{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T11:08:53Z","timestamp":1768475333989,"version":"3.49.0"},"reference-count":60,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2023,4,11]],"date-time":"2023-04-11T00:00:00Z","timestamp":1681171200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["SFRH\/BD\/146378\/2019"],"award-info":[{"award-number":["SFRH\/BD\/146378\/2019"]}]},{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["UIDB\/04326\/2020"],"award-info":[{"award-number":["UIDB\/04326\/2020"]}]},{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["UIDP\/04326\/2020"],"award-info":[{"award-number":["UIDP\/04326\/2020"]}]},{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["LA\/P\/0101\/2020"],"award-info":[{"award-number":["LA\/P\/0101\/2020"]}]},{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["EMBRC.PT ALG-01-0145-FEDER-022121"],"award-info":[{"award-number":["EMBRC.PT ALG-01-0145-FEDER-022121"]}]},{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"],"award-info":[{"award-number":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"]}]},{"name":"Portuguese national funds from FCT","award":["SFRH\/BD\/146378\/2019"],"award-info":[{"award-number":["SFRH\/BD\/146378\/2019"]}]},{"name":"Portuguese national funds from FCT","award":["UIDB\/04326\/2020"],"award-info":[{"award-number":["UIDB\/04326\/2020"]}]},{"name":"Portuguese national funds from FCT","award":["UIDP\/04326\/2020"],"award-info":[{"award-number":["UIDP\/04326\/2020"]}]},{"name":"Portuguese national funds from FCT","award":["LA\/P\/0101\/2020"],"award-info":[{"award-number":["LA\/P\/0101\/2020"]}]},{"name":"Portuguese national funds from FCT","award":["EMBRC.PT ALG-01-0145-FEDER-022121"],"award-info":[{"award-number":["EMBRC.PT ALG-01-0145-FEDER-022121"]}]},{"name":"Portuguese national funds from FCT","award":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"],"award-info":[{"award-number":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["SFRH\/BD\/146378\/2019"],"award-info":[{"award-number":["SFRH\/BD\/146378\/2019"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["UIDB\/04326\/2020"],"award-info":[{"award-number":["UIDB\/04326\/2020"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["UIDP\/04326\/2020"],"award-info":[{"award-number":["UIDP\/04326\/2020"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["LA\/P\/0101\/2020"],"award-info":[{"award-number":["LA\/P\/0101\/2020"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["EMBRC.PT ALG-01-0145-FEDER-022121"],"award-info":[{"award-number":["EMBRC.PT ALG-01-0145-FEDER-022121"]}]},{"name":"CRESC Algarve 2020 and COMPETE 2020","award":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"],"award-info":[{"award-number":["ZEBRABLOOM-ALG-01-0247-FEDER-039896"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Type I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.<\/jats:p>","DOI":"10.3390\/ijms24087035","type":"journal-article","created":{"date-parts":[[2023,4,11]],"date-time":"2023-04-11T02:02:33Z","timestamp":1681178553000},"page":"7035","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Type I Diabetes in Zebrafish Reduces Sperm Quality and Increases Insulin and Glucose Transporter Transcripts"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0804-5014","authenticated-orcid":false,"given":"Patr\u00edcia","family":"Diogo","sequence":"first","affiliation":[{"name":"Faculty of Sciences and Technology (FCT), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Necton-Companhia Portuguesa de Culturas Marinhas S.A, Belamandil s\/n, 8700-152 Olh\u00e3o, Portugal"}]},{"given":"Gil","family":"Martins","sequence":"additional","affiliation":[{"name":"Faculty of Sciences and Technology (FCT), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve, 8005-139 Faro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9658-0895","authenticated-orcid":false,"given":"M\u00e1rcio","family":"Sim\u00e3o","sequence":"additional","affiliation":[{"name":"Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve, 8005-139 Faro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9410-4772","authenticated-orcid":false,"given":"Ana","family":"Marreiros","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Algarve Biomedical Center (ABC), University of Algarve, 8005-139 Faro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2753-7874","authenticated-orcid":false,"given":"Ana Catarina","family":"Eufr\u00e1sio","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), 4200-135 Porto, Portugal"}]},{"given":"Elsa","family":"Cabrita","sequence":"additional","affiliation":[{"name":"Faculty of Sciences and Technology (FCT), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9582-1957","authenticated-orcid":false,"given":"Paulo Jorge","family":"Gavaia","sequence":"additional","affiliation":[{"name":"Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"},{"name":"Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve, 8005-139 Faro, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1002\/(SICI)1096-9136(199807)15:7<539::AID-DIA668>3.0.CO;2-S","article-title":"Definition, Diagnosis and Classification of Diabetes Mellitus and Its Complications. Part 1: Diagnosis and Classification of Diabetes Mellitus. Provisional Report of a WHO Consultation","volume":"15","author":"Alberti","year":"1998","journal-title":"Diabet. