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Fresh and frozen-thawed samples were analyzed using CASA and flow cytometry, measuring motility, viability, acrosome integrity, mitochondrial membrane potential and integrity, and multiple reactive oxygen species outputs. Cryopreservation reduced motility and mitochondrial parameters while shifting ROS and superoxide patterns, and incubation further increased mitochondrial damage and superoxide production.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/mitochondrial-function-and-reactive-oxygen-species-dynamics-in-italian-mediterranean-buffalo-semen-following-cryopreservation-and-post-thaw-incubation/412854/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/mitochondrial-function-and-reactive-oxygen-species-dynamics-in-italian-mediterranean-buffalo-semen-following-cryopreservation-and-post-thaw-incubation/412854.png","ImageObject",300,407,{"name":92,"@type":93},"A glass of water","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-10-31","2026-09-28",true,{"@type":102,"interactionType":103,"userInteractionCount":44},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What is the study purpose regarding IMB semen?","Question",{"text":112,"@type":113},"To investigate how physiological parameters and redox-related biomarkers differ between fresh and frozen-thawed Italian Mediterranean buffalo semen, and to clarify links between mitochondrial function and sperm performance.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"How were sperm samples analyzed in the study?",{"text":117,"@type":113},"Fresh and frozen-thawed semen were assessed using CASA and flow cytometry, including motility, viability, acrosome integrity, mitochondrial membrane potential and integrity, and reactive oxygen species and superoxide production.",{"name":119,"@type":110,"acceptedAnswer":120},"What are the key findings after cryopreservation and incubation?",{"text":121,"@type":113},"Cryopreservation significantly decreased total and progressive motility, H2O2 production, and mitochondrial membrane potential. After 3 h incubation, mitochondrial damage and superoxide production increased while total ROS production decreased.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},412854,1791098062,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":44,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":139,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":41,"preview_page_count":4},962090760832,"https://ap-avatar.wpscdn.com/davatar_155a257f0dc6eb9ab79c44ca47cae57d","TYPE Original Research PUBLISHED 18 December 2025 DOI 10. 3389/fvets.2025.1733446  \nOPEN ACCESS  \nEDITED BY  \nDaniel Mota-Rojas,  \nMetropolitan Autonomous University, Mexico  \nREVIEWED BY  \nRupali Rautela,  \nCentral Institute for Research on Cattle, India Manal Bakeer,  \nCairo University, Egypt  \n*CORRESPONDENCE  \nGiada Loddo  \n [giada.loddo@phd.unipd.it](giada.loddo@phd.unipd.it)  \nRECEIVED 27 October 2025  \nREVISED 21 November 2025  \nACCEPTED 26 November 2025  \nPUBLISHED 18 December 2025  \nCITATION  \nLoddo G, Gelain ME, Gabai G, D’Andrea A, Montanari E, Milani C and Giaretta E (2025) Mitochondrial function and reactive oxygen species dynamics in Italian Mediterranean buffalo semen following cryopreservation and post-thaw incubation.  \nFront. Vet. Sci. 12:1733446 .  \ndoi: 10.3389/fvets.2025.1733446  \nCOPYRIGHT  \n© 2025 Loddo, Gelain, Gabai, D’Andrea, Montanari, Milani and Giaretta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.  \nMitochondrial function and reactive oxygen species dynamics in Italian Mediterranean buffalo semen following cryopreservation and post-thaw incubation  \nGiada Loddo1*, Maria Elena Gelain1 , Gianfranco Gabai1 , Asia D’Andrea2 , Elisabetta Montanari2 , Chiara Milani3 and Elisa Giaretta1  \n1 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy,  \n2 Intermizoo S. p.A, Caorle, Italy, 3 Department of Animal Medicine, Production and Health (MAPS), University of Padova, Legnaro, Italy  \nIntroduction: The current understanding of physiological parameters and redox balance in buffalo bull semen is limited and derived from various breeds. Moreover, the effects of cryopreservation in various buffalo breeds remain unclear.  \nMethods: This study aimed to investigate the relationships between physiological parameters and compare fresh (F) and frozen-thawed (T) semen in Italian Mediterranean buffalos (IMB; 7, bulls) . Buffalo ejaculates were collected using an artificial vagina and cryopreserved using a standard protocol. Both F and T were analyzed by CASA and flow cytometry Semen parameters assessed included motility, viability (using PI or SG counterstains for each assay), acrosome integrity (PSA), mitochondrial membrane potential (JC1), mitochondrial integrity (MT), intracytoplasmic (DHE) and mitochondrial (MX) superoxide production, and other intracellular reactive oxygen species (CR) . The T samples were assessed immediately after thawing (T0) and following 3 h incubation at 37◦ C (T3) .  \nResults: Results showed significant correlations (p \u003C 0 .05) between total motility (TM) and progressive motility (PM) with mitochondrial membrane potential (MMP) and mitochondrial integrity (MI) . The TM, PM, MI, and MMP positively correlated (p \u003C 0.05) with total reactive oxygen species (ROS) production and negatively with superoxide production. Cryopreservation significantly decreased TM from 92 .1 ± 5.66% to 72 .61 ± 18.62%(p \u003C 0.05), H2 O2 production from 23.02 ± 7.42% to 11 .49 ± 8.85%(p \u003C 0.05), and MMP from 83 .29 ± 11. 20 to 56.98 ± 15.87%(p \u003C 0.05) . After 3 h incubation, T semen exhibited increased mitochondrial damage and superoxide production, with decreased total ROS production.  \nDiscussion: In conclusion, cryopreservation and subsequent incubation significantly affect mitochondrial functions, which strongly correlate with sperm motility. A deeper understanding of sperm energy metabolism and its relationship with redox regulation could allow for the optimization of current assisted reproductive technologies (ART), as these factors play a crucial role in sperm  \nFrontiers in Veterin","cbCaivUaWHioBkE6","https://ap.wps.com/l/cbCaivUaWHioBkE6","pdf",293398,12,"English","# Introduction\n## Study background and knowledge gaps\n# Methods\n## Sample collection and cryopreservation\n## Flow cytometry and CASA assessments\n## Timepoints: T0 and T3\n# Results\n## Correlations between motility and mitochondrial metrics\n## Effects of cryopreservation on ROS and mitochondrial markers\n## Changes after post-thaw incubation\n# Discussion\n## Interpretation and implications for ART","[{\"question\":\"What is the study purpose regarding IMB semen?\",\"answer\":\"To investigate how physiological parameters and redox-related biomarkers differ between fresh and frozen-thawed Italian Mediterranean buffalo semen, and to clarify links between mitochondrial function and sperm performance.\"},{\"question\":\"How were sperm samples analyzed in the study?\",\"answer\":\"Fresh and frozen-thawed semen were assessed using CASA and flow cytometry, including motility, viability, acrosome integrity, mitochondrial membrane potential and integrity, and reactive oxygen species and superoxide production.\"},{\"question\":\"What are the key findings after cryopreservation and incubation?\",\"answer\":\"Cryopreservation significantly decreased total and progressive motility, H2O2 production, and mitochondrial membrane potential. After 3 h incubation, mitochondrial damage and superoxide production increased while total ROS production decreased.\"}]","Mitochondrial function and reactive oxygen species dynamics in Italian Mediterranean buffalo semen following cryopreservation and post-thaw incubation | PDF",1790593028]