368338392c24607195e4c14a3272563b pmc_10968118.pdf 7cdee0e4bd2270c0bd5fd74afd85cbf0151287b6 pmc_10968118.pdf 2dac33486fda14af11888d39d4b85ff0be417e4e53aaed363dc468baf5a76b76 pmc_10968118.pdf Title: Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen–Thawed Horse Sperm Subject: Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen–thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen–thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP. Keywords: horse; semen; seminal plasma; sperm; cryopreservation; antioxidant; DNA fragmentation; lipid peroxidation Author: Jaime Catalán, Iván Yánez-Ortiz, Marc Torres-Garrido, Jordi Ribas-Maynou, Marc Llavanera, Isabel Barranco, Marc Yeste and Jordi Miró Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Mar 8 02:31:41 2024 CET ModDate: Fri Mar 8 02:34:32 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3098269 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NXCNLN+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 IUNTZG+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 XUNRBY+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 CWEWJB+CMSY10 Type 1 Builtin yes yes no 23 0 ZVHLWA+VnURWPalladioL Type 1 Custom yes yes no 27 0 ENXZUH+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 71 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 103 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 106 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 112 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 115 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 118 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 130 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 133 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 139 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 142 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 145 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 157 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 160 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 169 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 172 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 188 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 191 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 197 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 200 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 203 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 222 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 225 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 231 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 234 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 237 0 BJKIHE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 254 0 BJKIIF+PalatinoLinotype CID TrueType Identity-H yes yes yes 257 0 BJKIIG+PalatinoLinotype TrueType WinAnsi yes yes no 263 0 BJKIII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 266 0 BJKIPG+PalatinoLinotype TrueType MacRoman yes yes no 269 0 NYDKVC+VnURWPalladioL-Italic Type 1 Custom yes yes no 286 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-04-19 02:43:46 CEST RepresentationInformation: pmc_10968118.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-04-09 02:35:31 CEST Size: 3098269 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 716 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: Reagents and Suppliers Destination: subsection.2.1 Item: Title: Animals and Samples Destination: subsection.2.2 Item: Title: Isolation of SP Destination: subsection.2.3 Item: Title: Evaluation of Antioxidant Activity Levels of SP Destination: subsection.2.4 Children: Item: Title: Enzymatic Antioxidants Destination: subsubsection.2.4.1 Item: Title: Non-Enzymatic Antioxidants Destination: subsubsection.2.4.2 