d6f98ec2ef00970ff619908d5904d5b7 animals-14-01264-v2.pdf e2a71cff786d730a861694259d9cf7d03ff97755 animals-14-01264-v2.pdf dfeb838aae9d2f5bfe0b7887e6cb6823b1d46cd302f8456936c3627e9ca95445 animals-14-01264-v2.pdf Title: ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions Subject: ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity. Keywords: ProAKAP4; AKAP4; stallion; spermatozoa; motility; mitochondrial activity Author: Marta Dordas-Perpinyà, Iván Yánez-Ortiz, Nicolas Sergeant, Vincent Mevel, Jaime Catalán, Jean-François Bruyas, Jordi Miró and Lamia Briand-Amirat Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri Apr 26 10:33:15 2024 CEST ModDate: Fri Apr 26 11:18:04 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1598381 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NEBEKD+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 YCXYMW+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 ESTJVB+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 AKBUPW+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 FYZIQU+CMSY10 Type 1 Builtin yes yes yes 32 0 EUNBMT+VnURWPalladioL Type 1 Custom yes yes yes 37 0 SZMRXJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 73 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 84 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 97 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 101 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 105 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 109 0 BCDIEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 116 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 129 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 133 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 137 0 BCDIEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 141 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 152 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 156 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 160 0 BCDIEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 164 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 179 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 183 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 187 0 BCDIEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 191 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-05-29 02:39:28 CEST RepresentationInformation: animals-14-01264-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-05-24 11:17:14 CEST Size: 1598381 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 396 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: Semen Samples Destination: subsection.2.1 Item: Title: Experimental Groups Destination: subsection.2.2 Item: Title: Motility Analysis Destination: subsection.2.3 Item: Title: ProAKAP4 ELISA Destination: subsection.2.4 Item: Title: Mitochondrial Membrane Potential Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Correlation between proAKAP4, Motility Descriptors and Mitochondrial Membrane Potential Destination: subsection.3.1 Item: Title: Evolution of proAKAP4, Motility Descriptors and Mitochondrial Membrane Potential over 3 h Destination: subsection.3.2 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions Author: Marta Dordas-Perpinyà, Iván Yánez-Ortiz, Nicolas Sergeant, Vincent Mevel, Jaime Catalán, Jean-François Bruyas, Jordi Miró and Lamia Briand-Amirat Subject: ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity. Keywords: ProAKAP4; AKAP4; stallion; spermatozoa; motility; mitochondrial activity Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri Apr 26 10:33:15 CEST 2024 ModDate: Fri Apr 26 11:18:04 CEST 2024 ID: 0x3a7e994ab9282837c8fbc2ec04e4bed6, 0x3a7e994ab9282837c8fbc2ec04e4bed6 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: YCXYMW+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 231 FontDescriptor: