895e9e4933bd761524e2a251bc63c3a9 pmc_34944229.pdf 1124eae6be0317067a9e4bbc4a813991eabb50b6 pmc_34944229.pdf 775e4513d56f9b10d2351e1d961fa33892593226c185e83fc6d6384e0e2ea332 pmc_34944229.pdf Title: The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters Subject: In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant. Keywords: cryopreservation; seminal plasma; incubation; buck sperm; season; age; melatonin Author: Uchechi Linda Ohaneje, Uchebuchi Ike Osuagwuh, Manuel Alvarez-Rodríguez, Iván Yánez-Ortiz, Abigail Tabarez and María Jesús Palomo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Dec 6 07:15:44 2021 CET ModDate: Mon Dec 6 07:17:45 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 315929 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SDGJWK+VnURWPalladioL Type 1 Custom yes yes no 10 0 FMSVDF+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 OVMWLB+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 LHZYXX+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 JGBJTI+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 SZMRXJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 66 0 LRJSKZ+VnURWPalladioL-Italic Type 1 Custom yes yes no 81 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 85 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 89 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 02:53:08 CEST RepresentationInformation: pmc_34944229.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:06:32 CEST Size: 315929 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 285 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Reagents and Media Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Preparation of Extenders Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Semen Collection, Processing, and Freezing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Thawing and Sperm Washing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Incubation of Washed Sperm Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Plasma and Acrosome Membrane Integrity and Mitochondrial Function Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Analysis of DNA Integrity: Sperm Chromatin Structural Assay (SCSA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Analysis of Sperm Motility and Kinetic Parameters Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Info: Title: The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters Author: Uchechi Linda Ohaneje, Uchebuchi Ike Osuagwuh, Manuel Alvarez-Rodríguez, Iván Yánez-Ortiz, Abigail Tabarez and María Jesús Palomo Subject: In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant. 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