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Ram semen quality can be assessed by flow cytometry several hours after post-fixation

Published online by Cambridge University Press:  13 October 2020

Jaromír Vašíček Open the ORCID record for Jaromír Vašíček [Opens in a new window] ,
Andrea Svoradová ,
Andrej Baláži ,
Rastislav Jurčík ,
Marián Macháč  and
Peter Chrenek
Jaromír Vašíček*
Affiliation:
Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Science, Nitra, Slovak Republic
Andrea Svoradová
Affiliation:
Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic
Andrej Baláži
Affiliation:
Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic
Rastislav Jurčík
Affiliation:
Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic
Marián Macháč
Affiliation:
Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Science, Nitra, Slovak Republic
Peter Chrenek
Affiliation:
Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Science, Nitra, Slovak Republic University of Science and Technology, Department of Animal Biochemistry and Biotechnology, Bydgoszcz, Poland
*
Author for correspondence: Jaromír Vašíček, Research Institute for Animal Production in Nitra, NPPC, Lužianky, Slovak Republic. Tel: +421 37 6546 600. E-mail: [email protected]
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Summary

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Keywords

Flow cytometryFluorescent markersFormaldehyde fixationQuality analysisRam semen
Type
Research Article
Information
Zygote , Volume 29 , Issue 2 , April 2021 , pp. 130 - 137
Copyright
© The Author(s), 2020. Published by Cambridge University Press

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