{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T07:58:14Z","timestamp":1791359894481,"version":"4.3.1"},"reference-count":24,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,2,18]],"date-time":"2025-02-18T00:00:00Z","timestamp":1739836800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004901","name":"FAPEMIG (Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de Minas Gerais)","doi-asserted-by":"publisher","award":["APQ-01881-22"],"award-info":[{"award-number":["APQ-01881-22"]}],"id":[{"id":"10.13039\/501100004901","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004901","name":"FAPEMIG (Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de Minas Gerais)","doi-asserted-by":"publisher","award":["306482\/2022-3"],"award-info":[{"award-number":["306482\/2022-3"]}],"id":[{"id":"10.13039\/501100004901","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004901","name":"FAPEMIG (Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de Minas Gerais)","doi-asserted-by":"publisher","award":["001"],"award-info":[{"award-number":["001"]}],"id":[{"id":"10.13039\/501100004901","id-type":"DOI","asserted-by":"publisher"}]},{"name":"CNPq (Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico)","award":["APQ-01881-22"],"award-info":[{"award-number":["APQ-01881-22"]}]},{"name":"CNPq (Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico)","award":["306482\/2022-3"],"award-info":[{"award-number":["306482\/2022-3"]}]},{"name":"CNPq (Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico)","award":["001"],"award-info":[{"award-number":["001"]}]},{"name":"CAPES (Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superiorl)","award":["APQ-01881-22"],"award-info":[{"award-number":["APQ-01881-22"]}]},{"name":"CAPES (Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superiorl)","award":["306482\/2022-3"],"award-info":[{"award-number":["306482\/2022-3"]}]},{"name":"CAPES (Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superiorl)","award":["001"],"award-info":[{"award-number":["001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Sperm chromatin analysis is a crucial tool for investigating fertility in bulls, both in the field and in in vitro fertilization. This study aimed to identify efficient, low-cost, and easy-to-apply methods for detecting chromatin integrity alterations. Frozen semen samples from four bulls with varying results in in vitro embryo production (IVEP) were used. The sperm chromatin from these samples was evaluated using different methods: differences in the staining intensity with toluidine blue and the Feulgen reaction (FR), assessed both visually and computationally; visual evaluation of smears stained with acridine orange (AO) using epifluorescence and confocal microscopy; and evaluation using transmission electron microscopy (TEM) of sections positively stained with uranyl and lead. The same samples were utilized for IVEP. The results obtained using each method were correlated with one another and with the cleavage and embryonic development rates achieved in IVEPs. The odds ratio was employed to compare the chromatin alteration identification rates of the studied methods. Overall, chromatin integrity alterations identified using the AO and TEM methods exhibited correlations with the cleavage (r = \u22121.00) and embryonic development rates (r = \u22121.00), respectively. Among the methods tested, TEM and AO were the most effective for identifying chromatin integrity alterations that interfere with IVEP efficiency.<\/jats:p>","DOI":"10.3390\/vetsci12020184","type":"journal-article","created":{"date-parts":[[2025,2,18]],"date-time":"2025-02-18T12:16:37Z","timestamp":1739880997000},"page":"184","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Methods Used to Assess Bull Sperm Chromatin Integrity and Its Correlation with In Vitro Embryo Production Efficiency"],"prefix":"10.3390","volume":"12","author":[{"given":"Matheus Vicente","family":"Silva","sequence":"first","affiliation":[{"name":"Faculdade de Medicina Veterin\u00e1ria, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38400-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lays Oliveira","family":"Rocha","sequence":"additional","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38400-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruno Augusto Nassif","family":"Traven\u00e7olo","sequence":"additional","affiliation":[{"name":"Faculdade de Computa\u00e7\u00e3o, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38400-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ednaldo Carvalho","family":"Guimar\u00e3es","sequence":"additional","affiliation":[{"name":"Instituto de Matem\u00e1tica e Estat\u00edstica, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38400-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9320-7278","authenticated-orcid":false,"given":"Marcelo Emilio","family":"Beletti","sequence":"additional","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38400-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.bbagrm.2013.08.004","article-title":"Chromatin dynamics during spermiogenesis","volume":"1839","author":"Rathke","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.anireprosci.2007.01.005","article-title":"Biology of sperm chromatin structure and relationship to male fertility and embryonic survival","volume":"101","author":"Hengstberger","year":"2007","journal-title":"Anim. Reprod. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/j.freeradbiomed.2013.07.044","article-title":"DNA oxidative damage in mammalian spermatozoa: Where and why is the male nucleus affected?","volume":"65","author":"Noblanc","year":"2013","journal-title":"Free Radic. Biol. