Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

The main objective of this study was to investigate boar-to-boar variations in the quality characteristics of sperm from the sperm-rich fractions (SRFs) and whole ejaculates (WEs) following freezing–thawing. Several sperm attributes, such as motility patterns analyzed by the computer-assisted sperm analysis (CASA) system, mitochondrial function, membrane integrity, and DNA fragmentation were used to compare the cryo-survival of sperm from SRFs and WEs from boars with good and poor semen freezability (GSF and PSF, respectively). In this study, boars with post-thaw total motility (TMOT) more than 30% (>30%) were classified as having GSF, while those with post-thaw TMOT less than 30% (<30%) were classified as having PSF. Principal component analysis 1 (PCA1), which is the main component of the sample variation, explained approximately 75% of the variance between the GSF and PSF groups, reaffirming the reliability of post-thaw TMOT as a reliable criterion used to classify the animals. Most of the post-thaw sperm parameters of the SRFs and WEs were positively correlated. Furthermore, scatter plot analyses show stronger relationships between the analyzed post-thaw parameters of the frozen–thawed (FT) sperm of SRFs than those of WEs. Individual boar variations or the sperm source had marked effects on the quality characteristics of FT sperm. The higher TMOT, velocity straight line (VSL), and velocity average path (VAP) of FT sperm were more enhanced in the SRFs compared with the WEs of the PSF group. Furthermore, the mitochondrial function, membrane integrity, and DNA fragmentation of FT sperm were markedly higher in the SRFs than in the WEs, particularly for the poor freezability boars. We suggest that the freezability potential of sperm of the GSF group does not differ significantly between the SRFs and WEs, reaffirming that boar variability is an important factor that affects the cryo-survival of sperm.