Item: Title: Determination of the Oxidative Stress Index (OSI) Destination: subsection.2.5 Item: Title: Sperm Cryopreservation Destination: subsection.2.6 Item: Title: Assessment of Sperm Motility Destination: subsection.2.7 Item: Title: Sperm Functionality Analysis Destination: subsection.2.8 Children: Item: Title: Assessment of Plasma Membrane Integrity (SYBR14/PI) Destination: subsubsection.2.8.1 Item: Title: Assessment of Acrosome Integrity (PNA-FITC/PI) Destination: subsubsection.2.8.2 Item: Title: Assessment of Mitochondrial Membrane Potential (JC-1) Destination: subsubsection.2.8.3 Item: Title: Intracellular Reactive Oxygen Species: Total ROS (H2DCFDA/PI) and O2- (HE/YO-PRO-1) Destination: subsubsection.2.8.4 Item: Title: Plasma Membrane Lipid Disorder (M540/YO-PRO-1) Destination: subsubsection.2.8.5 Item: Title: Membrane Lipid Peroxidation (LPO) Destination: subsubsection.2.8.6 Item: Title: Evaluation of DNA Fragmentation Destination: subsection.2.9 Children: Item: Title: Preparation of Semen Agarose Plates and Lysis of Samples Destination: subsubsection.2.9.1 Item: Title: Electrophoresis and Dehydration Destination: subsubsection.2.9.2 Item: Title: Staining and Imaging Destination: subsubsection.2.9.3 Item: Title: Comet Analysis Destination: subsubsection.2.9.4 Item: Title: Statistical Analyses Destination: subsection.2.10 Children: Item: Title: Classification of Ejaculates Based on Their Cryotolerance Destination: subsubsection.2.10.1 Item: Title: Comparison of LPO and DNA Fragmentation between Good- (GFEs) and Poor-Freezability Ejaculates (PFEs) Destination: subsubsection.2.10.2 Item: Title: Correlations of Membrane LPO and DNA Fragmentation with Post-Thaw Sperm Quality Parameters and Activity Levels of Enzymatic and Non-Enzymatic Antioxidants and the OSI of SP Destination: subsubsection.2.10.3 Item: Title: Results Destination: section.3 Children: Item: Title: Classification of Horse Ejaculates into GFE and PFE Groups According to Their Cryotolerance Destination: subsection.3.1 Item: Title: Lipid Membrane Peroxidation Destination: subsection.3.2 Item: Title: DNA Fragmentation Destination: subsection.3.3 Item: Title: Correlations of the Degree of LPO and Sperm DNA Fragmentation with Sperm Motility Parameters after Thawing Destination: subsection.3.4 Item: Title: Correlations of Membrane LPO and Sperm DNA Fragmentation with Sperm Functionality Parameters after Thawing Destination: subsection.3.5 Item: Title: Correlations of the Degree of Lipid Peroxidation and Sperm DNA Fragmentation of Horse Sperm after Thawing with the Levels of Antioxidant Activity (Enzymatic and Non-Enzymatic) and the OSI of SP Destination: subsection.3.6 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen Thawed Horse Sperm Author: Jaime Catalán, Iván Yánez-Ortiz, Marc Torres-Garrido, Jordi Ribas-Maynou, Marc Llavanera, Isabel Barranco, Marc Yeste and Jordi Miró Subject: Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP. Keywords: horse; semen; seminal plasma; sperm; cryopreservation; antioxidant; DNA fragmentation; lipid peroxidation Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Mar 08 02:31:41 CET 2024 ModDate: Fri Mar 08 02:34:32 CET 2024 ID: 0xa6313b4d93ab778b48aa8a97f2fc2fcd, 0xa6313b4d93ab778b48aa8a97f2fc2fcd Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ENXZUH+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: ENXZUH+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XUNRBY+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: XUNRBY+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CWEWJB+CMSY10 FontSubset: true FirstChar: 0 LastChar: 112 FontDescriptor: FontName: CWEWJB+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: NXCNLN+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 