a0634328e8d85a85467c8b2b63d4b931 pmc_33171996.pdf 03f7f4ff6a8f104ac970264b5eb50cfd444e3ebc pmc_33171996.pdf 75a92dd557e49531da39e9e5100eae0684598367a5bc5f2b7faa75a24ea1b423 pmc_33171996.pdf Title: Validation of an Alternative Feather Sampling Method to Measure Corticosterone Subject: The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird’s skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland–Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement. Keywords: feather corticosterone; comparative study; plucked feathers; cut feathers; Domestic Goose; Mulard Duck; animal welfare Author: Marielu Voit, Roswitha Merle, Katrin Baumgartner, Lorenzo von Fersen, Lukas Reese, Mechthild Ladwig-Wiegard, Hermann Will, Oriol Tallo-Parra, Annaïs Carbajal, Manel Lopez-Bejar and Christa Thöne-Reineke Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Fri Nov 6 09:10:02 2020 CET ModDate: Fri Nov 6 09:10:02 2020 CET Custom Metadata: yes 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: 2124589 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JCXYCL+URWPalladioL-Ital Type 1 Custom yes yes no 67 0 ZLAMNQ+URWPalladioL-Bold Type 1 Custom yes yes no 68 0 APXYVG+URWPalladioL-Roma Type 1 Custom yes yes no 69 0 ESSDCZ+VnURWPalladioL Type 1 Custom yes yes no 70 0 FATHTC+Rpxr Type 1 Builtin yes yes no 71 0 SZMRXJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 76 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 93 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 94 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 95 0 TGMEOR+Rpcxr Type 1 Builtin yes yes no 234 0 HHMDGK+Pxsy Type 1 Builtin yes yes no 250 0 JFKFCF+PalatinoLinotype CID TrueType Identity-H yes yes yes 275 0 JFKFDG+PalatinoLinotype TrueType WinAnsi yes yes no 276 0 JFKGBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 277 0 JFKGDE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 278 0 JFKFDG+PalatinoLinotype TrueType WinAnsi yes yes no 311 0 JFKGBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 312 0 JFKGDE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 313 0 JFKFDG+PalatinoLinotype TrueType WinAnsi yes yes no 328 0 JFKGBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 329 0 JFKGDE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 330 0 JFKFCF+PalatinoLinotype CID TrueType Identity-H yes yes yes 356 0 JFKFDG+PalatinoLinotype TrueType WinAnsi yes yes no 357 0 JFKGBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 358 0 JFKGDE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 359 0 JFKFCF+PalatinoLinotype CID TrueType Identity-H yes yes yes 379 0 JFKFDG+PalatinoLinotype TrueType WinAnsi yes yes no 380 0 JFKGBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 381 0 JFKGDE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 382 0 MFIGJL+Rpxb Type 1 Builtin yes yes no 414 0 ZEJRDS+URWPalladioL-BoldItal Type 1 Custom yes yes no 415 0 LGZKLX+Rpxi Type 1 Builtin yes yes no 524 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 04:17:51 CEST RepresentationInformation: pmc_33171996.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:43:32 CEST Size: 2124589 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〠 Item: Title: きちぴづひどちぬび〠ちのつ〠きづぴとはつび〠 Children: Item: Title: こふぢなづっぴび〠 Item: Title: えふびぢちのつひべ〠 Item: Title: こちねばぬどので〠ぐひはぴはっはぬ〠 Item: Title: ぁのちぬべびどび〠はて〠ぴとづ〠ぃはひぴどっはびぴづひはのづ〠がづぶづぬ〠どの〠うづちぴとづひび〠 Item: Title: こぴちぴどびぴどっちぬ〠ぁのちぬべびどび〠 Item: Title: げづびふぬぴび〠 Item: Title: いどびっふびびどはの〠 Item: Title: ぃはのっぬふびどはのび〠 Item: Title: げづてづひづのっづび Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: JCXYCL+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 228 FontDescriptor: FontName: