Prostanoids as markers of oxidative stress in stallion semen

Abstract

Artificial insemination is vital for the equine breeding industry, but standard semen assessment before processing does not reliably predict semen quality after chilled storage. Reducing storage temperature can induce oxidative stress (OS) in spermatozoa in turn reducing fertilizing potential by damaging sperm membranes through lipid peroxidation. Isoprostanes have emerged as established biomarkers for lipid peroxidation and are generated in OS associated pathologies. A full prostanoid (isoprostanes, prostaglandins, and their metabolites) profile of stallion seminal plasma has never been compared to the ability to endure reduced temperatures associated with the chilling process. Semen from 9 stallions was collected with an artificial vagina and extended to achieve 25-35 × 106 sperm/ml using EquiPlus, followed by measuring total (TM) and progressive motility (PM) via CASA, viability (V) with a nucleocounter, DNA integrity (DI) using a DNA integrity kit, and the number of sperm morphological defects using eosin-nigrosin staining. Extended semen aliquots were prepared at 24°C and underwent passive cooling to be stored at 5°C for repeat evaluations every 24h. Aliquots of raw (time 0 after collection) and extended (time 0, 24h, 48h, 72h, 96h) semen were centrifuged, and seminal plasma (SP) rich samples were stored at -80°C for prostanoid extraction and identification (n = 13) via chromatography tandem mass spectrometry (6500+ Triple Quadrupole Mass Spectrometer - Applied Biosystems Sciex, Australia). Stallion ejaculates were categorised by their chilling ability (good chillers had >30% PM, n=5, poor chillers had <30% PM, n=4, after 24h cooling, recognised as being indicative of fertilising ability), and effects of semen characteristics and chilling ability on SP prostanoid concentrations were analysed. Viability (P<0.05), TM and PM (P<0.01) were higher on 0h than 24h and 48h in all samples. However, TM, PM, DNA integrity or viability did not differ between good or poor chillers at 0h, yet TM, PM, and viability were lower in poor compared to good chillers at 24h and 48h (P<0.001). Across all timepoints, 8-F3t-IsoP negatively correlated to PM (R=-0.33; P<0.05), 8-iso PGF2α positively correlated to DNA integrity (R=0.45), and 20-F4t-NeuroP positively correlated to viability (R=0.38; P<0.05). At 24h 5-F2t-IsoP, a classic marker for OS, was higher in SP from poor compared to good chillers (P=0.039) confirmed by the strong negative correlation with chilling ability (R=-0.772; P=0.015). Thus, we present preliminary data to identify four prostanoids in SP that correlate with functional sperm parameters before and during cooling, suggesting individual prostanoids may be predictive SP biomarkers for processed and chilled equine ejaculate quality. Further studies will continue to characterise key prostanoids as OS markers in ejaculates and may allow development of targeted novel OS biosensors.