150 FontDescriptor: FontName: NXCNLN+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZVHLWA+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: ZVHLWA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: NYDKVC+VnURWPalladioL-Italic FontSubset: true FirstChar: 161 LastChar: 243 FontDescriptor: FontName: NYDKVC+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IUNTZG+URWPalladioL-Roma FontSubset: true FirstChar: 1 LastChar: 252 FontDescriptor: FontName: IUNTZG+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 117, 279, 152, 288 Annotation: Subtype: Link Rect: 34, 267, 137, 276 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 175, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 118, 660, 129, 674 Annotation: Subtype: Link Rect: 236, 660, 247, 674 Annotation: Subtype: Link Rect: 468, 660, 479, 674 Annotation: Subtype: Link Rect: 118, 648, 129, 661 Annotation: Subtype: Link Rect: 210, 648, 221, 661 Annotation: Subtype: Link Rect: 307, 648, 318, 661 Annotation: Subtype: Link Rect: 406, 648, 417, 661 Annotation: Subtype: Link Rect: 375, 93, 382, 102 ActionDest: 280 Annotation: Subtype: Link Rect: 448, 80, 455, 89 ActionDest: 280 Annotation: Subtype: Link Rect: 127, 17, 273, 28 Annotation: Subtype: Link Rect: 392, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 377, 87, 396 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 271, 735, 278, 744 ActionDest: 280 Annotation: Subtype: Link Rect: 303, 710, 310, 719 ActionDest: 280 Annotation: Subtype: Link Rect: 313, 710, 320, 719 ActionDest: 280 Annotation: Subtype: Link Rect: 507, 685, 514, 694 ActionDest: 280 Annotation: Subtype: Link Rect: 517, 685, 529, 694 ActionDest: 280 Annotation: Subtype: Link Rect: 537, 660, 544, 669 ActionDest: 280 Annotation: Subtype: Link Rect: 544, 660, 556, 669 ActionDest: 280 Annotation: Subtype: Link Rect: 387, 648, 399, 657 ActionDest: 280 Annotation: Subtype: Link Rect: 326, 623, 338, 631 ActionDest: 280 Annotation: Subtype: Link Rect: 340, 623, 352, 631 ActionDest: 283 Annotation: Subtype: Link Rect: 407, 585, 419, 594 ActionDest: 283 Annotation: Subtype: Link Rect: 224, 560, 235, 569 ActionDest: 283 Annotation: Subtype: Link Rect: 227, 535, 239, 544 ActionDest: 283 Annotation: Subtype: Link Rect: 457, 509, 469, 519 ActionDest: 283 Annotation: Subtype: Link Rect: 469, 509, 481, 519 ActionDest: 283 Annotation: Subtype: Link Rect: 379, 472, 385, 481 ActionDest: 280 Annotation: Subtype: Link Rect: 205, 434, 217, 443 ActionDest: 283 Annotation: Subtype: Link Rect: 483, 422, 495, 431 ActionDest: 283 Annotation: Subtype: Link Rect: 377, 384, 389, 393 ActionDest: 283 Annotation: Subtype: Link Rect: 392, 384, 404, 393 ActionDest: 283 Annotation: Subtype: Link Rect: 253, 359, 260, 368 ActionDest: 280 Annotation: Subtype: Link Rect: 261, 359, 273, 368 ActionDest: 283 Annotation: Subtype: Link Rect: 276, 359, 288, 368 ActionDest: 283 Annotation: Subtype: Link Rect: 295, 334, 307, 343 ActionDest: 283 Annotation: Subtype: Link Rect: 308, 334, 320, 343 ActionDest: 283 Annotation: Subtype: Link Rect: 336, 309, 348, 318 ActionDest: 283 Annotation: Subtype: Link Rect: 349, 309, 361, 318 ActionDest: 283 Annotation: Subtype: Link Rect: 361, 309, 373, 318 ActionDest: 283 Annotation: Subtype: Link Rect: 359, 284, 371, 293 ActionDest: 283 Annotation: Subtype: Link Rect: 372, 283, 384, 293 ActionDest: 283 Annotation: Subtype: Link Rect: 387, 283, 399, 293 ActionDest: 283 Annotation: Subtype: Link Rect: 506, 271, 518, 280 ActionDest: 283 Annotation: Subtype: Link Rect: 519, 271, 531, 280 ActionDest: 283 Annotation: Subtype: Link Rect: 531, 271, 543, 280 ActionDest: 283 Annotation: Subtype: Link Rect: 544, 271, 556, 280 ActionDest: 283 Annotation: Subtype: Link Rect: 298, 246, 310, 255 ActionDest: 283 