4d61cfd6ac6ea4752ddd9065b9abed81 pmc_33207812.pdf 5532dd07a69ecbd21416bd34fde76dfff0b73482 pmc_33207812.pdf 7d8a2e3b14165ad8feaabe05034938ee1812f5dde7b82332ed4ccc2d5c7c8bbc pmc_33207812.pdf Title: Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils Subject: This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding. Keywords: sperm; fresh semen; donkey; single layer centrifugation; sperm–PMN binding Author: Marion Papas, Jaime Catalán, Sandra Recuero, Jane M. Morrell, Marc Yeste and Jordi Miró Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Tue Nov 17 04:58:01 2020 CET ModDate: Tue Nov 17 07:22:30 2020 CET Custom Metadata: no 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: 1560520 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ENXZUH+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 XNITEO+Pxsy Type 1 Builtin yes yes no 15 0 VYLLMY+VnURWPalladioL Type 1 Custom yes yes no 19 0 VRUTNV+URWPalladioL-Ital Type 1 Custom yes yes no 23 0 VMSPKG+URWPalladioL-Bold Type 1 Custom yes yes no 28 0 LEMFQN+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 OFLXAT+Rpxr Type 1 Builtin yes yes no 36 0 QTLELQ+Pxbsy Type 1 Builtin yes yes no 40 0 SZMRXJ+TimesNewRomanPS-BoldItalicMT Type 1C 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no 273 0 LLFOFH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 276 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 04:17:42 CEST RepresentationInformation: pmc_33207812.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:43:33 CEST Size: 1560520 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 522 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Animals Destination: subsection.2.1 Item: Title: Experimental Design Destination: subsection.2.2 Item: Title: Flow Cytometry Destination: subsection.2.3 Children: Item: Title: Evaluation of Sperm Membrane Integrity (SYBR14/PI) Destination: subsubsection.2.3.1 Item: Title: Evaluation of Sperm Membrane Lipid Disorder (M540/YO-PRO-1) Destination: subsubsection.2.3.2 Item: Title: Evaluation of Mitochondrial Membrane Potential (JC1) Destination: subsubsection.2.3.3 Item: Title: Evaluation of Intracellular Calcium Levels (Fluo3/PI and Rhod5/YO-PRO-1) Destination: subsubsection.2.3.4 Item: Title: Evaluation of Intracellular Reactive Oxygen Species (ROS) Levels: H2O2 and O2- (H2DCFDA/PI and HE/YO-PRO-1) Destination: subsubsection.2.3.5 Item: Title: Evaluation of Sperm Motility Destination: subsection.2.4 Item: Title: Evaluation of Sperm PMN Binding Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Integrity and Lipid Disorder of Sperm Plasma Membrane Destination: subsection.3.1 Item: Title: Mitochondrial Membrane Potential Destination: subsection.3.2 Item: Title: Intracellular Calcium Levels Destination: subsection.3.3 Item: Title: Intracellular Levels of Peroxides and Superoxides Destination: subsection.3.4 Item: Title: Sperm Motility Destination: subsection.3.5 Item: Title: Motile Sperm Subpopulations Destination: subsection.3.6 Item: Title: Sperm-PMN Interaction Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils Author: Marion Papas, Jaime Catalán, Sandra Recuero, Jane M. Morrell, Marc Yeste and Jordi Miró Subject: This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding. 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