Mehmed Berk Toker, Ahmet Ümit Sabancı, Gülcan Avcı, Ahmet Aktar, Barış Denk, Özge Bari, Gözde Rabia Özalp
{"title":"Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.","authors":"Mehmed Berk Toker, Ahmet Ümit Sabancı, Gülcan Avcı, Ahmet Aktar, Barış Denk, Özge Bari, Gözde Rabia Özalp","doi":"10.1089/bio.2023.0073","DOIUrl":null,"url":null,"abstract":"<p><p>Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (<i>p</i> < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.</p>","PeriodicalId":1,"journal":{"name":"Accounts of Chemical Research","volume":null,"pages":null},"PeriodicalIF":16.4000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Accounts of Chemical Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1089/bio.2023.0073","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/24 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.
期刊介绍：
Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance.
Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
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