

“…Since permeable protectants easily pass through the cell membrane, they form an osmotic gradient that draws water out of the cell, thereby preventing the formation of intracellular ice crystals [ 162 ]. However, these reactions can have toxic effects on the sperm plasma membrane due to lipid peroxidation as well as the stress created during processing [ 178 ]. In contrast, non-permeable cryoprotectants cannot pass through the cell membrane due to their high molecular weight, and extracellular solute accumulation allows the intracellular water to be discharged; thus, dehydration occurs in the cell.…”