{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T23:15:45Z","timestamp":1763507745072,"version":"3.40.5"},"reference-count":38,"publisher":"Cambridge University Press (CUP)","issue":"2","license":[{"start":{"date-parts":[[2020,10,13]],"date-time":"2020-10-13T00:00:00Z","timestamp":1602547200000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/www.cambridge.org\/core\/terms"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Zygote"],"published-print":{"date-parts":[[2021,4]]},"abstract":"<jats:title>Summary<\/jats:title><jats:p>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\u2122-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX\u2122 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, <jats:italic>P<\/jats:italic> &lt; 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.<\/jats:p>","DOI":"10.1017\/s0967199420000581","type":"journal-article","created":{"date-parts":[[2020,10,13]],"date-time":"2020-10-13T07:42:10Z","timestamp":1602574930000},"page":"130-137","source":"Crossref","is-referenced-by-count":7,"title":["Ram semen quality can be assessed by flow cytometry several hours after post-fixation"],"prefix":"10.1017","volume":"29","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4144-8584","authenticated-orcid":false,"given":"Jarom\u00edr","family":"Va\u0161\u00ed\u010dek","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Svoradov\u00e1","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrej","family":"Bal\u00e1\u017ei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rastislav","family":"Jur\u010d\u00edk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mari\u00e1n","family":"Mach\u00e1\u010d","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"Chrenek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"56","published-online":{"date-parts":[[2020,10,13]]},"reference":[{"key":"S0967199420000581_ref12","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.top36"},{"key":"S0967199420000581_ref36","first-page":"359","article-title":"The assessment of cryopreservation on the quality of endangered oravka rooster spermatozoa using casa and cytometry","volume":"39","author":"Svoradov\u00e1","year":"2018","journal-title":"CryoLetters"},{"key":"S0967199420000581_ref10","doi-asserted-by":"publisher","DOI":"10.1155\/2016\/8213071"},{"key":"S0967199420000581_ref32","doi-asserted-by":"publisher","DOI":"10.1385\/1-59259-811-0:313"},{"key":"S0967199420000581_ref22","first-page":"41","article-title":"Dilution factor affects the ability of ram sperm to survive cryopreservation","volume":"51","author":"Kul\u00edkov\u00e1","year":"2018","journal-title":"Slovak J Anim Sci"},{"key":"S0967199420000581_ref25","doi-asserted-by":"publisher","DOI":"10.1111\/j.1439-0531.2010.01622.x"},{"key":"S0967199420000581_ref33","doi-asserted-by":"publisher","DOI":"10.1177\/44.12.8985128"},{"volume-title":"Slovak National Animal Breeds","year":"2019","author":"Chrenek","key":"S0967199420000581_ref7"},{"key":"S0967199420000581_ref2","first-page":"1000157","article-title":"An efficient technique to detect sperm reactive oxygen species: the CellROX deep red fluorescent probe","volume":"4","author":"Alves","year":"2015","journal-title":"Biochem Physiolol"},{"key":"S0967199420000581_ref35","first-page":"69","article-title":"Viability assessment of fresh chicken blastodermal cells by flow cytometry","volume":"50","author":"Svoradov\u00e1","year":"2017","journal-title":"Slovak J Anim Sci"},{"key":"S0967199420000581_ref31","doi-asserted-by":"publisher","DOI":"10.1002\/0471142956.cy0934s53"},{"volume-title":"The Handbook: A Guide to Fluorescent Probes and Labeling Technologies","year":"2005","author":"Haughland","key":"S0967199420000581_ref18"},{"key":"S0967199420000581_ref38","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2010.01.028"},{"key":"S0967199420000581_ref16","doi-asserted-by":"publisher","DOI":"10.1111\/j.1745-7262.2008.00399.x"},{"key":"S0967199420000581_ref19","doi-asserted-by":"publisher","DOI":"10.1016\/0022-1759(95)00172-7"},{"key":"S0967199420000581_ref26","first-page":"55","article-title":"Overview of cell fixation and permeabilization","volume":"34","author":"Melan","year":"1994","journal-title":"Methods Mol Biol"},{"key":"S0967199420000581_ref29","doi-asserted-by":"publisher","DOI":"10.1111\/andr.12419"},{"key":"S0967199420000581_ref3","first-page":"53","article-title":"Comparison of three different methods for the analysis of ram sperm concentration","volume":"53","author":"Bal\u00e1\u017ei","year":"2020","journal-title":"Slovak J Anim Sci"},{"key":"S0967199420000581_ref14","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-642-57049-0_14"},{"key":"S0967199420000581_ref13","doi-asserted-by":"publisher","DOI":"10.1530\/REP-13-0191"},{"key":"S0967199420000581_ref9","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0138777"},{"key":"S0967199420000581_ref30","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.b.21506"},{"key":"S0967199420000581_ref11","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2012.4886"},{"key":"S0967199420000581_ref21","doi-asserted-by":"publisher","DOI":"10.1210\/jc.2007-2616"},{"key":"S0967199420000581_ref28","doi-asserted-by":"publisher","DOI":"10.1177\/1087057112468765"},{"key":"S0967199420000581_ref8","doi-asserted-by":"publisher","DOI":"10.1002\/eji.201970107"},{"key":"S0967199420000581_ref4","first-page":"365","article-title":"Dead cell stains in flow cytometry: a comprehensive analysis","volume":"74","author":"Bradford","year":"2008","journal-title":"Cytometry B Clin Cytom"},{"key":"S0967199420000581_ref17","doi-asserted-by":"publisher","DOI":"10.1155\/2016\/1687657"},{"key":"S0967199420000581_ref15","doi-asserted-by":"publisher","DOI":"10.1111\/jnc.12267"},{"key":"S0967199420000581_ref20","first-page":"271","article-title":"Stoichiometry of immunocytochemical staining reactions","volume":"2001","author":"Jacobberger","year":"2000","journal-title":"Methods Cell Biol"},{"key":"S0967199420000581_ref27","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.112.105650"},{"key":"S0967199420000581_ref1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-776-7_14"},{"key":"S0967199420000581_ref37","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-385493-3.00004-8"},{"key":"S0967199420000581_ref5","article-title":"Labeling mitochondria with MitoTracker dyes","author":"Chazotte","year":"2011","journal-title":"Cold Spring Harbor Protocols"},{"key":"S0967199420000581_ref23","doi-asserted-by":"publisher","DOI":"10.1186\/s12958-015-0008-1"},{"key":"S0967199420000581_ref24","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.23268"},{"key":"S0967199420000581_ref34","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.b.20135"},{"key":"S0967199420000581_ref6","first-page":"135","article-title":"Current situation in the gene bank of animal genetic resources in Slovakia","volume":"50","author":"Chrenek","year":"2017","journal-title":"Slovak J Anim Sci"}],"container-title":["Zygote"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.cambridge.org\/core\/services\/aop-cambridge-core\/content\/view\/S0967199420000581","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T08:42:48Z","timestamp":1615452168000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.cambridge.org\/core\/product\/identifier\/S0967199420000581\/type\/journal_article"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,10,13]]},"references-count":38,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2021,4]]}},"alternative-id":["S0967199420000581"],"URL":"https:\/\/doi.org\/10.1017\/s0967199420000581","relation":{},"ISSN":["0967-1994","1469-8730"],"issn-type":[{"type":"print","value":"0967-1994"},{"type":"electronic","value":"1469-8730"}],"subject":[],"published":{"date-parts":[[2020,10,13]]}}}