Sperm DNA damage in relation to lipid peroxidation following freezing-thawing of boar semen

Abstract

This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based extenders after single and repeated freezing and thawing. As well as an analysis of sperm motion characteristics, mitochondrial function, membrane integrity, and lipid peroxidation (LPO) were assessed simultaneously with the measurements of sperm DNA damage. Consistent positive significant correlations were found between sperm DNA damage and LPO after freezing-thawing. Comet assay measurements showed that cryo-induced sperm DNA damage was more marked in the cryoprotectant-free extender, irrespective of freezing cycle. The frequency of sperm cells with damaged DNA increased with repeated freezing and thawing in the cryoprotectant-based extenders. Except for sperm DNA damage, there were no consistent associations between post-thaw sperm LPO and sperm quality characteristics. It could be suggested that the increased LPO of membrane phospholipids is associated with higher susceptibility of boar spermatozoa to cryoinduced DNA damage. ______________________________________________________________________________________ Keywords: Comet assay measurements, cryopreservation, extenders, spermatozoa # Corresponding author: fraser@uwm.edu.pl Evidence has shown that reduced fertilizing ability of frozen-thawed spermatozoa is because of compromised viability, DNA integrity, and increased peroxidative damage (Thompson et al., 2009;Fraser et al., 2010;Hamilton et al., 2016;Fraser et al., 2016). Intact sperm DNA integrity is essential for fertilization and embryo development (Evenson, 2016). Accumulating evidence has shown that sperm DNA damage is a useful marker for post-thaw semen quality (Fraser et al., 2010;Gosálvez et al., 2011;Gürler et al., 2016). Cryo-induced oxidative stress, associated with excess reactive oxygen species (ROS), has a negative effect on sperm nuclear DNA integrity (Peris et al., 2007; Thompson et al., 2009;Gürler et al., 2016). Moreover, lipid peroxidation (LPO) cascade is initiated by oxidative challenge on the membrane phospholipids of spermatozoa, which significantly compromises their function (Neild et al., 2005;Whitaker et al., 2012;Hamilton et al., 2016). Studies on the relationships between DNA damage and LPO of frozen-thawed boar spermatozoa are limited. This study therefore investigated the relationships between sperm DNA damage and LPO following freezing-thawing of boar semen in a cryoprotectant-free extender or in cryoprotectantbased extenders supplemented with whole hen egg yolk (HEY), ostrich egg yolk (OEY) and lipoprotein fractions isolated from OEY (LPFo). Repeated freezing and thawing of semen samples was performed to assess its effect on post-thaw sperm DNA integrity.Sperm-rich fractions (SRFs) were collected from five Polish Large White boars (average age 18 months), using the gloved-hand tech...

Effects of supplemental seminal plasma on cryopreserved boar sperm quality

“…The viability values found in the thawed semen are similar to those reported by Roca et al (2006). Similar to the current results, decreased spermatozoa with intact acrosome after freezing-thawing have been reported by other authors (Trzcińska et al, 2015;Fraser et al, 2017). This damage was more evident in cells from ejaculates diluted with BTS compared with semen that was not diluted.…”

“…Freeze-thawed boar semen has limited commercial use because during the freezing-thawing process, 50% of the spermatozoa lose their fertilizing capacity (Qian et al, 2016) as a result of thermal shock, which compromises viability and integrity of DNA and increased lipid peroxidation of membrane phospholipids (Fraser et al, 2017). Cryodamage is not restricted to sperm membrane, but affects the integrity of sperm chromatin (nucleoprotein structure and DNA) and mitochondrial function, involves degradation of mRNAs, and impairs the function of certain proteins (Yeste, 2016).…”

N‐acetyl‐ l ‐cysteine and alpha lipoic acid are protective supplement on human sperm parameters in cryopreservation of asthenoteratozoospermia patients

“…The different study has been shown that ROS level has a positive correlation with the reduction sperm parameters, DNA damage, DNA fragmentation and chromatin deficiency (Saeednia et al, 2015). Since DNA damage leads to sperm dysfunction and male infertility, it is necessary to evaluate sperm chromatin after the cryopreservation process (Fraser et al, 2017). The result of many study indicated that are significant relationship between reduced amount antioxidants and low quality of sperm parameters and male infertility.…”

Assessment of Ageing-Dependent Effects on Sperm Functions following Semen Cryopreservation

“…1,2 Cryo-induced damage to spermatozoa is attributed to membrane deterioration caused mainly by the formation of intracellular ice and increased oxidative stress, resulting in a loss of the sperm fertilizing ability. 3 It is noteworthy that ageing-dependent processes in frozen-thawed semen occur in different sperm compartments, and are associated with membrane modifications of specific lipid-protein interactions, activation of an apoptosis-like mechanism and reduced genomic integrity, which consequently compromise sperm cryo-survival. 4,5 Moreover, ageing is a natural process occurring in sperm cells during semen preservation, and there is no single sperm test that can effectively predict the fertilizing capacity of semen following the different reproductive technologies.…”