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.diabres.2013.11.002","article-title":"Global Estimates of Diabetes Prevalence for 2013 and Projections for 2035","volume":"103","author":"Guariguata","year":"2014","journal-title":"Diabetes Res. Clin. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"102","DOI":"10.2337\/diabetes.52.1.102","article-title":"\u03b2-Cell Deficit and Increased \u03b2-Cell Apoptosis in Humans with Type 2 Diabetes","volume":"52","author":"Butler","year":"2003","journal-title":"Diabetes"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1038\/nature05482","article-title":"Mechanisms Linking Obesity to Insulin Resistance and Type 2 Diabetes","volume":"444","author":"Kahn","year":"2006","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.plipres.2008.10.002","article-title":"Dietary Fats and Prevention of Type 2 Diabetes","volume":"48","author":"Willett","year":"2009","journal-title":"Prog. Lipid Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e3075","DOI":"10.1002\/dmrr.3075","article-title":"Incidence and Trend of Type 1 Diabetes and the Underlying Environmental Determinants","volume":"35","author":"Xia","year":"2019","journal-title":"Diabetes\/Metab. Res. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"948","DOI":"10.4103\/1008-682X.150844","article-title":"The Effects of Diabetes on Male Fertility and Epigenetic Regulation during Spermatogenesis","volume":"17","author":"Ding","year":"2015","journal-title":"Asian J. Androl."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1038\/nature12111","article-title":"The Zebrafish Reference Genome Sequence and Its Relationship to the Human Genome","volume":"496","author":"Howe","year":"2013","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1111\/wrr.12633","article-title":"Nifurpirinol: A More Potent and Reliable Substrate Compared to Metronidazole for Nitroreductase-Mediated Cell Ablations","volume":"26","author":"Bergemann","year":"2018","journal-title":"Wound Repair Regen."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1016\/j.mod.2006.11.005","article-title":"Targeted Ablation of Beta Cells in the Embryonic Zebrafish Pancreas Using E. Coli Nitroreductase","volume":"124","author":"Pisharath","year":"2007","journal-title":"Mech. Dev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"432","DOI":"10.1111\/wrr.12536","article-title":"Improved Regeneration and de Novo Bone Formation in a Diabetic Zebrafish Model Treated with Paricalcitol and Cinacalcet","volume":"25","author":"Carvalho","year":"2017","journal-title":"Wound Repair Regen."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.mce.2014.08.005","article-title":"Sperm Glucose Transport and Metabolism in Diabetic Individuals","volume":"396","author":"Dias","year":"2014","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1002\/mrd.20222","article-title":"Involvement of the Pentose Phosphate Pathway and Redox Regulation in Fertilization in the Mouse","volume":"70","author":"Urner","year":"2005","journal-title":"Mol. Reprod. Dev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1466","DOI":"10.1126\/science.1108190","article-title":"Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility","volume":"308","author":"Anway","year":"2005","journal-title":"Science"},{"key":"ref_15","first-page":"157","article-title":"The Effect of Maternal Diabetes on the Wnt-PCP Pathway during Embryogenesis as Reflected in the Developing Mouse Eye","volume":"8","author":"Cano","year":"2015","journal-title":"Dis. Model. Mech."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/S1472-6483(10)60602-5","article-title":"Increased Concentrations of the Oxidative DNA Adduct 7,8-Dihydro-8-Oxo-2-Deoxyguanosine in the Germ-Line of Men with Type 1 Diabetes","volume":"16","author":"Agbaje","year":"2008","journal-title":"Reprod. Biomed. Online"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"253","DOI":"10.4161\/spmg.21362","article-title":"Impact of Obesity on Male Fertility, Sperm Function and Molecular Composition","volume":"2","author":"Palmer","year":"2012","journal-title":"Spermatogenesis"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1111\/j.2047-2927.2013.00071.x","article-title":"High-Energy Diets May Induce a Pre-Diabetic State Altering Testicular Glycolytic Metabolic Profile and Male Reproductive Parameters","volume":"1","author":"Rato","year":"2013","journal-title":"Andrology"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Cabrita, E., Robles, V., and Herr\u00e1ez, M. (2009). Methods in Reproductive Aquaculture: Marine and Freshwater Species, CRC Press. [1st ed.].","DOI":"10.1201\/9780849380549"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1016\/j.theriogenology.2009.07.001","article-title":"Freezability Prediction of Boar Ejaculates Assessed by Functional Sperm Parameters and Sperm Proteins","volume":"72","author":"Casas","year":"2009","journal-title":"Theriogenology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1002\/jand.2002.23.1.71","article-title":"Sperm Cryopreservation for Men With Nonmalignant, Systemic Diseases: A Descriptive Study","volume":"23","author":"Ranganathan","year":"2002","journal-title":"J. Androl."