Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa

Context: While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6 (3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. Aims: The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. Methods: Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6 (3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. Key results: DiOC6 (3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6 (3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. Conclusions: JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6 (3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. Implications: Both DiOC6 (3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.

The authors acknowledge the support from the Ministries of Science and Innovation (AGL201788329-R, PID2020-113320RB-I00 and FPU18/00666) and of Universities (EST19/00389), Spain and the Regional Government of Catalonia, Spain (2017-SGR-1229).