Annotation: Subtype: Link Rect: 313, 246, 325, 255 ActionDest: 283 Annotation: Subtype: Link Rect: 354, 221, 366, 230 ActionDest: 283 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 367, 392, 379, 401 ActionDest: 290 Annotation: Subtype: Link Rect: 541, 365, 555, 377 ActionDest: 68 Annotation: Subtype: Link Rect: 526, 355, 538, 364 ActionDest: 290 Annotation: Subtype: Link Rect: 358, 254, 370, 263 ActionDest: 283 Annotation: Subtype: Link Rect: 371, 254, 383, 263 ActionDest: 290 Annotation: Subtype: Link Rect: 383, 254, 395, 263 ActionDest: 290 Annotation: Subtype: Link Rect: 533, 127, 544, 136 ActionDest: 283 Annotation: Subtype: Link Rect: 545, 127, 556, 136 ActionDest: 290 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 545, 647, 556, 657 ActionDest: 290 Annotation: Subtype: Link Rect: 495, 522, 506, 531 ActionDest: 290 Annotation: Subtype: Link Rect: 296, 484, 308, 493 ActionDest: 290 Annotation: Subtype: Link Rect: 309, 484, 321, 493 ActionDest: 290 Annotation: Subtype: Link Rect: 266, 434, 278, 443 ActionDest: 290 Annotation: Subtype: Link Rect: 279, 434, 291, 443 ActionDest: 290 Annotation: Subtype: Link Rect: 495, 396, 507, 405 ActionDest: 290 Annotation: Subtype: Link Rect: 508, 396, 520, 405 ActionDest: 290 Annotation: Subtype: Link Rect: 544, 295, 556, 305 ActionDest: 290 Annotation: Subtype: Link Rect: 361, 283, 373, 292 ActionDest: 290 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 245, 673, 257, 682 ActionDest: 290 Annotation: Subtype: Link Rect: 215, 608, 229, 619 ActionDest: 78 Annotation: Subtype: Link Rect: 232, 608, 246, 619 ActionDest: 84 Annotation: Subtype: Link Rect: 544, 559, 556, 568 ActionDest: 283 Annotation: Subtype: Link Rect: 254, 170, 266, 179 ActionDest: 290 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 543, 597, 555, 606 ActionDest: 290 Annotation: Subtype: Link Rect: 407, 446, 419, 455 ActionDest: 290 Annotation: Subtype: Link Rect: 444, 295, 455, 304 ActionDest: 290 Annotation: Subtype: Link Rect: 196, 107, 208, 116 ActionDest: 290 Annotation: Subtype: Link Rect: 254, 82, 266, 91 ActionDest: 290 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 543, 484, 555, 493 ActionDest: 290 Annotation: Subtype: Link Rect: 401, 471, 413, 481 ActionDest: 290 Annotation: Subtype: Link Rect: 500, 333, 512, 342 ActionDest: 290 Annotation: Subtype: Link Rect: 339, 132, 351, 141 ActionDest: 290 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 510, 195, 522, 204 ActionDest: 290 Annotation: Subtype: Link Rect: 522, 195, 534, 204 ActionDest: 290 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 319, 660, 331, 669 ActionDest: 290 Annotation: Subtype: Link Rect: 372, 272, 379, 284 ActionDest: 93 Annotation: Subtype: Link Rect: 368, 197, 375, 209 ActionDest: 93 Annotation: Subtype: Link Rect: 397, 134, 404, 146 ActionDest: 93 Annotation: Subtype: Link Rect: 371, 96, 378, 108 ActionDest: 93 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 216, 707, 223, 719 ActionDest: 178 Annotation: Subtype: Link Rect: 216, 289, 223, 301 ActionDest: 210 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 216, 189, 223, 201 ActionDest: 242 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 452, 348, 464, 357 ActionDest: 290 Annotation: Subtype: Link Rect: 406, 323, 418, 332 ActionDest: 293 Annotation: Subtype: Link Rect: 421, 323, 433, 332 ActionDest: 293 Annotation: Subtype: Link Rect: 465, 298, 477, 307 ActionDest: 293 Annotation: Subtype: Link Rect: 480, 298, 492, 307 ActionDest: 293 Annotation: Subtype: Link Rect: 215, 273, 227, 282 ActionDest: 293 Annotation: Subtype: Link Rect: 230, 273, 242, 282 ActionDest: 293 Annotation: Subtype: Link Rect: 461, 260, 473, 269 ActionDest: 293 Annotation: Subtype: Link Rect: 474, 260, 486, 269 ActionDest: 293 Annotation: