0a669fc305c2dcc1f5dd4c68dd8767c0 NIM_a2025v15p3353.pdf ebea9035cbbfb95a90691967221701dedd377cf9 NIM_a2025v15p3353.pdf 1b0d05b443497207cb0d8a4e9178266be33a477b59c53fe563a8435238b6cf19 NIM_a2025v15p3353.pdf Title: Red LED Light Irradiation Increases the Resistance Against Environmental Stress of Frozen Bovine Sperm Thawed in Suboptimal Conditions Subject: This work sought to test the positive effect of red LED light on frozen bovine sperm resistance to thawing in suboptimal conditions. Moreover, a preliminary study explores whether this improvement could have any repercussions on in vivo fertilizing ability. Thus, frozen bull semen straws (n = 16) were thawed (a) with irradiation for 5 min at 20 C (2 min light, 1 min darkness, 2 min light; PHOTO); (b) without irradiation for 5 min at 20 C (ET); or (c) through immersion into a water bath at 38 C for 40 s as standard control (CONTROL). Sperm quality and preliminary, purely descriptive AI trials were performed. The PHOTO samples demonstrated significantly (p < 0.05) improved percentages of intact acrosomes, progressive motility, DNA condensation and fragmentation, and viable sperm with high ROS/superoxides. The viability of PHOTO samples decreased significantly (p < 0.05) when compared with the ET ones. Overall results of both the PHOTO and ET samples were poorer than those of the CONTROL. Otherwise, the PHOTO straws yielded greater pregnancy rates (64.0% vs. 49.4% in CONTROL) when evaluated at two different farms. The results suggest that irradiating cryopreserved bovine sperm during thawing in suboptimal conditions could improve AI pregnancy rates, although more in vivo studies are needed to support this conclusion. Keywords: red light; irradiation; cryopreserved sperm; artificial insemination Author: Olga Blanco-Prieto, Carolina Maside, Andrea Gruzmacher, Manuel Ortiz, Marcelo Ratto, Francisco Javier Urra, Tomás Vera, Pablo Strobel, Jaime Catalán, Beatrice Mislei, Diego Bucci, Marc Yeste, Joan E. Rodríguez-Gil and Alfredo Ramírez-Reveco Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Thu Nov 20 12:28:31 2025 CET ModDate: Thu Nov 20 12:39:03 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1877278 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CTWYMC+URWPalladioL-Roma Type 1 Custom yes yes yes 173 0 DARXAD+CMSY10 Type 1 Builtin yes yes yes 174 0 HFLUYY+URWPalladioL-Ital Type 1 Custom yes yes yes 175 0 TJGYCB+URWPalladioL-Bold Type 1 Custom yes yes yes 176 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 241 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 293 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 294 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 302 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 303 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 341 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 342 0 EUQOAW+EURM7 Type 1 Builtin yes yes yes 374 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 383 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 384 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 395 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 396 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 426 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 427 0 OHIMLK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 450 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 451 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 471 0 OHIMLI+PalatinoLinotype TrueType WinAnsi yes yes no 494 0 DRZBZG+VnURWPalladioL Type 1 Custom yes yes yes 614 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-12-19 05:12:13 CET RepresentationInformation: NIM_a2025v15p3353.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-12-18 13:26:01 CET Size: 1877278 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 714 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Animals and Samples Item: Title: Evaluation of Post-Thaw Sperm Integrity and Functionality Children: Item: Title: Sperm Viability Item: Title: Acrosome Integrity Item: Title: Sperm Motility Item: Title: Chromatin Condensation Item: Title: Sperm DNA Fragmentation Item: Title: Antioxidant Capacity Item: Title: Nitro Blue Tetrazolium Chloride Test Item: Title: Mitochondrial Membrane Potential Item: Title: Flow Cytometry Analyses Children: Item: Title: Evaluation of Intracellular ROS Levels (DHE/YO-PRO-1 and CM-H2DCFDA/PI) Item: Title: Mitochondria-Associated ROS Production (MitoSOX/Mitotracker Deep Red/YO-PRO-1) Item: Title: Artificial Insemination Trials Item: Title: Statistical Analyses Item: Title: Results Children: Item: Title: Thawing Dynamics of Sperm Straws Item: Title: Effects of Red-Light Irradiation on Sperm Viability and Acrosome Integrity Item: Title: Effects of Red-Light Irradiation on Sperm Motility Item: Title: Effects of Red-Light Irradiation on Motile Sperm Subpopulations Item: Title: Effects of Red-Light Irradiation on Chromatin Condensation and DNA Fragmentation Item: Title: Effects of Red-Light Irradiation on ROS Levels and Overall Antioxidant Capacity Item: Title: Effects of Red-Light Irradiation on Mitochondrial Function Item: Title: Effects of Red-Light Irradiation on In Vivo Reproductive Performance Item: Title: Discussion Item: Title: Conclusions Item: Title: Patents Item: Title: References Info: Title: Red LED Light Irradiation Increases the Resistance Against Environmental Stress of Frozen Bovine Sperm Thawed in Suboptimal Conditions Author: Olga Blanco-Prieto, Carolina Maside, Andrea Gruzmacher, Manuel Ortiz, Marcelo Ratto, Francisco Javier Urra, Tomás Vera, Pablo Strobel, Jaime Catalán, Beatrice Mislei, Diego Bucci, Marc Yeste, Joan E. Rodríguez-Gil and Alfredo Ramírez-Reveco Subject: This work sought to test the positive effect of red LED light on frozen bovine sperm resistance to thawing in suboptimal conditions. Moreover, a preliminary study explores whether this improvement could have any repercussions on in vivo fertilizing ability. Thus, frozen bull semen straws (n = 16) were thawed (a) with irradiation for 5 min at 20 C (2 min light, 1 min darkness, 2 min light; PHOTO); (b) without irradiation for 5 min at 20 C (ET); or (c) through immersion into a water bath at 38 C for 40 s as standard control (CONTROL). Sperm quality and preliminary, purely descriptive AI trials were performed. The PHOTO samples demonstrated significantly (p < 0.05) improved percentages of intact acrosomes, progressive motility, DNA condensation and fragmentation, and viable sperm with high ROS/superoxides. The viability of PHOTO samples decreased significantly (p < 0.05) when compared with the ET ones. Overall results of both the PHOTO and ET samples were poorer than those of the CONTROL. Otherwise, the PHOTO straws yielded greater pregnancy rates (64.0% vs. 49.4% in CONTROL) when evaluated at two different farms. The results suggest that irradiating cryopreserved bovine sperm during thawing in suboptimal conditions could improve AI pregnancy rates, although more in vivo studies are needed to support this conclusion. 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