9b43beef28981a7776a8b105667aab1e animals-16-02185.pdf 4072be41bf144c17190457223631d42dd2bcaf16 animals-16-02185.pdf 3ab503154866598bcb410bca2e74e0b92d4a759964603f35c2e9d27c6d9639cc animals-16-02185.pdf Title: DNase Promotes the Release of Sperm Trapped by NETs and Induces Important Changes in Donkey Sperm Motility Patterns Subject: A deeper understanding of the donkey’s reproductive strategy is essential to improve reproductive efficiency in this species. In the jenny’s uterus, sperm selection relies on the post-insemination endometrial reaction, characterized by the influx of polymorphonuclear neutrophils (PMNs), which form extracellular traps (NETs) that capture spermatozoa. Of these, some are liberated through the action of a deoxyribonuclease (DNase) present in the seminal plasma. This study aimed to develop an in vitro model to evaluate the effects of NETs and DNase action on donkey sperm viability and motility. For this purpose, donkey ejaculates were collected, PMNs were isolated from the jenny’s blood and free spermatozoa were selected using a Silica gel filtration (EquiPure®, EP, Nidacon International AB, Mölndal, Sweden) to form five experimental groups: control (semen), SPZ-PMN, SPZ-PMN-DNase, SPZ-PMN-EP, SPZ-PMN-DNase-EP. Sperm viability and motility were assessed in all experimental groups using eosin-nigrosin staining and CASA sperm analysis system, respectively, at 1, 2 and 3 h of incubation. Sperm viability remained stable in the groups containing PMN, while it significantly (p < 0.05) decreased in the control group, suggesting a protective effect of PMN on sperm viability. Regarding kinetic parameters, spermatozoa initially captured by NETs and later liberated by DNase exhibited higher velocity (VCL, VSL, VAP), linearity (LIN), straightness (STR) and oscillation (WOB). These changes in sperm motility may be related to the physiological changes occurring before sperm capacitation. This study can improve the understanding of the unique reproductive strategy of donkeys and could be useful to improve in vitro sperm capacitation and embryo production. Keywords: donkey; sperm; neutrophil extracellular traps (NETs); deoxyribonuclease (DNase); polymorphonuclear neutrophils (PMNs) Author: Sandra Llorens, Sebastià Company, Luisina Chapero, Iván Yánez-Ortiz, Augusto Carluccio, Ippolito De Amicis, Jaime Catalán and Jordi Miró Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jul 14 08:57:11 2026 CEST ModDate: Tue Jul 14 09:17:38 2026 CEST Custom Metadata: yes Metadata Stream: no 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: 1644569 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 148 0 WNWTFR+VnURWPalladioL-Bold Type 1 Custom yes yes yes 149 0 ZLXUAG+URWPalladioL-Bold Type 1 Custom yes yes yes 150 0 RWJFFP+URWPalladioL-Roma Type 1 Custom yes yes yes 151 0 XMAAKJ+URWPalladioL-Ital Type 1 Custom yes yes yes 152 0 SIJIIJ+CMSY10 Type 1 Builtin yes yes yes 230 0 GPKMID+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 219 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 220 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 221 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 265 0 GPKOHP+ArialMT TrueType WinAnsi yes yes no 257 0 GPKMID+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 258 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 259 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 260 0 GPKMID+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 303 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 304 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 305 0 GPKMID+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 331 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 332 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 333 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 365 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 371 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 387 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 388 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 405 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 406 0 URYLRZ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 456 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 412 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 413 0 GPKMHB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 450 0 GPKMIC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 451 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-09-30 03:57:14 CEST RepresentationInformation: animals-16-02185.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-09-29 14:33:17 CEST Size: 1644569 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 676 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 Item: Title: Isolation of PMNs Item: Title: Semen Collection and Process Item: Title: Evaluation of Sperm Viability Item: Title: Evaluation of Sperm Motility Item: Title: DNase Item: Title: Experimental Groups Item: Title: Statistical Analysis Item: Title: Results Children: Item: Title: Sperm Viability Item: Title: Sperm Motility Children: Item: Title: Total and Progressive Motility Item: Title: Velocity Parameters Item: Title: Linearity Item: Title: Straightness Item: Title: Wobble Item: Title: Lateral Head Displacement Item: Title: Beat Cross Frequency Item: Title: Motile Sperm Subpopulation Study Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: DNase Promotes the Release of Sperm Trapped by NETs and Induces Important Changes in Donkey Sperm Motility Patterns Author: Sandra Llorens, Sebastià Company, Luisina Chapero, Iván Yánez-Ortiz, Augusto Carluccio, Ippolito De Amicis, Jaime Catalán and Jordi Miró Subject: A deeper understanding of the donkey’s reproductive strategy is essential to improve reproductive efficiency in this species. In the jenny’s uterus, sperm selection relies on the post-insemination endometrial reaction, characterized by the influx of polymorphonuclear neutrophils (PMNs), which form extracellular traps (NETs) that capture spermatozoa. Of these, some are liberated through the action of a deoxyribonuclease (DNase) present in the seminal plasma. This study aimed to develop an in vitro model to evaluate the effects of NETs and DNase action on donkey sperm viability and motility. For this purpose, donkey ejaculates were collected, PMNs were isolated from the jenny’s blood and free spermatozoa were selected using a Silica gel filtration (EquiPure®, EP, Nidacon International AB, Mölndal, Sweden) to form five experimental groups: control (semen), SPZ-PMN, SPZ-PMN-DNase, SPZ-PMN-EP, SPZ-PMN-DNase-EP. Sperm viability and motility were assessed in all experimental groups using eosin-nigrosin staining and CASA sperm analysis system, respectively, at 1, 2 and 3 h of incubation. Sperm viability remained stable in the groups containing PMN, while it significantly (p < 0.05) decreased in the control group, suggesting a protective effect of PMN on sperm viability. Regarding kinetic parameters, spermatozoa initially captured by NETs and later liberated by DNase exhibited higher velocity (VCL, VSL, VAP), linearity (LIN), straightness (STR) and oscillation (WOB). These changes in sperm motility may be related to the physiological changes occurring before sperm capacitation. This study can improve the understanding of the unique reproductive strategy of donkeys and could be useful to improve in vitro sperm capacitation and embryo production. 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