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.

Keywords: RNA-Seq; SNPs; freezability; genotype; variant calling.

Figure 1

Gene ontology (GO) of enriched terms associated with candidate genes (CGs) in boar spermatozoa. A. Biological process categories; B. molecular function categories; C. cellular component categories. The values in the parenthesis indicate the number of inputs and the percentage of CGs enriched to the category.

Figure 2

Genotyping cluster plots of A. APPL1, B. OXSR1, C. FBXO16, D. RAB3C, E. PLBD1, and F. SARS genes using the Kompetitive Allele Specific PCR (KASP) assay. Samples shown in pink were considered questionable genotypes as they were not assigned to the cluster. NTC, non-template control. Visualization of the genotype data was performed with the SNPViewer software (http://results.lgcgenomics.com/software/snpviewer).

Figure 3

Effect of polymorphisms in A. APPL1, B. CYP7B1, C. RAB3C, D. OXSR1, E. FBXO16, and F. PLBD1 genes on post-thaw motility of boar spermatozoa. Data are presented as the mean ± SEM; ^a,b Differences between genotypes were significantly differed at p < 0.05.

Figure 4

Effect of PLBD1 gene polymorphism on post-thaw A. MMP, B. PMI, C. NAR acrosome integrity, and D. LPO of boar spermatozoa. Data are presented as the mean ± SEM. ^a,b Differences between genotypes were significantly differed at p < 0.05.

Figure 5

Effect of polymorphisms in A–B. APPL1, C–E. EML5, and F. FBXO16 genes on post-thaw membrane integrity of boar spermatozoa. Data are presented as the mean ± SEM. ^a,b Differences between genotypes were significantly different at p < 0.05.

Figure 6

Effect of polymorphisms in A–B. SARS, C–D. RAB3C, and E–F. PRICKLE1 genes on post-thaw membrane integrity of boar spermatozoa. Data are presented as the mean ± SEM. ^a,b Differences between genotypes were significantly different at p < 0.05.

Figure 7

Effect of freezability and gene polymorphisms in A–B. SCLTL1, C–D. MAP3K20, E. MS4A2, and F. ROBO1 genes on post-thaw quality of boar semen. Data are presented as the mean ± SEM. ^a,b Differences between genotypes were significantly different at p < 0.05; GSF—good semen freezability; PSF—poor semen freezability.