Gene Polymorphisms in Boar Spermatozoa and Their Associations with Post-Thaw Semen Quality

Abstract

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3’-untranslated regions (3’-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1, PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality.

Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals

“…The progressive advancement in OMICS techniques made possible to measure the link between gene polymorphism and sperm freeze-thawed activity. RNA-Seq datasets were used to identify SNPs, and a total of 40 SNPs were genotyped, while several polymorphisms in MS4A2, MAP3K20, and ROBO1 genes were significantly associated with sperm motility, membrane integrity, reduced cryo-induced lipid per-oxidase, and DNA damage in the boar spermatozoa (17). The genotyping frequencies are different among the genotype groups, while the Gene Ontology terminology (e.g., stress response) is relevant to polymorphisms, such as MAP3K20 (rs340643892), APPL1 (rs339379734), and MS4A2 (rs339836492) and play an important role in the cryopreservation stresses (113).…”

“…Spermatozoa contain a subset of RNAs, including mRNAs, non-coding RNAs [ncRNA including microRNAs (miRNAs)], mitochondrial (mtRNA), and ribosomal RNAs (rRNAs) that can be routinely isolated from the sperm of several species including bulls, horses, and pigs (16,72,118). This novel approach is based on sperm RNA-sequencing (RNAseq) data analysis, by comparing the mRNA profile between higher and lower post-thawing semen to identify marker genes for mammalian semen post-thawing (16,17).…”

“…Although it has been proved that some sperm protein markers are correlated with high cryo-tolerance, their function is reliant on the presence of mRNA (7). It has been recommended that spermatozoa RNAs evaluation provides valuable information on their biological function (16,17).…”

Regulatory Potential of Long Non-Coding RNAs (lncRNAs) in Boar Spermatozoa with Good and Poor Freezability

“…Remarkably, a significantly enriched KEGG pathway was not detected for ROBO2 , which is associated with the axon guidance pathway [ 80 ]. We have shown in our recent study that the ROBO1 is significantly associated with the axon guidance pathway [ 43 ], which appears to have a potential biological function in boar fertility [ 57 ]. It is noteworthy that ROBO2 receptors have been detected in the testis, and their interaction with a secretory protein (SLIT) contributes to the SLIT–ROBO signaling pathway that is implicated in cell adhesion and cell death [ 80 ].…”

“…ANOVA analysis was performed with the general linear modeling (GLM) procedure. The following linear model was used to analyze the effect of the boar on the fresh semen quality or PT semen quality traits, as described in a recent study [ 43 ].…”