Subtype: Link Rect: 360, 235, 372, 244 ActionDest: 283 Annotation: Subtype: Link Rect: 372, 235, 384, 244 ActionDest: 283 Annotation: Subtype: Link Rect: 384, 235, 396, 244 ActionDest: 283 Annotation: Subtype: Link Rect: 481, 210, 493, 219 ActionDest: 283 Annotation: Subtype: Link Rect: 493, 210, 505, 219 ActionDest: 283 Annotation: Subtype: Link Rect: 506, 210, 518, 219 ActionDest: 283 Annotation: Subtype: Link Rect: 518, 210, 530, 219 ActionDest: 290 Annotation: Subtype: Link Rect: 216, 97, 228, 106 ActionDest: 293 Annotation: Subtype: Link Rect: 228, 97, 240, 106 ActionDest: 293 Annotation: Subtype: Link Rect: 241, 97, 253, 106 ActionDest: 293 Annotation: Subtype: Link Rect: 419, 72, 426, 81 ActionDest: 280 Annotation: Subtype: Link Rect: 427, 72, 439, 81 ActionDest: 293 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 490, 735, 502, 744 ActionDest: 293 Annotation: Subtype: Link Rect: 349, 723, 361, 732 ActionDest: 293 Annotation: Subtype: Link Rect: 474, 723, 486, 732 ActionDest: 293 Annotation: Subtype: Link Rect: 243, 647, 255, 657 ActionDest: 293 Annotation: Subtype: Link Rect: 528, 647, 540, 657 ActionDest: 293 Annotation: Subtype: Link Rect: 543, 647, 555, 657 ActionDest: 293 Annotation: Subtype: Link Rect: 193, 635, 205, 644 ActionDest: 293 Annotation: Subtype: Link Rect: 208, 635, 220, 644 ActionDest: 296 Annotation: Subtype: Link Rect: 264, 635, 276, 644 ActionDest: 296 Annotation: Subtype: Link Rect: 201, 622, 213, 631 ActionDest: 290 Annotation: Subtype: Link Rect: 213, 622, 225, 631 ActionDest: 296 Annotation: Subtype: Link Rect: 228, 622, 240, 631 ActionDest: 296 Annotation: Subtype: Link Rect: 291, 622, 308, 631 ActionDest: 296 Annotation: Subtype: Link Rect: 311, 622, 328, 631 ActionDest: 296 Annotation: Subtype: Link Rect: 271, 597, 283, 606 ActionDest: 290 Annotation: Subtype: Link Rect: 284, 597, 296, 606 ActionDest: 293 Annotation: Subtype: Link Rect: 296, 597, 308, 606 ActionDest: 293 Annotation: Subtype: Link Rect: 309, 597, 325, 606 ActionDest: 296 Annotation: Subtype: Link Rect: 377, 597, 389, 606 ActionDest: 293 Annotation: Subtype: Link Rect: 390, 597, 407, 606 ActionDest: 296 Annotation: Subtype: Link Rect: 410, 597, 427, 606 ActionDest: 296 Annotation: Subtype: Link Rect: 462, 597, 474, 606 ActionDest: 293 Annotation: Subtype: Link Rect: 521, 597, 538, 606 ActionDest: 296 Annotation: Subtype: Link Rect: 538, 597, 555, 606 ActionDest: 296 Annotation: Subtype: Link Rect: 196, 585, 213, 594 ActionDest: 296 Annotation: Subtype: Link Rect: 213, 585, 230, 594 ActionDest: 296 Annotation: Subtype: Link Rect: 304, 585, 321, 594 ActionDest: 296 Annotation: Subtype: Link Rect: 366, 585, 383, 594 ActionDest: 296 Annotation: Subtype: Link Rect: 437, 572, 449, 581 ActionDest: 293 Annotation: Subtype: Link Rect: 449, 572, 461, 581 ActionDest: 293 Annotation: Subtype: Link Rect: 462, 572, 474, 581 ActionDest: 293 Annotation: Subtype: Link Rect: 474, 572, 486, 581 ActionDest: 293 Annotation: Subtype: Link Rect: 487, 572, 504, 581 ActionDest: 296 Annotation: Subtype: Link Rect: 504, 572, 521, 581 ActionDest: 296 Annotation: Subtype: Link Rect: 521, 572, 538, 581 ActionDest: 296 Annotation: Subtype: Link Rect: 539, 572, 556, 581 ActionDest: 296 Annotation: Subtype: Link Rect: 427, 547, 439, 556 ActionDest: 290 Annotation: Subtype: Link Rect: 199, 509, 216, 519 ActionDest: 296 Annotation: Subtype: Link Rect: 219, 509, 236, 519 ActionDest: 296 Annotation: Subtype: Link Rect: 319, 497, 336, 506 ActionDest: 299 Annotation: Subtype: Link Rect: 339, 497, 356, 506 ActionDest: 299 Annotation: Subtype: Link Rect: 396, 497, 413, 506 ActionDest: 296 Annotation: Subtype: Link Rect: 413, 497, 430, 506 ActionDest: 296 Annotation: Subtype: Link Rect: 431, 497, 447, 506 ActionDest: 299 