FontName: YCXYMW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: FYZIQU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 112 FontDescriptor: FontName: FYZIQU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: WBVKDJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 112 LastChar: 112 FontDescriptor: FontName: WBVKDJ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EUNBMT+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: EUNBMT+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ESTJVB+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: ESTJVB+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: NEBEKD+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: NEBEKD+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AKBUPW+URWPalladioL-Ital FontSubset: true FirstChar: 44 LastChar: 150 FontDescriptor: FontName: AKBUPW+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 317, 152, 326 Annotation: Subtype: Link Rect: 34, 307, 74, 314 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 125, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 149, 87 Annotation: Subtype: Link Rect: 34, 756, 165, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 168, 662, 179, 676 Annotation: Subtype: Link Rect: 273, 662, 284, 676 Annotation: Subtype: Link Rect: 531, 662, 542, 676 Annotation: Subtype: Link Rect: 203, 650, 214, 663 Annotation: Subtype: Link Rect: 345, 650, 356, 663 Annotation: Subtype: Link Rect: 116, 17, 251, 28 Annotation: Subtype: Link Rect: 409, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 414, 87, 433 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 251, 703, 258, 712 ActionDest: 202 Annotation: Subtype: Link Rect: 394, 678, 401, 687 ActionDest: 202 Annotation: Subtype: Link Rect: 255, 665, 262, 674 ActionDest: 202 Annotation: Subtype: Link Rect: 549, 627, 556, 636 ActionDest: 202 Annotation: Subtype: Link Rect: 271, 615, 278, 624 ActionDest: 202 Annotation: Subtype: Link Rect: 292, 590, 299, 599 ActionDest: 202 Annotation: Subtype: Link Rect: 262, 577, 269, 586 ActionDest: 202 Annotation: Subtype: Link Rect: 519, 577, 526, 586 ActionDest: 202 Annotation: Subtype: Link Rect: 527, 577, 534, 586 ActionDest: 202 Annotation: Subtype: Link Rect: 395, 565, 402, 574 ActionDest: 202 Annotation: Subtype: Link Rect: 252, 552, 259, 561 ActionDest: 202 Annotation: Subtype: Link Rect: 259, 552, 266, 561 ActionDest: 202 Annotation: Subtype: Link Rect: 406, 540, 413, 549 ActionDest: 202 Annotation: Subtype: Link Rect: 541, 540, 548, 549 ActionDest: 202 Annotation: Subtype: Link Rect: 549, 539, 556, 549 ActionDest: 202 Annotation: Subtype: Link Rect: 535, 502, 541, 511 ActionDest: 202 Annotation: Subtype: Link Rect: 398, 464, 405, 473 ActionDest: 202 Annotation: Subtype: Link Rect: 406, 464, 413, 473 ActionDest: 202 Annotation: Subtype: Link Rect: 232, 339, 238, 348 ActionDest: 202 Annotation: Subtype: Link Rect: 327, 314, 339, 323 ActionDest: 202 Annotation: Subtype: Link Rect: 335, 289, 342, 298 ActionDest: 202 Annotation: Subtype: Link Rect: 278, 276, 290, 285 ActionDest: 202 Annotation: Subtype: Link Rect: 543, 251, 555, 260 ActionDest: 202 Annotation: Subtype: Link Rect: 266, 238, 278, 247 ActionDest: 202 Annotation: Subtype: Link Rect: 281, 238, 293, 247 ActionDest: 202 Annotation: Subtype: Link Rect: 531, 226, 543, 235 ActionDest: 202 Annotation: Subtype: Link Rect: 544, 226, 556, 235 ActionDest: 202 Annotation: Subtype: Link Rect: 342, 213, 354, 222 ActionDest: 202 Annotation: Subtype: Link Rect: 355, 213, 367, 222 ActionDest: 202 Annotation: Subtype: Link Rect: 315, 201, 327, 210 ActionDest: 202 Annotation: Subtype: Link Rect: 327, 201, 339, 210 ActionDest: 202 Annotation: Subtype: Link Rect: 339, 201, 351, 210 ActionDest: 202 Annotation: Subtype: Link Rect: 302, 188, 314, 197 ActionDest: 202 Annotation: Subtype: Link Rect: 317, 188, 