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1016\/j.theriogenology.2009.05.013","article-title":"The Degree of Resistance to Freezing-Thawing Is Related to Specific Changes in the Structures of Motile Sperm Subpopulations and Mitochondrial Activity in Boar Spermatozoa","volume":"72","author":"Flores","year":"2009","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/s41598-018-36624-5","article-title":"The Proteome of Frozen-Thawed Pig Spermatozoa Is Dependent on the Ejaculate Fraction Source","volume":"9","author":"Li","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2692","DOI":"10.2527\/jas.2006-094","article-title":"Factors Influencing Boar Sperm Cryosurvival1","volume":"84","author":"Roca","year":"2006","journal-title":"J. Anim. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Dietrich, M.A., and Ciereszko, A. (2018). Proteomic Characterization of Fresh Spermatozoa and Supernatant after Cryopreservation in Relation to Freezability of Carp (Cyprinus carpio L) Semen. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0192972"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1210\/en.2004-1252","article-title":"Autocrine Regulation of Insulin Secretion in Human Ejaculated Spermatozoa","volume":"146","author":"Aquila","year":"2005","journal-title":"Endocrinology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1111\/j.1365-2605.2009.00971.x","article-title":"Insulin and IR-\u03b2 in Pig Spermatozoa: A Role of the Hormone in the Acquisition of Fertilizing Ability","volume":"33","author":"Carpino","year":"2010","journal-title":"Int. J. Androl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mce.2005.09.011","article-title":"Arguments Raised by the Recent Discovery That Insulin and Leptin Are Expressed in and Secreted by Human Ejaculated Spermatozoa","volume":"245","author":"Aquila","year":"2005","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"348","DOI":"10.2164\/jandrol.110.011197","article-title":"GLUTs and Mammalian Sperm Metabolism","volume":"32","author":"Bucci","year":"2011","journal-title":"J. Androl."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1177\/0148607104028005364","article-title":"The Glucose Transporter Families SGLT and GLUT: Molecular Basis of Normal and Aberrant Function","volume":"28","author":"Scheepers","year":"2004","journal-title":"J. Parenter. Enter. Nutr."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1016\/bs.mcb.2016.04.015","article-title":"Chapter 24\u2014New Frontiers for Zebrafish Management","volume":"135","author":"Lawrence","year":"2016","journal-title":"Methods Cell Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"9732780","DOI":"10.1155\/2016\/9732780","article-title":"Zebrafish: A Versatile Animal Model for Fertility Research","volume":"2016","author":"Hoo","year":"2016","journal-title":"BioMed Res. Int."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"dmm039370","DOI":"10.1242\/dmm.039370","article-title":"Spotlight on Zebrafish: The next Wave of Translational Research","volume":"12","author":"Patton","year":"2019","journal-title":"Dis. Model. Mech."},{"key":"ref_34","first-page":"1","article-title":"Andrological Characterization of the Patient with Diabetes Mellitus","volume":"34","author":"Vignera","year":"2009","journal-title":"Minerva Endocrinol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1080\/01926230701320337","article-title":"Apoptosis: A Review of Programmed Cell Death","volume":"35","author":"Elmore","year":"2007","journal-title":"Toxicol. Pathol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1016\/j.immuni.2005.02.004","article-title":"Granzyme A Induces Caspase-Independent Mitochondrial Damage, a Required First Step for Apoptosis","volume":"22","author":"Martinvalet","year":"2005","journal-title":"Immunity"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e70714","DOI":"10.7554\/eLife.70714","article-title":"Repression of Hypoxia-Inducible Factor-1 Contributes to Increased Mitochondrial Reactive Oxygen Species Production in Diabetes","volume":"11","author":"Zheng","year":"2022","journal-title":"Elife"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1016\/j.fertnstert.2009.06.006","article-title":"The Influence of Type 1 Diabetes Mellitus on Spermatogenic Gene Expression","volume":"92","author":"Mallidis","year":"2009","journal-title":"Fertil. Steril."