f5c7fb2fc59cf9e088d751562c12e2b1 biology_a2020v9p254.pdf 7d41d5dc0ab5df625a9b704ac593dbcdebd1550c biology_a2020v9p254.pdf ee8a25f66a9eb007486ea4a705f2e734940e5f2bf4b46e4f734f58a12e3ccc78 biology_a2020v9p254.pdf Title: Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure Subject: Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects. Keywords: photobiology; red-light; horse; semen; motility; mitochondrial activity Author: Jaime Catalán, Marion Papas, Sabrina Gacem, Yentel Mateo-Otero, Joan E. Rodríguez-Gil, Jordi Miró and Marc Yeste Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Sat Aug 29 11:50:57 2020 CEST ModDate: Sat Aug 29 11:50:57 2020 CEST 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: 1832067 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MHJXND+URWPalladioL-Ital Type 1 Custom yes yes no 130 0 DZABYN+URWPalladioL-Bold Type 1 Custom yes yes no 131 0 PRJKGQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 132 0 QTLELQ+Pxbsy Type 1 Builtin yes yes no 133 0 VTDBNT+URWPalladioL-Roma Type 1 Custom yes yes no 134 0 ZMEFUK+VnURWPalladioL Type 1 Custom yes yes no 135 0 OFLXAT+Rpxr Type 1 Builtin yes yes no 136 0 ESKEDM+Pxsy Type 1 Builtin yes yes no 137 0 GLCFZC+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 142 0 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yes no 612 0 RYVCUX+VnURWPalladioL-Italic Type 1 Custom yes yes no 756 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2021-05-01 02:38:35 CEST RepresentationInformation: biology_a2020v9p254.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-04-30 11:22:34 CEST Size: 1832067 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 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: ぉのぴひはつふっぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸲ Item: Title: きちぴづひどちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰きづぴとはつびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮ㄩ‾㸊⡜㌷㙜㌷㝜〰こふばばぬどづひびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぁのどねちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰ぅなちっふぬちぴづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰いづびどでのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Children: Item: Title: こふばばぬどづひびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぁのどねちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰ぅなちっふぬちぴづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰いづびどでのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Item: Title: ぁのどねちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰ぅなちっふぬちぴづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰いづびどでのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Item: Title: ぅへばづひどねづのぴちぬぜ〴ぜ〰いづびどでのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Item: Title: ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Item: Title: うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸱ Children: Item: Title: ぇづのづひちぬぜ〴ぜ〰ぉのてはひねちぴどはのぜ〴ぜ〰ちぢはふぴぜ〴ぜ〰うぬはぷぜ〴ぜ〰ぃべぴはねづぴひべぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸲ Item: Title: ぐぬちびねちぜ〴ぜ〰きづねぢひちのづぜ〴ぜ〰ぉのぴづでひどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸳ Item: Title: ぅぶちぬふちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰きどぴはっとはのつひどちぬぜ〴ぜ〰きづねぢひちのづぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴ぜ〰ぜ〵ぜ〰ね〬ぜ〴ぜ〰おぃ〱ぜ〵ㅜ〰ぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸵⸴ Item: Title: いぎぁぜ〴ぜ〰ぉのぴづでひどぴべぜ〴ぜ〰ぜ〵ぜ〰こぃこぁぜ〴ぜ〰ごづびぴぜ〵ㅜ〰ぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰いづぴづひねどのちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㜩‾㸊⡜㌷㙜㌷㝜〰いづぴづひねどのちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰く〲ぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴ぜ〰げちぴづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㠩‾㸊⡜㌷㙜㌷㝜〰こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: いづぴづひねどのちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㜩‾㸊⡜㌷㙜㌷㝜〰いづぴづひねどのちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰く〲ぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴ぜ〰げちぴづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㠩‾㸊⡜㌷㙜㌷㝜〰こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: いづぴづひねどのちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰く〲ぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴ぜ〰げちぴづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㠩‾㸊⡜㌷㙜㌷㝜〰こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: げづびふぬぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮ㄩ‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰ざどちぢどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㈩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰きどぴはっとはのつひどちぬぜ〴ぜ〰きづねぢひちのづぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴ぜ〰ぜ〵ぜ〰ねぜ〵ㅜ〰ぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㌩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㐩‾㸊⡜㌷㙜㌷㝜〰こばづひねぜ〴ぜ〰こふぢばはばふぬちぴどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㔩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Children: Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰ざどちぢどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㈩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰きどぴはっとはのつひどちぬぜ〴ぜ〰きづねぢひちのづぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴ぜ〰ぜ〵ぜ〰ねぜ〵ㅜ〰ぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㌩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㐩‾㸊⡜㌷㙜㌷㝜〰こばづひねぜ〴ぜ〰こふぢばはばふぬちぴどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㔩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰きどぴはっとはのつひどちぬぜ〴ぜ〰きづねぢひちのづぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴ぜ〰ぜ〵ぜ〰ねぜ〵ㅜ〰ぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㌩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㐩‾㸊⡜㌷㙜㌷㝜〰こばづひねぜ〴ぜ〰こふぢばはばふぬちぴどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㔩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰ぉひひちつどちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰こばづひねぜ〴ぜ〰きはぴどぬどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㐩‾㸊⡜㌷㙜㌷㝜〰こばづひねぜ〴ぜ〰こふぢばはばふぬちぴどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㔩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: こばづひねぜ〴ぜ〰こふぢばはばふぬちぴどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㔩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰ぉのぴひちっづぬぬふぬちひぜ〴ぜ〰ぁごぐぜ〴ぜ〰がづぶづぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㘩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰くへべでづのぜ〴ぜ〰ぃはのびふねばぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㌮㜩‾㸊⡜㌷㙜㌷㝜〰ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: ぅててづっぴびぜ〴ぜ〰はてぜ〴ぜ〰げづつ〭がどでとぴぜ〴ぜ〰こぴどねふぬちぴどはのぜ〴ぜ〰はのぜ〴ぜ〰いぎぁぜ〴ぜ〰うひちでねづのぴちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: いどびっふびびどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸵ Item: Title: ぃはのっぬふびどはのびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸵ Item: Title: げづてづひづのっづび Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: IYAOHD+PazoMath FontSubset: true FirstChar: 68 LastChar: 89 FontDescriptor: FontName: IYAOHD+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: TGMEOR+Rpcxr FontSubset: true FirstChar: 169 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