Relationships between metabolism of cryopreserved equine sperm determined by the Seahorse Analyzer and sperm characteristics measured by flow cytometry and computer assisted analysis of motility

Abstract

Metabolic activity of sperm has up to now mainly been determined by measuring mitochondrial membrane potential using flow cytometry, but these assays provide little bioenergetic information about sperm metabolism (Moraes et Meyers et al. Anim Reprod Sci. 2018; 194:71–80). Since a few years oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells can be examined using the Seahorse analyzer (Agilent Technologies, Inc., Santa Clara, CA, United States). Studies with this system have already been performed on sperm of humans (Freitas-Martins et al. 2024; Andrology. 2024 12(2):410-421), mice (Balbach et al. Biol Reprod. 2020 103:791-801) and bovine (Moraes et Anim Reprod Sci. Anim Reprod Sci. 2021; 194:71–80), but to our knowledge not in equine sperm. The aims of our studies were to examine intra- and interindividual variabilities of OCR and ECAR in cryopreserved equine sperm and to investigate relationships of these parameters with sperm characteristics determined by flow cytometric assays and computer assisted sperm analysis. Three cryopreserved ejaculates from each of 21 (n=63) warmblood stallions of the Holsteiner Verband (Elmshorn, Germany) were examined. The quality of the semen samples was characterized using different assays during a period of 120 min by measurements every 15 min at a temperature of 37°C. The sperm metabolism was examined using the Seahorse analyzer by measuring basal OCR and ECAR without manipulation performing the Cell Mito Stress. The total motility (TM) of the sperm was assessed by CASA. Furthermore, a flow cytometric assay was carried out to evaluate simultaneously the percentage of sperm with an intact plasma membrane and negative acrosomal status, sperm with a high esterase activity, high mitochondrial membrane potential, low intracellular Ca2+ level (Bucher et al., J Dairy Sci. 2019; 102(12):11652-11669). The SCSA™ was performed to determine the percentage of sperm with a high DNA fragmentation (%DFI) in each sperm sample. There were high variabilities in OCR (34 ± 28 pmol/min of 1 × 106 sperm) as well as in ECAR (1.89 ± 1.18 mpH/min of 1 × 106 sperm) with high inter- and intraindividual variabilities in OCR- and ECAR values within (CVs: OCR: 22.83 - 93.03; ECAR: 29.46 - 90.16) and between (CVs: 65.33; ECAR: 11.39) stallions. There were no high correlations between OCR- and ECAR values and flow cytometric as well as CASA parameters (r-values: min: -0.44; max 0.55). The results of our study show that there are high intra- and interindividual variabilities in oxygen consumption as well as extracellular acidification. As there were no high correlations between the metabolic parameters and other conventionally determined sperm parameters it can be concluded that the Seahorse Analyzer provides new information about sperm quality.