329, 197 ActionDest: 202 Annotation: Subtype: Link Rect: 487, 188, 499, 197 ActionDest: 202 Annotation: Subtype: Link Rect: 543, 188, 555, 197 ActionDest: 202 Annotation: Subtype: Link Rect: 193, 175, 205, 185 ActionDest: 202 Annotation: Subtype: Link Rect: 249, 175, 261, 185 ActionDest: 202 Annotation: Subtype: Link Rect: 297, 176, 309, 185 ActionDest: 206 Annotation: Subtype: Link Rect: 390, 175, 402, 185 ActionDest: 206 Annotation: Subtype: Link Rect: 410, 163, 422, 172 ActionDest: 202 Annotation: Subtype: Link Rect: 425, 163, 437, 172 ActionDest: 202 Annotation: Subtype: Link Rect: 425, 151, 437, 159 ActionDest: 202 Annotation: Subtype: Link Rect: 544, 138, 556, 147 ActionDest: 202 Annotation: Subtype: Link Rect: 367, 113, 379, 122 ActionDest: 202 Annotation: Subtype: Link Rect: 449, 75, 461, 84 ActionDest: 202 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 403, 706, 415, 715 ActionDest: 206 Annotation: Subtype: Link Rect: 414, 693, 426, 702 ActionDest: 202 Annotation: Subtype: Link Rect: 345, 275, 360, 287 ActionDest: 70 Annotation: Subtype: Link Rect: 203, 262, 218, 274 ActionDest: 78 Annotation: Subtype: Link Rect: 206, 250, 220, 262 ActionDest: 78 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 212, 527, 227, 539 ActionDest: 70 Annotation: Subtype: Link Rect: 334, 253, 346, 263 ActionDest: 206 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 496, 237, 503, 249 ActionDest: 81 Annotation: Subtype: Link Rect: 537, 237, 544, 249 ActionDest: 89 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 379, 100, 386, 112 ActionDest: 121 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 242, 452, 249, 464 ActionDest: 121 Annotation: Subtype: Link Rect: 549, 119, 556, 131 ActionDest: 171 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 206, 422, 218, 431 ActionDest: 202 Annotation: Subtype: Link Rect: 218, 422, 230, 431 ActionDest: 202 Annotation: Subtype: Link Rect: 231, 422, 243, 431 ActionDest: 202 Annotation: Subtype: Link Rect: 389, 422, 401, 431 ActionDest: 202 Annotation: Subtype: Link Rect: 401, 422, 413, 431 ActionDest: 206 Annotation: Subtype: Link Rect: 414, 422, 426, 431 ActionDest: 206 Annotation: Subtype: Link Rect: 467, 422, 479, 431 ActionDest: 202 Annotation: Subtype: Link Rect: 545, 422, 557, 431 ActionDest: 206 Annotation: Subtype: Link Rect: 193, 409, 205, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 242, 409, 254, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 285, 409, 297, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 330, 409, 342, 418 ActionDest: 202 Annotation: Subtype: Link Rect: 375, 409, 387, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 388, 409, 400, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 501, 409, 513, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 514, 409, 525, 418 ActionDest: 206 Annotation: Subtype: Link Rect: 496, 384, 508, 393 ActionDest: 202 Annotation: Subtype: Link Rect: 307, 309, 319, 318 ActionDest: 206 Annotation: Subtype: Link Rect: 499, 284, 511, 293 ActionDest: 206 Annotation: Subtype: Link Rect: 512, 284, 523, 293 ActionDest: 206 Annotation: Subtype: Link Rect: 459, 259, 471, 268 ActionDest: 202 Annotation: Subtype: Link Rect: 302, 184, 314, 192 ActionDest: 202 Annotation: Subtype: Link Rect: 392, 121, 404, 130 ActionDest: 202 Annotation: Subtype: Link Rect: 466, 121, 478, 130 ActionDest: 202 Annotation: Subtype: Link Rect: 479, 121, 491, 130 ActionDest: 202 Annotation: Subtype: Link Rect: 491, 121, 503, 130 ActionDest: 206 Annotation: Subtype: Link Rect: 504, 121, 516, 130 ActionDest: 206 Annotation: Subtype: Link Rect: 355, 108, 367, 117 ActionDest: 206 Annotation: Subtype: Link Rect: 484, 108, 496, 117 ActionDest: 206 Annotation: Subtype: Link Rect: 497, 108, 509, 117 ActionDest: 206 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 317, 743, 329, 752 