141 Preliminary investigation of kisspeptin gene expression and concentration in stallion semen

Abstract

The objective of this study was to determine if the reproductive hormone kisspeptin (Kiss1) is present within stallion spermatozoa and seminal plasma. Semen samples (n = 19) were collected between March and August from 12 mature (ages 5–25) light horse-type stallions using an artificial vagina. Immediately following collection, raw semen samples were aliquoted for 2 different experiments. For experiment one, 10 mL of raw semen (n = 19) were centrifuged, and the seminal plasma was decanted and stored at −20°C for later analysis of Kiss1 concentrations using a competitive equine ELISA kit (MyBioSource) at a sample dilution ratio of 1:50,000. For experiment 2, 5 mL of raw semen (n = 10) were layered on top of 5 mL of EquiPure and centrifuged. The EquiPure was removed and the purified sperm pellet was rinsed with phosphate buffered solution (PBS). The PBS was decanted, and the purified sperm pellet was flash frozen in dry ice and stored at −80°C for later analysis of Kiss1 gene expression via quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Total RNA was isolated from thawed sperm samples using phenol-chloroform extraction, treated with DNase, and RNA quantity assessedbefore reverse transcription and qRT-PCR using primers validated on equine tissues. Preliminary results revealed the presence of the Kiss1 protein in seminal plasma and Kiss1 transcript in spermatozoa. Mean concentration of Kiss1 in seminal plasma was 1,262 ng/mL. Analysis by one-way ANOVA revealed no significant difference (P = 0.425) in Kiss1 concentrations between stallions. Spearman's correlation analysis was performed, but no significant correlation between stallion age and concentration of Kiss1 in seminal plasma was detected (r = −0.345; P = 0.270). Amplification of the Kiss1 transcript in spermatozoa was observed at a mean cycle threshold of 32 in both Kiss1 and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our findings of the expression of Kiss1 in stallion semen agrees with published results in the human, mouse, rat, dog, and goat. Further investigation and understanding of the role of kisspeptin in semen production and testicular function could improve the management of stallion fertility.