Ram semen quality can be assessed by flow cytometry several hours after post-fixation
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 an...
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| Veröffentlicht in: | Zygote (Cambridge) 2021-04, Vol.29 (2), p.130-137 |
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| creator | Vašíček, Jaromír Svoradová, Andrea Baláži, Andrej Jurčík, Rastislav Macháč, Marián Chrenek, Peter |
| description | 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. |
| doi_str_mv | 10.1017/S0967199420000581 |
| format | Article |
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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.</description><identifier>ISSN: 0967-1994</identifier><identifier>EISSN: 1469-8730</identifier><identifier>DOI: 10.1017/S0967199420000581</identifier><identifier>PMID: 33046151</identifier><language>eng</language><publisher>England: Cambridge University Press</publisher><subject>Annexin V ; Antibodies ; Apoptosis ; Cell viability ; Cold shock ; Damage ; Dyes ; Farms ; Fixation ; Flow cytometry ; Fluorescent indicators ; Formaldehyde ; Laboratories ; Markers ; Microscopy ; Mitochondria ; Motility ; Physiology ; Quality assessment ; Reagents ; Semen ; Sheep ; Sperm ; Spermatozoa ; Stability analysis ; Stains & staining ; Statistical methods</subject><ispartof>Zygote (Cambridge), 2021-04, Vol.29 (2), p.130-137</ispartof><rights>The Author(s), 2020. Published by Cambridge University Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c329t-976994cd1ad63209bf17506a3315101492f05445a579d7cb10608bf1063eeb8e3</citedby><cites>FETCH-LOGICAL-c329t-976994cd1ad63209bf17506a3315101492f05445a579d7cb10608bf1063eeb8e3</cites><orcidid>0000-0003-4144-8584</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33046151$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vašíček, Jaromír</creatorcontrib><creatorcontrib>Svoradová, Andrea</creatorcontrib><creatorcontrib>Baláži, Andrej</creatorcontrib><creatorcontrib>Jurčík, Rastislav</creatorcontrib><creatorcontrib>Macháč, Marián</creatorcontrib><creatorcontrib>Chrenek, Peter</creatorcontrib><title>Ram semen quality can be assessed by flow cytometry several hours after post-fixation</title><title>Zygote (Cambridge)</title><addtitle>Zygote</addtitle><description>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.</description><subject>Annexin V</subject><subject>Antibodies</subject><subject>Apoptosis</subject><subject>Cell viability</subject><subject>Cold shock</subject><subject>Damage</subject><subject>Dyes</subject><subject>Farms</subject><subject>Fixation</subject><subject>Flow cytometry</subject><subject>Fluorescent indicators</subject><subject>Formaldehyde</subject><subject>Laboratories</subject><subject>Markers</subject><subject>Microscopy</subject><subject>Mitochondria</subject><subject>Motility</subject><subject>Physiology</subject><subject>Quality assessment</subject><subject>Reagents</subject><subject>Semen</subject><subject>Sheep</subject><subject>Sperm</subject><subject>Spermatozoa</subject><subject>Stability analysis</subject><subject>Stains & staining</subject><subject>Statistical methods</subject><issn>0967-1994</issn><issn>1469-8730</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNplkEtLxDAUhYMozjj6A9xIwI2batK8mqUMvmBAUGdd0vYWO7TNTJKq_fdmmNGFXi7cxfnu4XAQOqfkmhKqbl6JlopqzVMSR2T0AE0plzrJFCOHaLqVk60-QSferyKjlObHaMIY4ZIKOkXLF9NhDx30eDOYtgkjLk2PC8DGe4hb4WLEdWs_cTkG20FwY-Q_wJkWv9vBeWzqAA6vrQ9J3XyZ0Nj-FB3VpvVwtr8ztLy_e5s_Jovnh6f57SIpWapDopWM4cqKmkqylOiipkoQaRiL2QjlOq2J4FwYoXSlyoISSbIIEckAigzYDF3tfNfObgbwIe8aX0Lbmh7s4POURztFUsoievkHXcX0fUyXp4IzmVEhVKTojiqd9d5Bna9d0xk35pTk287zf53Hn4u981B0UP1-_JTMvgHAoXpJ</recordid><startdate>20210401</startdate><enddate>20210401</enddate><creator>Vašíček, Jaromír</creator><creator>Svoradová, Andrea</creator><creator>Baláži, Andrej</creator><creator>Jurčík, Rastislav</creator><creator>Macháč, Marián</creator><creator>Chrenek, Peter</creator><general>Cambridge University Press</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0003-4144-8584</orcidid></search><sort><creationdate>20210401</creationdate><title>Ram semen quality can be assessed by flow cytometry several hours after post-fixation</title><author>Vašíček, Jaromír ; Svoradová, Andrea ; Baláži, Andrej ; Jurčík, Rastislav ; Macháč, Marián ; Chrenek, Peter</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c329t-976994cd1ad63209bf17506a3315101492f05445a579d7cb10608bf1063eeb8e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Annexin V</topic><topic>Antibodies</topic><topic>Apoptosis</topic><topic>Cell viability</topic><topic>Cold shock</topic><topic>Damage</topic><topic>Dyes</topic><topic>Farms</topic><topic>Fixation</topic><topic>Flow cytometry</topic><topic>Fluorescent indicators</topic><topic>Formaldehyde</topic><topic>Laboratories</topic><topic>Markers</topic><topic>Microscopy</topic><topic>Mitochondria</topic><topic>Motility</topic><topic>Physiology</topic><topic>Quality assessment</topic><topic>Reagents</topic><topic>Semen</topic><topic>Sheep</topic><topic>Sperm</topic><topic>Spermatozoa</topic><topic>Stability analysis</topic><topic>Stains & staining</topic><topic>Statistical methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Vašíček, Jaromír</creatorcontrib><creatorcontrib>Svoradová, Andrea</creatorcontrib><creatorcontrib>Baláži, Andrej</creatorcontrib><creatorcontrib>Jurčík, Rastislav</creatorcontrib><creatorcontrib>Macháč, Marián</creatorcontrib><creatorcontrib>Chrenek, Peter</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>MEDLINE - Academic</collection><jtitle>Zygote (Cambridge)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vašíček, Jaromír</au><au>Svoradová, Andrea</au><au>Baláži, Andrej</au><au>Jurčík, Rastislav</au><au>Macháč, Marián</au><au>Chrenek, Peter</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Ram semen quality can be assessed by flow cytometry several hours after post-fixation</atitle><jtitle>Zygote (Cambridge)</jtitle><addtitle>Zygote</addtitle><date>2021-04-01</date><risdate>2021</risdate><volume>29</volume><issue>2</issue><spage>130</spage><epage>137</epage><pages>130-137</pages><issn>0967-1994</issn><eissn>1469-8730</eissn><abstract>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.</abstract><cop>England</cop><pub>Cambridge University Press</pub><pmid>33046151</pmid><doi>10.1017/S0967199420000581</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0003-4144-8584</orcidid></addata></record> |
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| identifier | ISSN: 0967-1994 |
| ispartof | Zygote (Cambridge), 2021-04, Vol.29 (2), p.130-137 |
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| source | Cambridge University Press Journals Complete |
| subjects | Annexin V Antibodies Apoptosis Cell viability Cold shock Damage Dyes Farms Fixation Flow cytometry Fluorescent indicators Formaldehyde Laboratories Markers Microscopy Mitochondria Motility Physiology Quality assessment Reagents Semen Sheep Sperm Spermatozoa Stability analysis Stains & staining Statistical methods |
| title | Ram semen quality can be assessed by flow cytometry several hours after post-fixation |
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