JCXYCL+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZLAMNQ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 246 FontDescriptor: FontName: ZLAMNQ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: APXYVG+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: APXYVG+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ESSDCZ+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: ESSDCZ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FATHTC+Rpxr FontSubset: true FirstChar: 11 LastChar: 61 FontDescriptor: FontName: FATHTC+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: TGMEOR+Rpcxr FontSubset: true FirstChar: 169 LastChar: 174 FontDescriptor: FontName: TGMEOR+Rpcxr Flags: Symbolic FontBBox: -212, -286, 1344, 973 FontFile: true Font: BaseFont: HHMDGK+Pxsy FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: HHMDGK+Pxsy Flags: Symbolic FontBBox: -21, -994, 1292, 912 FontFile: true Font: BaseFont: LGZKLX+Rpxi FontSubset: true FirstChar: 14 LastChar: 14 FontDescriptor: FontName: LGZKLX+Rpxi Flags: Symbolic FontBBox: -162, -280, 856, 733 FontFile: true Font: BaseFont: MFIGJL+Rpxb FontSubset: true FirstChar: 47 LastChar: 47 FontDescriptor: FontName: MFIGJL+Rpxb Flags: Symbolic FontBBox: -57, -266, 932, 729 FontFile: true Font: BaseFont: ZEJRDS+URWPalladioL-BoldItal FontSubset: true FirstChar: 65 LastChar: 115 FontDescriptor: FontName: ZEJRDS+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 75, 757, 206, 793 Annotation: Subtype: Link Rect: 474, 757, 519, 793 Annotation: Subtype: Link Rect: 373, 648, 384, 662 Annotation: Subtype: Link Rect: 468, 648, 479, 662 Annotation: Subtype: Link Rect: 175, 634, 186, 648 Annotation: Subtype: Link Rect: 319, 634, 330, 648 Annotation: Subtype: Link Rect: 466, 471, 518, 490 Annotation: Subtype: Link Rect: 170, 38, 243, 50 Annotation: Subtype: Link Rect: 403, 38, 519, 50 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 508, 553, 515, 562 ActionDest: 463 Annotation: Subtype: Link Rect: 294, 526, 301, 535 ActionDest: 463 Annotation: Subtype: Link Rect: 302, 526, 309, 535 ActionDest: 463 Annotation: Subtype: Link Rect: 160, 499, 167, 508 ActionDest: 463 Annotation: Subtype: Link Rect: 418, 486, 425, 495 ActionDest: 463 Annotation: Subtype: Link Rect: 425, 486, 432, 495 ActionDest: 463 Annotation: Subtype: Link Rect: 435, 485, 442, 495 ActionDest: 463 Annotation: Subtype: Link Rect: 142, 459, 149, 468 ActionDest: 463 Annotation: Subtype: Link Rect: 169, 404, 176, 414 ActionDest: 463 Annotation: Subtype: Link Rect: 335, 351, 342, 360 ActionDest: 463 Annotation: Subtype: Link Rect: 343, 350, 350, 360 ActionDest: 463 Annotation: Subtype: Link Rect: 491, 296, 503, 306 ActionDest: 463 Annotation: Subtype: Link Rect: 503, 297, 515, 306 ActionDest: 489 Annotation: Subtype: Link Rect: 426, 256, 438, 265 ActionDest: 489 Annotation: Subtype: Link Rect: 491, 242, 503, 252 ActionDest: 489 Annotation: Subtype: Link Rect: 503, 242, 515, 252 ActionDest: 489 Annotation: Subtype: Link Rect: 503, 229, 515, 238 ActionDest: 489 Annotation: Subtype: Link Rect: 471, 175, 478, 184 ActionDest: 463 Annotation: Subtype: Link Rect: 128, 121, 135, 130 ActionDest: 463 Annotation: Subtype: Link Rect: 126, 80, 137, 89 ActionDest: 489 Annotation: Subtype: Link Rect: 141, 80, 153, 89 ActionDest: 489 Annotation: Subtype: Link Rect: 503, 80, 515, 89 ActionDest: 489 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 113, 715, 125, 725 ActionDest: 489 Annotation: Subtype: Link Rect: 427, 675, 439, 684 ActionDest: 489 Annotation: Subtype: Link Rect: 439, 675, 451, 684 ActionDest: 489 Annotation: Subtype: Link Rect: 381, 607, 388, 617 ActionDest: 463 Annotation: Subtype: Link Rect: 393, 580, 400, 590 ActionDest: 463 Annotation: Subtype: Link