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/j.cbpa.2010.12.008","article-title":"Metabolism of Motile Zebrafish Sperm","volume":"158","author":"Ingermann","year":"2011","journal-title":"Comp. Biochem. Physiol. Part A Mol. Integr. Physiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"820","DOI":"10.1071\/RD17460","article-title":"Sperm Motility in Fish: Technical Applications and Perspectives through CASA-Mot Systems","volume":"30","author":"Gallego","year":"2018","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.bbagen.2011.11.006","article-title":"Effect of Insulin Deprivation on Metabolism and Metabolism-Associated Gene Transcript Levels of in Vitro Cultured Human Sertoli Cells","volume":"1820","author":"Oliveira","year":"2012","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3084","DOI":"10.1073\/pnas.1513212113","article-title":"Leptin Signaling Regulates Glucose Homeostasis, but Not Adipostasis, in the Zebrafish","volume":"113","author":"Michel","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.ygcen.2018.08.012","article-title":"Different Roles of Insulin Receptor a and b in Maintaining Blood Glucose Homeostasis in Zebrafish","volume":"269","author":"Gong","year":"2018","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1443","DOI":"10.1007\/s10695-018-0500-6","article-title":"Electric Ultrafreezer (\u2212150 \u00b0C) as an Alternative for Zebrafish Sperm Cryopreservation and Storage","volume":"44","author":"Diogo","year":"2018","journal-title":"Fish Physiol. Biochem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/S0303-7207(01)00408-7","article-title":"Analysis of Pancreatic Development in Living Transgenic Zebrafish Embryos","volume":"177","author":"Huang","year":"2001","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1844","DOI":"10.2337\/db08-0628","article-title":"Regeneration of the Pancreas in Adult Zebrafish","volume":"58","author":"Moss","year":"2009","journal-title":"Diabetes"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1095\/biolreprod.109.076299","article-title":"Histological and Stereological Evaluation of Zebrafish (Danio Rerio) Spermatogenesis with an Emphasis on Spermatogonial Generations1","volume":"81","author":"Leal","year":"2009","journal-title":"Biol. Reprod."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1089\/zeb.2009.0640","article-title":"Blood Sugar Measurement in Zebrafish Reveals Dynamics of Glucose Homeostasis","volume":"7","author":"Eames","year":"2010","journal-title":"Zebrafish"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1093\/ilar.53.2.192","article-title":"Anesthesia and Euthanasia in Zebrafish","volume":"53","author":"Matthews","year":"2012","journal-title":"ILAR J."},{"key":"ref_50","first-page":"785","article-title":"Evaluation of Rapid Cooling and Tricaine Methanesulfonate (MS222) as Methods of Euthanasia in Zebrafish (Danio rerio)","volume":"48","author":"Wilson","year":"2009","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.aquaculture.2009.02.027","article-title":"Optimization of Activation, Collection, Dilution, and Storage Methods for Zebrafish Sperm","volume":"290","author":"Jing","year":"2009","journal-title":"Aquaculture"},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Hagedorn, M., and Carter, V.L. (2011). Zebrafish Reproduction: Revisiting in Vitro Fertilization to Increase Sperm Cryopreservation Success. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0021059"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"McCurley, A.T., and Callard, G.V. (2008). Characterization of Housekeeping Genes in Zebrafish: Male-Female Differences and Effects of Tissue Type, Developmental Stage and Chemical Treatment. BMC Mol. Biol., 9.","DOI":"10.1186\/1471-2199-9-102"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments","volume":"55","author":"Bustin","year":"2009","journal-title":"Clin. Chem."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u2212\u0394\u0394CT Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.aquaculture.2012.09.024","article-title":"Sources of Variation in Flow Cytometric Analysis of Aquatic Species Sperm: The Effect of Cryoprotectants on Flow Cytometry Scatter Plots and Subsequent Population Gating","volume":"370\u2013371","author":"Daly","year":"2012","journal-title":"Aquaculture"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.cryobiol.2005.02.005","article-title":"Evaluation of Gilthead Sea Bream, Sparus Aurata, Sperm Quality after Cryopreservation in 5 mL Macrotubes","volume":"50","author":"Cabrita","year":"2005","journal-title":"Cryobiology"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1089\/zeb.2018.1660","article-title":"Selection Criteria of Zebrafish Male Donors for Sperm Cryopreservation","volume":"16","author":"Diogo","year":"2019","journal-title":"Zebrafish"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1111\/jfb.12042","article-title":"Assessment of DNA Damage in Sperm after Repeated Non-Invasive Sampling in Zebrafish Danio rerio","volume":"82","author":"Reinardy","year":"2013","journal-title":"J. Fish Biol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1080\/01621459.1963.10500845","article-title":"Hierarchical Grouping to Optimize an Objective Function","volume":"58","author":"Ward","year":"1963","journal-title":"J. Am. Stat. Assoc."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/8\/7035\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:13:36Z","timestamp":1760123616000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/8\/7035"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,11]]},"references-count":60,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["ijms24087035"],"URL":"https:\/\/doi.org\/10.3390\/ijms24087035","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,11]]}}}