Annotation: Subtype: Link Rect: 450, 497, 467, 506 ActionDest: 299 Annotation: Subtype: Link Rect: 244, 459, 261, 468 ActionDest: 299 Annotation: Subtype: Link Rect: 219, 409, 236, 418 ActionDest: 299 Annotation: Subtype: Link Rect: 320, 384, 337, 393 ActionDest: 299 Annotation: Subtype: Link Rect: 372, 384, 384, 393 ActionDest: 293 Annotation: Subtype: Link Rect: 384, 384, 401, 393 ActionDest: 299 Annotation: Subtype: Link Rect: 538, 384, 555, 393 ActionDest: 299 Annotation: Subtype: Link Rect: 197, 371, 214, 380 ActionDest: 299 Annotation: Subtype: Link Rect: 279, 371, 296, 380 ActionDest: 299 Annotation: Subtype: Link Rect: 340, 334, 357, 343 ActionDest: 299 Annotation: Subtype: Link Rect: 449, 258, 461, 267 ActionDest: 283 Annotation: Subtype: Link Rect: 462, 258, 474, 267 ActionDest: 283 Annotation: Subtype: Link Rect: 474, 258, 486, 267 ActionDest: 283 Annotation: Subtype: Link Rect: 487, 258, 499, 267 ActionDest: 283 Annotation: Subtype: Link Rect: 502, 259, 514, 267 ActionDest: 283 Annotation: Subtype: Link Rect: 195, 208, 207, 217 ActionDest: 293 Annotation: Subtype: Link Rect: 319, 208, 336, 217 ActionDest: 299 Annotation: Subtype: Link Rect: 463, 183, 475, 192 ActionDest: 293 Annotation: Subtype: Link Rect: 256, 158, 268, 167 ActionDest: 283 Annotation: Subtype: Link Rect: 269, 158, 281, 167 ActionDest: 283 Annotation: Subtype: Link Rect: 281, 158, 293, 167 ActionDest: 290 Annotation: Subtype: Link Rect: 294, 158, 306, 167 ActionDest: 290 Annotation: Subtype: Link Rect: 288, 95, 300, 104 ActionDest: 290 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 320, 736, 332, 744 ActionDest: 290 Annotation: Subtype: Link Rect: 234, 685, 251, 694 ActionDest: 299 Annotation: Subtype: Link Rect: 257, 635, 274, 644 ActionDest: 299 Annotation: Subtype: Link Rect: 274, 635, 291, 644 ActionDest: 299 Annotation: Subtype: Link Rect: 478, 622, 490, 631 ActionDest: 290 Annotation: Subtype: Link Rect: 491, 622, 508, 631 ActionDest: 299 Annotation: Subtype: Link Rect: 511, 622, 528, 631 ActionDest: 299 Annotation: Subtype: Link Rect: 460, 610, 476, 619 ActionDest: 299 Annotation: Subtype: Link Rect: 477, 610, 494, 619 ActionDest: 299 Annotation: Subtype: Link Rect: 319, 547, 331, 556 ActionDest: 283 Annotation: Subtype: Link Rect: 402, 497, 414, 506 ActionDest: 293 Annotation: Subtype: Link Rect: 538, 472, 555, 481 ActionDest: 303 Annotation: Subtype: Link Rect: 437, 422, 449, 431 ActionDest: 290 Annotation: Subtype: Link Rect: 540, 422, 557, 431 ActionDest: 303 Annotation: Subtype: Link Rect: 379, 384, 396, 393 ActionDest: 296 Annotation: Subtype: Link Rect: 370, 359, 382, 368 ActionDest: 283 Annotation: Subtype: Link Rect: 479, 346, 496, 355 ActionDest: 303 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 294, 303, 331, 314 Annotation: Subtype: Link Rect: 109, 281, 146, 291 Annotation: Subtype: Link Rect: 95, 246, 133, 256 Annotation: Subtype: Link Rect: 419, 222, 457, 233 Annotation: Subtype: Link Rect: 510, 199, 548, 210 Annotation: Subtype: Link Rect: 395, 165, 432, 176 Annotation: Subtype: Link Rect: 509, 153, 546, 164 Annotation: Subtype: Link Rect: 81, 131, 119, 141 Annotation: Subtype: Link Rect: 59, 108, 96, 118 Annotation: Subtype: Link Rect: 156, 85, 193, 95 Annotation: Subtype: Link Rect: 180, 61, 217, 72 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 122, 722, 160, 733 Annotation: Subtype: Link Rect: 158, 688, 196, 698 Annotation: Subtype: Link Rect: 520, 676, 557, 687 Annotation: Subtype: Link Rect: 95, 653, 133, 664 Annotation: Subtype: Link Rect: 253, 629, 291, 640 Annotation: Subtype: Link Rect: 338, 606, 376, 617 Annotation: Subtype: Link Rect: 86, 584, 124, 594 Annotation: Subtype: Link Rect: 210, 560, 247, 571 Annotation: Subtype: Link Rect: 285, 537, 322, 548 Annotation: