Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Abstract

Cryopreservation of epididymal spermatozoa may be the only opportunity to preserve valuable genetics of males in cases of unforeseen injury or death. Stallion epididymal spermatozoa have been cryopreserved successfully and it has been demonstrated that stallion epididymal spermatozoa are fertile, and pregnancies as well as live foals have been produced. As spermatozoal quality parameters like motility, morphology and viability have a major influence on fertility and pregnancy rates, it is of great interest to describe these and investigate the influence of seminal plasma on these parameters. Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and that have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa from the same stallions. Six sperm categories of each stallion (n= 4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, we could show that seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. In conclusion, we recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa. AFRIKAANS : Bevriesing van epididimale spermatozoa mag die enigste geleentheid wees om waardevolle genetika van manlike diere te bewaar in die geval van onverwagse dood of besering. Epididimale spermatozoa van hingste is al suksesvol bevries, is bewys vrugbaar te wees en dragtighede sowel as lewendige vullens is deur die gebruik daarvan gelewer. Aangesien maatstawwe van spermgehalte soos beweeglikheid, morfologie en lewensvatbaarheid ‘n beduidende invloed uitoefen op vrugbaarheid en dragtigheidsyfers, is dit van groot belang om hierdie maatstawwe te omskryf en die invloed van seminale plasma daarop te ondersoek. Vars en ontdooide maatstawwe (beweeglikheid, morfologie en lewensvatbaarheid) van hings epididimale spermatozoa wat of aan seminal plasma blootgestel is of nie, is vergelyk met vars en ontdooide maatstawwe van geejakuleerde spermatozoa. Ses spermkategorieë van elke hings (n=4) is geevalueer vir beweeglikheid, morfologie en lewensvatbaarheid. Die kategorieë was vars geejakuleerde spermatozoa (Fr-E), vars epididimale spermatozoa wat blootgestel is aan seminale plasma (Fr-SP+), vars epididimale spermatozoa sonder blootstelling aan seminale plasma (Fr-SP-), ontdooide geejakuleerde spermatozoa (Cr-E), ontdooide epididimale spermatozoa blootgestel aan seminale plasma voor bevriesing (Cr-SP+) en ontdooide epididimale spermatozoa wat nooit blootgestel is aan seminale plasma nie (Cr-SP-). Uitslae toon dat seminale plasma die aanvanklike beweeglikheid van vars epididimale hings spermatozoa stimuleer, terwyl die verskil in lynbeweeglikheid nie meer teenwoordig is na ontdooiing nie; dat lynbeweeglikheid van vars epididimale spermatozoa of ontdooide geejakuleerde hings spermatozoa nie altyd ‘n goeie aanduiding is vir lynbeweeglikheid na ontdooiing nie. Hierdie studie toon dat seminale plasma ‘n positiewe invloed het op die voorkoms van algehele spermdefekte, midstukreflekse en distale sitoplasmiese druppeltjies in ontdooide hings epididimale spermatozoa terwyl die voorkoms van midstuk reflekse waarskynlik verband hou met distale sitoplasmiese druppletjies. Verder kon ons ook aantoon dat seminale plasma geen invloed het op die lewensvatbaarheid van vars en ontdooide morfologies normale epididimale spermatozoa nie. Ten slotte beveel ons aan dat retrograadse spoeling met seminale plasma as spoelmedium gebruik word wanneer hings epididimale spermatozoa versamel en bevries word.