787c44ec52350c103f0d3514890836d2 pmc_34941829.pdf 97a7ac54ee7dcc617a43d32f8e92e1b4d48a8740 pmc_34941829.pdf c0c04ab2c48e5164af067aaf4c2e21006722d362ab3de3de1598c29205971091 pmc_34941829.pdf Title: Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm Subject: In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2-) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species. Keywords: sperm cryopreservation; reactive oxygen species (ROS); superoxide anion (O2-); reduced glutathione (GSH); antioxidants; donkey Author: Iván Yánez-Ortiz, Jaime Catalán, Ariadna Delgado-Bermúdez, Augusto Carluccio, Jordi Miró and Marc Yeste Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Dec 2 08:52:04 2021 CET ModDate: Thu Dec 2 08:55:35 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1881118 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- HBVOND+VnURWPalladioL Type 1 Custom yes yes no 10 0 NAYIHB+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 KDYWRT+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 HZVWWM+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 MEQNWG+CMSY10 Type 1 Builtin yes yes no 28 0 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HPKDGL+PalatinoLinotype CID TrueType Identity-H yes yes yes 312 0 HPKDGM+PalatinoLinotype TrueType WinAnsi yes yes no 318 0 HPKDGO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 321 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:28:11 CET RepresentationInformation: pmc_34941829.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-10 07:59:40 CET Size: 1881118 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 592 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Donkeys and Sperm Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Experimental Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Sperm Cryopreservation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Sperm Thawing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Sperm Motility Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Sperm Viability Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Evaluation of Sperm through Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Plasma Membrane Integrity Analysis (SYBR-14/PI) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Acrosome Membrane Integrity Analysis (PNA-FITC/PI) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Mitochondrial Membrane Potential (MMP) Analysis (JC-1) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Intracellular Reactive Oxygen Species (ROS) Levels Analysis: H2O2 (H2DCFDA/PI) and O2- (HE/YO-PRO-1) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Statistical Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Motile Sperm Subpopulations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Sperm Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Sperm Quality Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: Sperm Membrane Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Acrosome Membrane Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Mitochondrial Membrane Potential (MPP) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Intracellular Reactive Oxygen Species (ROS) Levels: H2O2 and O2- Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Info: Title: Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm Author: Iván Yánez-Ortiz, Jaime Catalán, Ariadna Delgado-Bermúdez, Augusto Carluccio, Jordi Miró and Marc Yeste Subject: In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2-) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species. 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629, 540, 640 Annotation: Subtype: Link Rect: 361, 606, 398, 617 Annotation: Subtype: Link Rect: 404, 606, 440, 617 Annotation: Subtype: Link Rect: 122, 584, 160, 594 Annotation: Subtype: Link Rect: 166, 584, 202, 594 Checksum: 4d9cdae8 Type: CRC32 Checksum: 787c44ec52350c103f0d3514890836d2 Type: MD5 Checksum: 97a7ac54ee7dcc617a43d32f8e92e1b4d48a8740 Type: SHA-1