Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa
Marc Llavanera
A B C * , Beatrice Mislei D , Olga Blanco-Prieto C , Vito Antonio Baldassarro C E , Yentel Mateo-Otero A B C , Marcella Spinaci C , Marc Yeste A B and Diego Bucci C
+ Author Affiliations
- Author Affiliations
A Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain.
B Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain.
C Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra, 50, Ozzano dell’Emilia, IT-40064 Bologna, Italy.
D National Institute of Artificial Insemination (AUB-INFA), University of Bologna, Via Gandolfi 16, Cadriano, IT-40057 Bologna, Italy.
E IRET Foundation, University of Bologna, Via Tolara di Sopra 41/E, Ozzano dell’Emilia, IT-40064 Bologna, Italy.
Reproduction, Fertility and Development 34(9) 679-688 https://doi.org/10.1071/RD21355
Published online: 1 April 2022
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing
Abstract
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.
Keywords: bull, DiOC6(3), flow cytometry, fluorescent probes, JC-1, mitochondria, MitoTracker, sperm.
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