Subtype: Link Rect: 122, 515, 160, 525 Annotation: Subtype: Link Rect: 230, 492, 268, 502 Annotation: Subtype: Link Rect: 235, 457, 272, 468 Annotation: Subtype: Link Rect: 519, 434, 557, 445 Annotation: Subtype: Link Rect: 419, 411, 457, 422 Annotation: Subtype: Link Rect: 336, 388, 374, 399 Annotation: Subtype: Link Rect: 284, 365, 322, 376 Annotation: Subtype: Link Rect: 452, 342, 489, 353 Annotation: Subtype: Link Rect: 407, 319, 444, 330 Annotation: Subtype: Link Rect: 345, 273, 382, 284 Annotation: Subtype: Link Rect: 519, 261, 557, 272 Annotation: Subtype: Link Rect: 59, 251, 95, 261 Annotation: Subtype: Link Rect: 243, 227, 281, 238 Annotation: Subtype: Link Rect: 375, 204, 413, 215 Annotation: Subtype: Link Rect: 440, 181, 477, 192 Annotation: Subtype: Link Rect: 493, 158, 530, 169 Annotation: Subtype: Link Rect: 330, 135, 367, 146 Annotation: Subtype: Link Rect: 107, 113, 145, 123 Annotation: Subtype: Link Rect: 421, 89, 458, 100 Annotation: Subtype: Link Rect: 464, 66, 501, 77 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 197, 699, 235, 710 Annotation: Subtype: Link Rect: 59, 665, 96, 675 Annotation: Subtype: Link Rect: 417, 641, 455, 652 Annotation: Subtype: Link Rect: 350, 606, 388, 617 Annotation: Subtype: Link Rect: 447, 583, 484, 594 Annotation: Subtype: Link Rect: 489, 560, 526, 571 Annotation: Subtype: Link Rect: 185, 527, 222, 537 Annotation: Subtype: Link Rect: 185, 504, 222, 514 Annotation: Subtype: Link Rect: 393, 480, 430, 491 Annotation: Subtype: Link Rect: 462, 457, 500, 468 Annotation: Subtype: Link Rect: 432, 435, 470, 445 Annotation: Subtype: Link Rect: 59, 412, 96, 422 Annotation: Subtype: Link Rect: 483, 388, 520, 399 Annotation: Subtype: Link Rect: 109, 366, 146, 376 Annotation: Subtype: Link Rect: 131, 343, 168, 353 Annotation: Subtype: Link Rect: 95, 308, 133, 318 Annotation: Subtype: Link Rect: 177, 274, 215, 284 Annotation: Subtype: Link Rect: 520, 250, 557, 261 Annotation: Subtype: Link Rect: 165, 216, 202, 226 Annotation: Subtype: Link Rect: 434, 181, 471, 192 Annotation: Subtype: Link Rect: 171, 147, 209, 157 Annotation: Subtype: Link Rect: 516, 135, 553, 146 Annotation: Subtype: Link Rect: 316, 112, 353, 123 Annotation: Subtype: Link Rect: 515, 89, 553, 100 Annotation: Subtype: Link Rect: 520, 77, 557, 88 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 466, 722, 503, 733 Annotation: Subtype: Link Rect: 59, 699, 96, 710 Annotation: Subtype: Link Rect: 102, 699, 138, 710 Annotation: Subtype: Link Rect: 59, 676, 96, 687 Annotation: Subtype: Link Rect: 102, 676, 138, 687 Annotation: Subtype: Link Rect: 446, 664, 484, 675 Annotation: Subtype: Link Rect: 104, 642, 141, 652 Annotation: Subtype: Link Rect: 143, 607, 181, 617 Annotation: Subtype: Link Rect: 187, 607, 223, 617 Annotation: Subtype: Link Rect: 113, 573, 151, 583 Annotation: Subtype: Link Rect: 441, 549, 479, 560 Annotation: Subtype: Link Rect: 477, 514, 514, 525 Annotation: Subtype: Link Rect: 377, 491, 415, 502 Annotation: Subtype: Link Rect: 181, 468, 218, 479 Annotation: Subtype: Link Rect: 474, 457, 512, 468 Annotation: Subtype: Link Rect: 485, 434, 522, 445 Annotation: Subtype: Link Rect: 122, 400, 160, 410 Annotation: Subtype: Link Rect: 131, 366, 168, 376 Annotation: Subtype: Link Rect: 474, 342, 511, 353 Annotation: Subtype: Link Rect: 520, 319, 557, 330 Annotation: Subtype: Link Rect: 227, 297, 265, 307 Annotation: Subtype: Link Rect: 271, 297, 307, 307 Annotation: Subtype: Link Rect: 59, 274, 96, 284 Annotation: Subtype: Link Rect: 104, 239, 141, 249 Annotation: Subtype: Link Rect: 147, 239, 183, 249 Annotation: Subtype: Link Rect: 331, 215, 368, 226 Annotation: Subtype: Link Rect: 483, 192, 521, 203 Annotation: Subtype: Link Rect: 201, 169, 239, 180 Annotation: Subtype: Link Rect: 