The demand for beef is increasing globally, necessitating improved reproductive efficiency in beef cattle. However, fertility has declined in recent decades, partly due to selection pressure on production traits and the pleiotropic effects of genes impacting both fertility and production. Genetic studies have primarily focused on female fertility, which has low heritability. Bull fertility, however, is moderately heritable and significantly influenced by genetics. Improving bull fertility could significantly enhance overall reproductive success. Scrotal circumference (SC) and sperm motility (SM) are heritable traits correlated with fertility and are good indicators for selecting fertile sires. While many GWAS have been conducted in purebred cattle, fewer studies have focused on crossbred cattle, which comprise a significant portion of the beef cattle population. This study aimed to identify SNPs associated with SC and SM in crossbred beef bulls using a weighted single-step genomic BLUP (WssGBLUP) approach and to identify positional candidate genes and their roles in bull fertility. This is the first study analyzing SC and SM simultaneously in Canadian crossbred beef cattle.

Previous research indicates a decline in beef cattle fertility due to intense selection pressure on production traits and the pleiotropic effects of genes affecting both fertility and production. Studies on female fertility have been more prevalent due to the ease of data collection, but these traits show low heritability (0.01-0.10). In contrast, bull fertility traits, such as scrotal circumference and sperm motility, show moderate to high heritability (0.05-0.22 and 0.29-0.60, respectively), making them suitable targets for genetic improvement. Scrotal circumference is positively associated with testis weight, sperm output, and semen quality, while sperm motility is a crucial factor in fertilization success. However, a negative genetic correlation exists between performance traits like feed efficiency and SC, requiring careful selection strategies. Relatively few genome-wide association studies (GWAS) have focused on crossbred beef cattle compared to purebred cattle, partly due to differences in genetic evaluation methods among breed associations. Existing GWAS have shown promising results with similar sample sizes, indicating the potential for further research.

This study utilized data from 265 crossbred beef bulls from the Ontario Beef Research Centre, with predominant breeds including Angus, Simmental, Piedmontese, Gelbvieh, Charolais, and Limousine. Scrotal circumference (SC) was measured using a looped tape, and sperm motility (SM) was assessed using CASA (computer-assisted semen analysis) after semen collection via electroejaculation. Genotyping was performed using the Affymetrix Genechip Bovine Genome High Density Array, resulting in 379,591 SNPs after quality control. A weighted single-step genomic BLUP (WssGBLUP) analysis was conducted using the BLUPF90 family of programs. This approach incorporates pedigree, phenotype, and genotype data, assigning weights to markers iteratively to update SNP solutions. The GWAS results were reported as the proportion of variance explained by non-overlapping genomic windows of 1 Mb. Windows explaining >1% of the genetic variance for SC and SM were selected for further analysis. Gene annotation was performed using GALLO, and QTL enrichment analysis was conducted to assess the overrepresentation of QTLs within the candidate windows. Functional candidate gene prioritization was performed using ToppGene, incorporating a trained gene list created using GUILDify based on genes associated with fertility-related keywords. NetworkAnalyst was used for protein-protein interaction network analysis and gene ontology (GO) enrichment analysis to identify functional relationships among prioritized candidate genes.

For scrotal circumference (SC), eight windows located on BTA9, BTA10, BTA20, BTA24, and BTA29 explained >13.19% of the total genetic variance. Thirty-two positional candidate genes were identified within these windows, with 14 prioritized for spermatic-related processes using ToppGene. These prioritized genes explained 9.76% of the total genetic variance for SC. QTL enrichment analysis revealed 38 significant QTLs (FDR-corrected p-value ≤ 0.05) associated with exterior conformation traits. For sperm motility (SM), five windows located on BTA9, BTA13, BTA20, and BTA24 explained >7.17% of the genetic variance, with 28 positional candidate genes identified. ToppGene analysis prioritized 14 genes for spermatic-related processes, which explained 7.16% of the total genetic variance for SM. QTL enrichment identified 13 significant QTLs associated with reproduction and exterior conformation. Functional analysis using NetworkAnalyst revealed significant GO terms associated with biological processes, molecular functions, and cellular components related to male fertility and reproduction. For SC, MAP3K1 and VIP were prioritized, both previously linked to male reproduction. For SM, SOD2, TCP1, PACRG, SPEF2, and PRLR were prioritized, all previously associated with male fertility regulation. Several genes were found to be involved in the regulation of both male and female fertility traits. Analysis of the interaction networks of prioritized genes revealed significant GO terms related to the MAPK cascade, spermatid differentiation, hormone secretion, and other key aspects of male reproduction.

The findings of this study provide valuable insights into the genetic architecture of male fertility in beef cattle. The identification of specific genomic regions and positional candidate genes associated with SC and SM, including previously identified fertility genes and novel candidates, contributes significantly to our understanding of the genetic basis of these important traits. The identified genes and their functional enrichment in pathways crucial for spermatogenesis, sperm maturation, and hormone regulation support their role in influencing male fertility. The overlap between QTLs associated with fertility traits and other economically important traits like body conformation highlights the complexity of the genetic architecture and the need for integrated breeding strategies. While the study sample size is comparable to other studies in this field, future studies with larger sample sizes and validation studies could strengthen the results. The identification of key genes provides opportunities for developing more efficient genomic prediction models for these traits. This can help breeders to select for improved fertility, increase overall reproductive efficiency, and enhance the overall productivity of beef cattle.

This study identified several genomic regions and candidate genes significantly associated with scrotal circumference and sperm motility in crossbred beef bulls. Key findings highlight the roles of genes like MAP3K1 and VIP for SC and SOD2, TCP1, PACRG, SPEF2, and PRLR for SM. These genes and their related pathways are crucial for male fertility. The study also revealed overlapping effects on other economically important traits, emphasizing the need for integrated breeding strategies. Further research is warranted to validate these findings and investigate causal mutations, which could lead to improved genomic selection tools for bull fertility.

The relatively modest sample size (265 bulls) may have limited the power to detect some smaller effects. The crossbred nature of the population, while representative of many beef cattle operations, introduces genetic complexity that may affect the generalizability of findings to specific purebred populations. The study focused on two fertility-related traits, and the identified genes may not represent the full complexity of genetic influences on male fertility.