Does the addition of butorphanol have an effect on pharmacologically induced ejaculation in severely injured stallions?

Abstract

suggested that stimulation of sperm hyperactivity requires calcium influx through a specific cation channel (CatSper) localized on the principal piece of the sperm tail. To study the molecular processes underlying hyperactivation of stallion sperm, it is essential to verify that the genes for the CatSper complex are expressed within the stallion's reproductive tract and that the protein is present onmature sperm. In this study testicular and epididymal tissues were harvested from eight 3-year old DutchWarmblood stallions with acceptable semen quality, immediately after elective castration; in addition, prostate tissue was collected from twomature stallions shortly after euthanasia for medical or surgical problems unrelated to their reproductive function. Prostate tissue was included because binding of prostatederived vesicles (prostasomes) to sperm has been reported to improve their ability to undergo sustained calcium influx (Ren, 2011). The mRNA was isolated, and cDNA was produced from testicular, epididymidal and prostatic tissue using commercial kits. RT-PCR primers were designed for the four subunits of CatSper (CatSper 1, 2, 3 and 4) using the appropriate sequences in the equine genome. Gene expression in the various tissues was then compared using quantitative (real-time) RT-PCR, with relative expression calculated with respect to three stable housekeeping genes (GAPDH, PGK1 and RPL32) using GeNorm analysis. In addition, an antibody against CatSper-1 (H-300, sc-33153: Santa Cruz Biotechnology) was used to verify the presence of CatSper protein (and by inference the cation channel) in stallion spermatozoa and testis by means of dot blots and western blots. Results showed that gene expression for all four Catsper sub-units was high in testicular tissue but more than a factor 100 lower in epididymal tissue (P < 0.001). Interestingly, all four CatSper sub-units were also expressed at low levels in the prostate. CatSper 1 was expressed at a lower level than CatSper 2, 3 and 4 in all tissues examined (P< 0.05). Nevertheless, immunoblotting studies demonstrated that CatSper-1 proteinwas present in stallion testicular tissue and spermatozoa. Having demonstrated expression of the genes for CatSper 1, 2, 3 and 4 in the testicles and prostate of stallions, and the presence of CatSper protein on mature stallion sperm, future studies will focus on the role of CatSper in the induction of hyperactivated motility and zona pellucida penetration.