206, 135, 243, 146 Annotation: Subtype: Link Rect: 520, 112, 557, 123 Annotation: Subtype: Link Rect: 59, 101, 95, 111 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 250, 722, 288, 733 Annotation: Subtype: Link Rect: 242, 699, 280, 710 Annotation: Subtype: Link Rect: 59, 676, 96, 687 Annotation: Subtype: Link Rect: 223, 653, 261, 664 Annotation: Subtype: Link Rect: 267, 653, 303, 664 Annotation: Subtype: Link Rect: 328, 618, 365, 629 Annotation: Subtype: Link Rect: 468, 595, 505, 606 Annotation: Subtype: Link Rect: 510, 572, 548, 583 Annotation: Subtype: Link Rect: 378, 549, 416, 560 Annotation: Subtype: Link Rect: 303, 514, 341, 525 Annotation: Subtype: Link Rect: 420, 491, 458, 502 Annotation: Subtype: Link Rect: 464, 491, 500, 502 Annotation: Subtype: Link Rect: 281, 468, 318, 479 Annotation: Subtype: Link Rect: 295, 434, 332, 445 Annotation: Subtype: Link Rect: 90, 400, 128, 410 Annotation: Subtype: Link Rect: 134, 400, 170, 410 Annotation: Subtype: Link Rect: 293, 377, 330, 387 Annotation: Subtype: Link Rect: 232, 343, 269, 353 Annotation: Subtype: Link Rect: 95, 308, 132, 318 Annotation: Subtype: Link Rect: 168, 273, 205, 284 Annotation: Subtype: Link Rect: 345, 238, 382, 249 Annotation: Subtype: Link Rect: 334, 215, 372, 226 Annotation: Subtype: Link Rect: 135, 182, 173, 192 Annotation: Subtype: Link Rect: 59, 147, 96, 157 Annotation: Subtype: Link Rect: 338, 123, 375, 134 Annotation: Subtype: Link Rect: 341, 100, 378, 111 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 329, 722, 366, 733 Annotation: Subtype: Link Rect: 333, 687, 370, 698 Annotation: Subtype: Link Rect: 59, 653, 96, 664 Annotation: Subtype: Link Rect: 519, 618, 557, 629 Annotation: Subtype: Link Rect: 59, 607, 95, 617 Annotation: Subtype: Link Rect: 477, 583, 515, 594 Annotation: Subtype: Link Rect: 521, 583, 557, 594 Annotation: Subtype: Link Rect: 243, 560, 280, 571 Annotation: Subtype: Link Rect: 472, 537, 509, 548 Annotation: Subtype: Link Rect: 519, 514, 557, 525 Annotation: Subtype: Link Rect: 59, 504, 95, 514 Annotation: Subtype: Link Rect: 59, 469, 96, 479 Annotation: Subtype: Link Rect: 102, 469, 138, 479 Annotation: Subtype: Link Rect: 257, 445, 294, 456 Annotation: Subtype: Link Rect: 300, 445, 336, 456 Annotation: Subtype: Link Rect: 228, 411, 266, 422 Annotation: Subtype: Link Rect: 272, 411, 308, 422 Annotation: Subtype: Link Rect: 220, 388, 258, 399 Annotation: Subtype: Link Rect: 264, 388, 300, 399 Annotation: Subtype: Link Rect: 451, 365, 488, 376 Annotation: Subtype: Link Rect: 495, 365, 531, 376 Annotation: Subtype: Link Rect: 59, 331, 96, 341 Annotation: Subtype: Link Rect: 104, 308, 141, 318 Annotation: Subtype: Link Rect: 520, 284, 557, 295 Annotation: Subtype: Link Rect: 223, 250, 261, 261 Annotation: Subtype: Link Rect: 220, 228, 257, 238 Annotation: Subtype: Link Rect: 264, 228, 300, 238 Annotation: Subtype: Link Rect: 399, 204, 436, 215 Annotation: Subtype: Link Rect: 442, 204, 478, 215 Annotation: Subtype: Link Rect: 95, 170, 133, 180 Annotation: Subtype: Link Rect: 139, 170, 175, 180 Annotation: Subtype: Link Rect: 95, 136, 133, 146 Annotation: Subtype: Link Rect: 139, 136, 175, 146 Annotation: Subtype: Link Rect: 59, 113, 96, 123 Annotation: Subtype: Link Rect: 261, 89, 299, 100 Annotation: Subtype: Link Rect: 305, 89, 341, 100 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 520, 722, 557, 733 Annotation: Subtype: Link Rect: 86, 688, 123, 698 Annotation: Subtype: Link Rect: 519, 664, 557, 675 Annotation: Subtype: Link Rect: 59, 653, 95, 664 Checksum: 62d8b688 Type: CRC32 Checksum: 368338392c24607195e4c14a3272563b Type: MD5 Checksum: 7cdee0e4bd2270c0bd5fd74afd85cbf0151287b6 Type: SHA-1 Checksum: 2dac33486fda14af11888d39d4b85ff0be417e4e53aaed363dc468baf5a76b76 Type: SHA-256