A combination of biomarkers for predicting stallion sperm fertility.

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3-5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24-30 h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.