2 Association between sperm metabolites and field fertility in beef bulls

Increased knowledge on causes of bull subfertility and the development of potential diagnostic tools are of great importance to lessen the economic loss experienced by reproductive failure in beef cattle. Currently, the main method to evaluate the fertility potential of a bull is through a breeding soundness exam (BSE). However, a BSE does not provide reveal the intracellular processes that may be dysfunctional in normal-appearing sperm cells from subfertile males. Metabolomic analysis of sperm from bulls with diverging field fertility may provide insights on sperm metabolism that are associated with pregnancy outcomes. Our objective was to perform metabolomic analyses of sperm from bulls with differing field fertility and evaluate the differences in metabolome profiles between high- and low-fertility bulls. Angus bulls (n = 15) were classified as having high (n = 8) or low (n = 7) fertility, with the average number of services being 7668 ± 10 389 and 7868 ± 6511, and the average fertility deviation from the mean (fertility index) being 1.85 ± 0.67 and −3.96 ± 2.08, respectively. Frozen-thawed semen straws (n = 10 per bull) underwent a Percoll gradient sperm purification process. A 100-µL aliquot of each sample containing 4 million sperm underwent five rounds of freezing and thawing in liquid nitrogen for cell lysis. Samples were stored at −80°C until analysis. Metabolomic analysis was performed through ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry at the University of Tennessee Biological and Small Molecule Mass Spectrometry Core. Pearson correlation was used to analyze the relationship between metabolite peak area (abundance) and fertility index. We also compared mean metabolite abundance between the four highest-fertility bulls (2.30 ± 0.71 fertility index) and the four lowest-fertility bulls (−5.25 ± 2.84 fertility index), using ANOVA. Significance was determined when P ≤ 0.05. There were 93 unique metabolites present in sperm samples, and 75 metabolites were present in greater than 50% of samples. No correlation (P ≥ 0.10) was found between metabolites and field fertility index. When high- and low-fertility groups were evaluated individually, there was a positive correlation (P ≤ 0.03) between high fertility and the metabolites IMP, N-acetylglucosamine 1/6-phosphate, orotate, and phosphoenolpyruvate. The metabolites adenosine, hypoxanthine, xanthine, and tricarballylic acid were negatively correlated (P ≤ 0.05) with low fertility. Further, between the highest- and lowest-fertility bulls in this study, pyroglutamic acid (P = 0.05) and N-acetylglutamate (P = 0.01) had greater abundance in the lowest-fertility bulls compared with the highest-fertility bulls. In summary, although further analyses and validation of findings are necessary, the use of metabolites as a fertility marker to identify and remove subfertile bulls from a breeding population has promising future implications.

This project was supported by the NAAB Doak Graduate Fellowship from the National Association of Animal Breeders and the state of Tennessee through the UT Institute of Agriculture and Department of Animal Science.