Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm
Authors: Bulkeley Evelyn , Santistevan Anthony C. , Varner Dickson , Meyers Stuart 
Published: September 2022

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation‐produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24‐h cool‐stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co‐stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24‐h cool‐stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.