The application of image cytometry to viability assessment in dual fluorescence‐stained fish spermatozoa
Identifieur interne : 001079 ( Main/Merge ); précédent : 001078; suivant : 001080The application of image cytometry to viability assessment in dual fluorescence‐stained fish spermatozoa
Auteurs : Martin Flajšhans [République tchèque] ; Jacky Cosson [France] ; Marek Rodina [République tchèque] ; Otomar Linhart [République tchèque]Source :
- Cell Biology International [ 1065-6995 ] ; 2004-12.
English descriptors
- KwdEn :
- Acipenser baerii, Animals, Cell Survival (physiology), Cyprinus carpio, Dual fluorescent staining, Epifluorescence, Fertility (physiology), Fish spermatozoa, Fishes (physiology), Fluorescent Dyes, Image Cytometry (methods), Image cytometry, Image overlay, Male, Microscopy, Fluorescence, Organic Chemicals, Phase contrast microscopy, Propidium, Silurus glanis, Sperm Count (methods), Spermatozoa (cytology), Spermatozoa (physiology), Tinca tinca.
- MESH :
- chemical : Fluorescent Dyes, Organic Chemicals, Propidium.
- cytology : Spermatozoa.
- methods : Image Cytometry, Sperm Count.
- physiology : Cell Survival, Fertility, Fishes, Spermatozoa.
- Animals, Male, Microscopy, Fluorescence.
Abstract
The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual threshholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32–113, 61–105, 48–104 and 29–91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56–94.59%, 93.92–97.02%, 76.14–97.76% and 79.45–83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.
Url:
DOI: 10.1016/j.cellbi.2004.07.014
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Fluorescence</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual threshholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32–113, 61–105, 48–104 and 29–91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56–94.59%, 93.92–97.02%, 76.14–97.76% and 79.45–83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.</div></front></TEI><double doi="10.1016/j.cellbi.2004.07.014"><ISTEX><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">The application of image cytometry to viability assessment in dual fluorescence‐stained fish spermatozoa</title><author><name sortKey="Flajshans, Martin" sort="Flajshans, Martin" uniqKey="Flajshans M" first="Martin" last="Flajšhans">Martin Flajšhans</name></author><author><name sortKey="Cosson, Jacky" sort="Cosson, Jacky" uniqKey="Cosson J" first="Jacky" last="Cosson">Jacky Cosson</name></author><author><name sortKey="Rodina, Marek" sort="Rodina, Marek" uniqKey="Rodina M" first="Marek" last="Rodina">Marek Rodina</name></author><author><name sortKey="Linhart, Otomar" sort="Linhart, Otomar" uniqKey="Linhart O" first="Otomar" last="Linhart">Otomar 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tchèque</country></affiliation></author><author><name sortKey="Cosson, Jacky" sort="Cosson, Jacky" uniqKey="Cosson J" first="Jacky" last="Cosson">Jacky Cosson</name><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>Centre National de la Recherche Scientifique, UMR 7009, Université Paris 6, Station Marine, 06230 Villefranche sur Mer</wicri:regionArea><placeName><region type="region" nuts="2">Provence-Alpes-Côte d'Azur</region><settlement type="city">Villefranche sur Mer</settlement></placeName></affiliation></author><author><name sortKey="Rodina, Marek" sort="Rodina, Marek" uniqKey="Rodina M" first="Marek" last="Rodina">Marek Rodina</name><affiliation wicri:level="1"><country xml:lang="fr">République tchèque</country><wicri:regionArea>Joint Laboratory of Genetics, Physiology and Reproduction of Fish, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic and University of South Bohemia České Budějovice, Research Institute of Fish Culture and Hydrobiology, 38925 Vodňany</wicri:regionArea><wicri:noRegion>38925 Vodňany</wicri:noRegion></affiliation></author><author><name sortKey="Linhart, Otomar" sort="Linhart, Otomar" uniqKey="Linhart O" first="Otomar" last="Linhart">Otomar Linhart</name><affiliation wicri:level="1"><country xml:lang="fr">République tchèque</country><wicri:regionArea>Joint Laboratory of Genetics, Physiology and Reproduction of Fish, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic and University of South Bohemia České Budějovice, Research Institute of Fish Culture and Hydrobiology, 38925 Vodňany</wicri:regionArea><wicri:noRegion>38925 Vodňany</wicri:noRegion></affiliation><affiliation wicri:level="1"><country xml:lang="fr">République tchèque</country><wicri:regionArea>Department of Fisheries, Agriculture Faculty, University of South Bohemia České Budějovice, Studentská 13, 370 05 České Budějovice</wicri:regionArea><wicri:noRegion>370 05 České Budějovice</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j">Cell Biology International</title><idno type="ISSN">1065-6995</idno><idno type="eISSN">1095-8355</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2004-12">2004-12</date><biblScope unit="volume">28</biblScope><biblScope unit="issue">12</biblScope><biblScope unit="page" from="955">955</biblScope><biblScope unit="page" to="959">959</biblScope></imprint><idno type="ISSN">1065-6995</idno></series><idno type="istex">68527B3F9EF080E508B5969A2CB37DD7316F5B85</idno><idno type="DOI">10.1016/j.cellbi.2004.07.014</idno><idno type="ArticleID">CBIN2289</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1065-6995</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Acipenser baerii</term><term>Cyprinus carpio</term><term>Dual fluorescent staining</term><term>Epifluorescence</term><term>Fish spermatozoa</term><term>Image cytometry</term><term>Image overlay</term><term>Phase contrast microscopy</term><term>Silurus glanis</term><term>Tinca tinca</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual threshholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32–113, 61–105, 48–104 and 29–91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56–94.59%, 93.92–97.02%, 76.14–97.76% and 79.45–83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.</div></front></TEI></ISTEX><PubMed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa.</title><author><name sortKey="Flajshans, Martin" sort="Flajshans, Martin" uniqKey="Flajshans M" first="Martin" last="Flajshans">Martin Flajshans</name><affiliation wicri:level="1"><nlm:affiliation>Joint Laboratory of Genetics, Physiology and Reproduction of Fish, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Czech Republic. flajshans@vurh.jcu.cz</nlm:affiliation><country xml:lang="fr">République tchèque</country><wicri:regionArea>Joint Laboratory of Genetics, Physiology and Reproduction of Fish, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech 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Survival</term><term>Fertility</term><term>Fishes</term><term>Spermatozoa</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Male</term><term>Microscopy, Fluorescence</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.</div></front></TEI></PubMed></double></record>Pour manipuler ce document sous Unix (Dilib)
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