Fol. Biol. 2023, 69, 127-132

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa

Filipp Georgijevič Savvulidi1, Martin Ptáček1, Anežka Málková1, Elena Golovina2, Jan Pytlík1, Martina Janošíková1, Szabolcs Nagy3, Joăo Pedro Barbas4, Luděk Stádník1, Karina Savvulidi Vargová2

1Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic
2Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic
3Institute of Animal Sciences, Hungarian University of Agriculture and Life Sciences, Georgikon Campus, Keszthely, Hungary
4Instituto Nacional de Investigaçăo Agrária e Veterinária, Quinta da Fonte Boa, Vale de Santarém, Portugal

Received October 2023
Accepted December 2023

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

Supplementary material: Supplementary Table 1


This study was supported by the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic with the S grant; from the European Regional Development Fund – Project “Centre for the investigation of synthesis and transformation of nutritional substances in the food chain in interaction with potentially harmful substances of anthropogenic origin: comprehensive assessment of soil contamination risks for the quality of agricultural products” (No. CZ.02.1.01/0.0/0.0/16_019/0000845); with Project NAZV QK1910156 – New approaches for the preservation of endangered livestock populations; with SGS grants Nos. SV21-6-21320, SV23-7-21320; with grant START/MED/089, and with grant PRIMUS/17/MED/16.


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