Metabolomic analysis of seminal plasma in stallions belonging to two different horse breeds

Seminal plasma is a biofluid responsible for survival and fertil- ization ability of spermatozoa during natural mating [Sichtar et al. Animals. 2019; 9: 414]. However, its effect on spermatozoa during storage is still not fully understood [Kareskoski and Katila. Animal Reproduction Science. 2008, 107: 249–256]. In recent years, metabolomics was applied to animal breeding next generation phenotyping approach to predict the breeding value and fertility of animals [Kalaiselvi et al., Int J Curr Res. 2019,11:1972–80]. When assessing fertility, breed was found to influence semen quality and fertility [Gottschalk et al., Theriogenology. 2016, 85:1375–81]. Therefore, we aimed to investigate the metabolomic profile of seminal plasma in American Quarter Horse (AQH) and Italian Draft Horse (IDH) stallions. Twelve healthy stallions, AQH (n 1⁄4 6, mean age 5.2 years, mean BW 500 kg), and IDH (n 1⁄4 6, mean age 5.5 years, mean BW 700 Kg) were subjected to two semen collections, 30 days apart. Semen quality analyses (gel-free volume, spermatozoa con- centration, progressive motility) were performed by the same experienced professional. Aliquots (2ml) of seminal plasma were obtained by centrifugation (4,000 X g for 10 min) and stored at -20C until metabolomic analysis performed by 1H-NMR [Laghi et al. Journal of Applied Physiology. 2017, japplphysiol.01131.2017]. Mares were inseminated with fresh semen (AQH n1⁄4 82 mares, IDH n 1⁄4 98 mares). Semen characteristics differed between IDH and AQH (p<0.05). The 1st cycle conception rate was 63% for AQH and 27% for IDH (p1⁄40.001) and the overall pregnancy rate reached 94% in AQH and 75% in IDH (p1⁄40.03). Metabolomic analysis quantified 56 molecules in equine seminal plasma, with 19 metabolites showing different concentrations in IDH compared to AQH (p<0.05). Cystine, isoleucine, fumarate, hippurate, 2-hydrox- yisobutyrate, citrate and glucose were found to be the most char- acterizing of the IDH stallions. Significant correlations (p<0.05) were also found between seminal plasma metabolites and semen characteristics irrespective to horse breed. Citrate and isoleucine were found negatively correlated with spermatozoa progressive motility (citrate: r 1⁄4 -0.86, isoleucine: r 1⁄4 -0.72), spermatozoa concentration (citrate: r 1⁄4 -0.49, isoleucine: r 1⁄4 -0.75) and 1st cycle fertility rate (citrate: r 1⁄4 -0.44, isoleucine: r 1⁄4 -0.40). Citrate has previously been identified as possible biomarker for male infer- tility. Citrate limits sperm capacitation and spontaneous acrosome reaction and is found in high concentrations in infertile men. Furthermore, citrate and isoleucine were considered differential biomarkers for high-fertility bulls exhibiting low seminal plasma levels of these metabolites [Kumar et al. Molecular Reproduction & Development. 2015]. The different seminal plasma composition of IDH stallions might contribute to their lower 1st cycle fertility rate when compared to AQH stallions.