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1007\/s10815-011-9631-8","article-title":"Sperm DNA integrity assays: Diagnostic and prognostic challenges and implications in management of infertility","volume":"28","author":"Shamsi","year":"2011","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.anireprosci.2016.01.017","article-title":"The Sperm Chromatin Structure Assay (SCSA\u00ae) and Other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility","volume":"169","author":"Evenson","year":"2016","journal-title":"Anim. Reprod. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"32","DOI":"10.5194\/aab-48-32-2005","article-title":"Effect of quality of sperm chromatin structure on in-vitro production of cattle embryos","volume":"48","author":"Bochenek","year":"2005","journal-title":"Arch. Anim. Breed."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.theriogenology.2017.11.006","article-title":"Effect of bovine sperm chromatin integrity evaluated using three different methods on in vitro fertility","volume":"107","author":"Castro","year":"2018","journal-title":"Theriogenology"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.fertnstert.2007.06.018","article-title":"Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates","volume":"90","author":"Lin","year":"2008","journal-title":"Fertil. Steril."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1186\/BF03547107","article-title":"Studies on DNA contente, dry mass and optical area of ejaculated spermatozoal heads from bulls with normal and lowered fertility","volume":"7","author":"Gledhill","year":"1966","journal-title":"Acta Vet. Scand."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1530\/jrf.0.1010531","article-title":"Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei","volume":"101","author":"Dobrinski","year":"1994","journal-title":"J. Reprod. Fertil."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1016\/j.theriogenology.2008.07.005","article-title":"Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability","volume":"70","author":"Fernandes","year":"2008","journal-title":"Theriogenology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1007\/BF00493438","article-title":"Induced metachromasia in bull spermatozoa","volume":"74","author":"Mello","year":"1982","journal-title":"Histochemistry"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1095\/biolreprod.114.124255","article-title":"Sperm protamine-status correlates to the fertility of breeding bulls","volume":"92","author":"Dogan","year":"2015","journal-title":"Biol. Reprod."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Hiraiwa, S.R., Alves, P.H.M., Traven\u00e7olo, B.A.N., Martins, M.C., and Beletti, M.E. (2021). Classification of the sperm chromatin compaction alterations in bulls (bos taurus) and its correlation with the efficiency of the in vitro production of embryos. Biosci. J., 37.","DOI":"10.14393\/BJ-v37n0a2021-47967"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/S0015-0282(16)47963-X","article-title":"A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence","volume":"42","author":"Tejada","year":"1984","journal-title":"Fertil. Steril."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/j.1439-0531.2008.01301.x","article-title":"Sperm Chromatin Stability During In Vitro Manipulation of Beef Bull Semen","volume":"45","author":"Lymberopoulos","year":"2010","journal-title":"Reprod. Dom. Anim."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"171","DOI":"10.3109\/19396368.2016.1155242","article-title":"The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic","volume":"62","author":"Moretti","year":"2016","journal-title":"Syst. Biol. Reprod. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1002\/j.1939-4640.1993.tb03263.x","article-title":"The nuclear status of human sperm cells by TEM image cytometry: Nuclear shape and chromatin texture in semen samples from fertile and infertile men","volume":"14","author":"Auger","year":"1993","journal-title":"J. Androl."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.cryobiol.2019.02.005","article-title":"Vitrification induces critical subcellular damages in ram spermatozoa","volume":"87","author":"Arando","year":"2019","journal-title":"Cryobiology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"110297","DOI":"10.1016\/j.vetpar.2024.110297","article-title":"The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility","volume":"331","author":"Blandon","year":"2024","journal-title":"Vet. Parasitol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"904","DOI":"10.1095\/biolreprod64.3.904","article-title":"Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: Correlation with morphological criteria and developmental capacity after in vitro fertilization culture","volume":"64","author":"Stojkovic","year":"2001","journal-title":"Biol. Reprod."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1016\/j.theriogenology.2003.10.016","article-title":"Comparison between the toluidina blue stain and the Feulgen reaction for evalution of rabbit sperm chromatin condensation and their relationship whit sperm morphology","volume":"62","author":"Beletti","year":"2004","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zini, A., and Agarwal, A. (2011). Acridine Orange Test for Assessment of Human Sperm DNA Integrity. Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction, Springer Science. Chapter 13.","DOI":"10.1007\/978-1-4419-6857-9"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1016\/S0093-691X(98)00027-2","article-title":"Cytogenetic analysis of bovine parthenotes after spontaneous activation in vitro","volume":"49","author":"Lechniak","year":"1998","journal-title":"Theriogenology"}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/12\/2\/184\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:37:11Z","timestamp":1760027831000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/12\/2\/184"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,18]]},"references-count":24,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,2]]}},"alternative-id":["vetsci12020184"],"URL":"https:\/\/doi.org\/10.3390\/vetsci12020184","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,18]]}}}