ActionDest: 202 Annotation: Subtype: Link Rect: 422, 706, 434, 715 ActionDest: 202 Annotation: Subtype: Link Rect: 434, 706, 446, 715 ActionDest: 202 Annotation: Subtype: Link Rect: 447, 706, 459, 715 ActionDest: 206 Annotation: Subtype: Link Rect: 544, 580, 556, 589 ActionDest: 206 Annotation: Subtype: Link Rect: 495, 480, 507, 489 ActionDest: 202 Annotation: Subtype: Link Rect: 334, 442, 341, 451 ActionDest: 202 Annotation: Subtype: Link Rect: 330, 392, 342, 401 ActionDest: 206 Annotation: Subtype: Link Rect: 200, 367, 211, 376 ActionDest: 206 Annotation: Subtype: Link Rect: 259, 342, 271, 351 ActionDest: 206 Annotation: Subtype: Link Rect: 245, 329, 257, 338 ActionDest: 206 Annotation: Subtype: Link Rect: 499, 317, 506, 326 ActionDest: 202 Annotation: Subtype: Link Rect: 484, 292, 496, 301 ActionDest: 202 Annotation: Subtype: Link Rect: 368, 254, 380, 263 ActionDest: 202 Annotation: Subtype: Link Rect: 215, 229, 227, 238 ActionDest: 206 Annotation: Subtype: Link Rect: 366, 191, 378, 200 ActionDest: 202 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 188, 718, 200, 727 ActionDest: 202 Annotation: Subtype: Link Rect: 244, 593, 256, 602 ActionDest: 206 Annotation: Subtype: Link Rect: 256, 593, 268, 602 ActionDest: 206 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 492, 727, 529, 738 Annotation: Subtype: Link Rect: 438, 715, 475, 726 Annotation: Subtype: Link Rect: 386, 704, 424, 715 Annotation: Subtype: Link Rect: 95, 670, 132, 680 Annotation: Subtype: Link Rect: 283, 646, 320, 657 Annotation: Subtype: Link Rect: 190, 624, 227, 634 Annotation: Subtype: Link Rect: 188, 600, 226, 611 Annotation: Subtype: Link Rect: 59, 566, 96, 576 Annotation: Subtype: Link Rect: 419, 542, 457, 553 Annotation: Subtype: Link Rect: 142, 509, 180, 519 Annotation: Subtype: Link Rect: 176, 485, 213, 496 Annotation: Subtype: Link Rect: 505, 462, 543, 473 Annotation: Subtype: Link Rect: 500, 427, 538, 438 Annotation: Subtype: Link Rect: 186, 393, 224, 404 Annotation: Subtype: Link Rect: 520, 370, 557, 381 Annotation: Subtype: Link Rect: 150, 336, 188, 346 Annotation: Subtype: Link Rect: 434, 266, 471, 277 Annotation: Subtype: Link Rect: 224, 232, 261, 243 Annotation: Subtype: Link Rect: 240, 209, 277, 220 Annotation: Subtype: Link Rect: 403, 186, 440, 197 Annotation: Subtype: Link Rect: 225, 151, 263, 162 Annotation: Subtype: Link Rect: 449, 128, 487, 139 Annotation: Subtype: Link Rect: 191, 71, 228, 82 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 198, 718, 235, 729 Annotation: Subtype: Link Rect: 361, 695, 399, 706 Annotation: Subtype: Link Rect: 520, 672, 557, 683 Annotation: Subtype: Link Rect: 55, 649, 108, 660 Annotation: Subtype: Link Rect: 126, 627, 164, 637 Annotation: Subtype: Link Rect: 320, 591, 358, 602 Annotation: Subtype: Link Rect: 344, 522, 381, 533 Annotation: Subtype: Link Rect: 495, 499, 532, 510 Annotation: Subtype: Link Rect: 398, 476, 435, 487 Annotation: Subtype: Link Rect: 179, 453, 373, 464 Annotation: Subtype: Link Rect: 375, 430, 412, 441 Annotation: Subtype: Link Rect: 254, 407, 291, 418 Annotation: Subtype: Link Rect: 59, 385, 96, 395 Annotation: Subtype: Link Rect: 102, 385, 138, 395 Annotation: Subtype: Link Rect: 142, 362, 180, 372 Annotation: Subtype: Link Rect: 394, 338, 432, 349 Annotation: Subtype: Link Rect: 334, 315, 372, 326 Annotation: Subtype: Link Rect: 252, 281, 290, 292 Annotation: Subtype: Link Rect: 59, 247, 96, 257 Annotation: Subtype: Link Rect: 349, 223, 387, 234 Annotation: Subtype: Link Rect: 59, 190, 96, 200 Annotation: Subtype: Link Rect: 104, 155, 141, 165 Annotation: Subtype: Link Rect: 59, 121, 96, 131 Checksum: 0744f930 Type: CRC32 Checksum: d6f98ec2ef00970ff619908d5904d5b7 Type: MD5 Checksum: e2a71cff786d730a861694259d9cf7d03ff97755 Type: SHA-1 Checksum: dfeb838aae9d2f5bfe0b7887e6cb6823b1d46cd302f8456936c3627e9ca95445 Type: SHA-256