Skip to main content
ARS Home » Plains Area » Miles City, Montana » Livestock and Range Research Laboratory » Research » Publications at this Location » Publication #419635
Research Project: Identifying and Mitigating Factors that Limit Beef Production Efficiency

Location: Livestock and Range Research Laboratory

Title: Improved evaluation of mitochondrial membrane potential in bovine spermatozoa using JC-1 with flow cytometry

Author
item Zezeski, Abigail
item HAMILTON, LAUREN - University Of Missouri
item Geary, Thomas

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2025
Publication Date: 4/14/2025
Citation: Zezeski, A.L., Hamilton, L.E., Geary, T.W. 2025. Improved evaluation of mitochondrial membrane potential in bovine spermatozoa using JC-1 with flow cytometry. Biology of Reproduction. https://doi.org/10.1093/biolre/ioaf081.
DOI: https://doi.org/10.1093/biolre/ioaf081

Interpretive Summary: The most common method of measuring energy potential of sperm cells from bulls is with the dye, JC-1. The JC-1 dye will emit a green signal when mitochondria (energy factory of sperm) is depolarized and orange when mitochondria are polarized. The red-orange signal is the signal of interest and indicates sperm have potential to create energy for swimming. The JC-1 dye must be excited to emit these color signals and the most common method of exciting it has been the use of a laser having a 488 wavelength. This old method of exciting JC-1 makes detection of the green and orange signals difficult. This paper presents more robust methods of exciting the JC-1 dye to better measure energy potential of sperm and adds a viability dye to make sure responses of only living sperm are measured. In addition, these new methods of measuring energy potential of sperm are highly correlated with sperm motility measures. Thus, we report a better method for identifying the sperm with the greatest potential for fertilization.

Technical Abstract: One of the most popular methods for measuring mitochondrial membrane potential via flow cytometry in sperm cells is through the use of the fluorescent dye 5,5,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). The JC-1 dye is unique in that it will fluoresce green (525nm, monomers) or red-orange (595nm, J-aggregates) when excited to indicate depolarized or polarized mitochondria. While JC-1 can be multiplexed with viability dyes such as propidium iodide and SYBR 14 for microscopy, doing so for conventional flow cytometry has proved difficult due to overlap of the emission spectra between dyes. The objective of this new protocol is to improve the accuracy of conventional flow cytometry JC1 J-aggregate quantification by using a 405nm or 532nm laser for excitation coupled with the viability dye calcein violet (CV). Quantification of live J-aggregates when excited with a 405nm or 532nm laser is more strongly correlated to progressive motility (405 nm: r = 0.73, P < 0.0001; 532 nm: r = 0.52, P = 0.0002) and viability (405 nm: r = 0.93, P < 0.0001; 532 nm: r = 0.74, P < 0.0001) than quantification with the traditional 488nm laser excitation and dual emission of 595nm and 525nm (progressive motility: r = 0.05, P = 0.72; viability: r = 0.21, P = 0.14). The use of a 405 nm or a 532 nm laser requires no compensation. This allows for clear identification of the polarized J-aggregates, and when combined with CV, may help identify which spermatozoa have the greatest fertilization potential.