Rect: 256, 553, 268, 562 ActionDest: 489 Annotation: Subtype: Link Rect: 269, 553, 281, 562 ActionDest: 489 Annotation: Subtype: Link Rect: 222, 526, 234, 535 ActionDest: 489 Annotation: Subtype: Link Rect: 503, 513, 515, 522 ActionDest: 489 Annotation: Subtype: Link Rect: 146, 486, 158, 495 ActionDest: 489 Annotation: Subtype: Link Rect: 159, 486, 171, 495 ActionDest: 489 Annotation: Subtype: Link Rect: 147, 459, 154, 468 ActionDest: 463 Annotation: Subtype: Link Rect: 154, 459, 166, 468 ActionDest: 489 Annotation: Subtype: Link Rect: 169, 459, 181, 468 ActionDest: 489 Annotation: Subtype: Link Rect: 224, 432, 231, 441 ActionDest: 463 Annotation: Subtype: Link Rect: 279, 297, 291, 306 ActionDest: 489 Annotation: Subtype: Link Rect: 291, 297, 303, 306 ActionDest: 489 Annotation: Subtype: Link Rect: 160, 188, 172, 198 ActionDest: 489 Annotation: Subtype: Link Rect: 173, 188, 185, 198 ActionDest: 516 Annotation: Subtype: Link Rect: 322, 148, 334, 157 ActionDest: 516 Annotation: Subtype: Link Rect: 466, 121, 478, 130 ActionDest: 516 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 155, 729, 167, 738 ActionDest: 516 Annotation: Subtype: Link Rect: 167, 729, 179, 738 ActionDest: 516 Annotation: Subtype: Link Rect: 182, 729, 194, 738 ActionDest: 516 Annotation: Subtype: Link Rect: 222, 688, 234, 698 ActionDest: 489 Annotation: Subtype: Link Rect: 235, 688, 247, 698 ActionDest: 516 Annotation: Subtype: Link Rect: 295, 648, 307, 657 ActionDest: 516 Annotation: Subtype: Link Rect: 326, 607, 338, 617 ActionDest: 489 Annotation: Subtype: Link Rect: 338, 607, 350, 617 ActionDest: 516 Annotation: Subtype: Link Rect: 419, 580, 431, 590 ActionDest: 489 Annotation: Subtype: Link Rect: 431, 580, 443, 590 ActionDest: 516 Annotation: Subtype: Link Rect: 444, 580, 456, 590 ActionDest: 516 Annotation: Subtype: Link Rect: 244, 526, 256, 535 ActionDest: 516 Annotation: Subtype: Link Rect: 256, 526, 268, 535 ActionDest: 516 Annotation: Subtype: Link Rect: 430, 499, 442, 508 ActionDest: 489 Annotation: Subtype: Link Rect: 488, 163, 500, 172 ActionDest: 489 Annotation: Subtype: Link Rect: 503, 163, 515, 172 ActionDest: 489 Annotation: Subtype: Link Rect: 164, 123, 176, 132 ActionDest: 489 Annotation: Subtype: Link Rect: 176, 123, 188, 132 ActionDest: 489 Annotation: Subtype: Link Rect: 304, 82, 316, 91 ActionDest: 516 Annotation: Subtype: Link Rect: 319, 82, 331, 91 ActionDest: 516 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 468, 715, 475, 725 ActionDest: 463 Annotation: Subtype: Link Rect: 476, 715, 488, 725 ActionDest: 489 Annotation: Subtype: Link Rect: 488, 715, 500, 725 ActionDest: 516 Annotation: Subtype: Link Rect: 503, 715, 515, 725 ActionDest: 516 Annotation: Subtype: Link Rect: 125, 619, 137, 628 ActionDest: 516 Annotation: Subtype: Link Rect: 355, 359, 362, 371 ActionDest: 231 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 403, 349, 410, 358 ActionDest: 463 Annotation: Subtype: Link Rect: 233, 292, 240, 301 ActionDest: 463 Annotation: Subtype: Link Rect: 240, 292, 252, 301 ActionDest: 516 Annotation: Subtype: Link Rect: 182, 265, 194, 274 ActionDest: 516 Annotation: Subtype: Link Rect: 428, 225, 435, 234 ActionDest: 463 Annotation: Subtype: Link Rect: 436, 225, 448, 234 ActionDest: 516 Annotation: Subtype: Link Rect: 448, 225, 460, 234 ActionDest: 516 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 338, 524, 350, 533 ActionDest: 547 Annotation: Subtype: Link Rect: 444, 511, 451, 520 ActionDest: 463 Annotation: Subtype: Link Rect: 451, 511, 458, 520 ActionDest: 463 Annotation: Subtype: Link Rect: 459, 511, 471, 520 ActionDest: 489 Annotation: Subtype: Link Rect: 471, 511, 483, 520 ActionDest: 547 Annotation: Subtype: Link Rect: 339, 443, 351, 452 ActionDest: 547 Annotation: Subtype: Link Rect: 160, 416, 172, 425 ActionDest: 547 Annotation: Subtype: Link Rect: 448, 400, 454, 412 ActionDest: 266 Annotation: Subtype: Link Rect: 457, 400, 464, 412 ActionDest: 303 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 188, 86, 200, 95 ActionDest: 547 Annotation: Subtype: Link Rect: 200, 86, 212, 95 ActionDest: 547 Annotation: Subtype: Link Rect: 378, 70, 385, 82 ActionDest: 349 Annotation: Subtype: Link Rect: 405, 70, 412, 82 ActionDest: 349 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 328, 615, 340, 624 ActionDest: 547 Annotation: Subtype: Link Rect: 340, 615, 352, 624 ActionDest: 547 Annotation: Subtype: Link Rect: 209, 572, 215, 584 ActionDest: 411 Annotation: Subtype: Link Rect: 273, 531, 281, 543 ActionDest: 266 Annotation: Subtype: Link Rect: 448, 531, 455, 543 ActionDest: 303 Annotation: Subtype: Link Rect: 124, 504, 131, 516 ActionDest: 349 Annotation: Subtype: Link Rect: 278, 477, 285, 489 ActionDest: 349 Page: Sequence: 11 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 153, 601, 165, 611 ActionDest: 547 Annotation: Subtype: Link Rect: 165, 601, 177, 611 ActionDest: 547 Annotation: Subtype: Link Rect: 103, 574, 115, 584 ActionDest: 547 Annotation: Subtype: Link Rect: 321, 412, 333, 421 ActionDest: 547 Annotation: Subtype: Link Rect: 336, 412, 348, 421 ActionDest: 547 Annotation: Subtype: Link Rect: 196, 355, 203, 367 ActionDest: 411 Annotation: Subtype: Link Rect: 121, 331, 128, 340 ActionDest: 463 Annotation: Subtype: Link Rect: 165, 182, 177, 192 ActionDest: 547 Annotation: Subtype: Link Rect: 137, 166, 144, 178 ActionDest: 303 Annotation: Subtype: Link Rect: 322, 166, 329, 178 ActionDest: 266 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 288, 699, 295, 711 ActionDest: 349 Annotation: Subtype: Link Rect: 109, 659, 116, 671 ActionDest: 349 Annotation: Subtype: Link Rect: 112, 391, 119, 400 ActionDest: 463 Annotation: Subtype: Link Rect: 119, 391, 126, 400 ActionDest: 463 Annotation: Subtype: Link Rect: 127, 391, 139, 400 ActionDest: 489 Annotation: Subtype: Link Rect: 139, 391, 151, 400 ActionDest: 547 Annotation: Subtype: Link Rect: 127, 310, 138, 319 ActionDest: 516 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 504, 634, 516, 644 ActionDest: 516 Annotation: Subtype: Link Rect: 330, 361, 367, 372 Annotation: Subtype: Link Rect: 373, 361, 409, 372 Annotation: Subtype: Link Rect: 460, 335, 496, 346 Annotation: Subtype: Link Rect: 332, 309, 369, 320 Annotation: Subtype: Link Rect: 376, 309, 412, 320 Annotation: Subtype: Link Rect: 136, 271, 174, 281 Annotation: Subtype: Link Rect: 180, 271, 216, 281 Annotation: Subtype: Link Rect: 287, 245, 324, 256 Annotation: Subtype: Link Rect: 290, 193, 328, 204 Annotation: Subtype: Link Rect: 136, 168, 174, 178 Annotation: Subtype: Link Rect: 363, 141, 401, 152 Annotation: Subtype: Link Rect: 100, 91, 137, 101 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 353, 727, 390, 738 Annotation: Subtype: Link Rect: 265, 689, 303, 699 Annotation: Subtype: Link Rect: 275, 663, 312, 673 Annotation: Subtype: Link Rect: 365, 637, 402, 648 Annotation: Subtype: Link Rect: 435, 598, 471, 609 Annotation: Subtype: Link Rect: 412, 572, 450, 583 Annotation: Subtype: Link Rect: 312, 546, 349, 557 Annotation: Subtype: Link Rect: 480, 521, 516, 531 Annotation: Subtype: Link Rect: 326, 495, 519, 506 Annotation: Subtype: Link Rect: 97, 482, 256, 493 Annotation: Subtype: Link Rect: 387, 430, 425, 441 Annotation: Subtype: Link Rect: 356, 404, 393, 415 Annotation: Subtype: Link Rect: 136, 366, 174, 376 Annotation: Subtype: Link Rect: 100, 328, 137, 338 Annotation: Subtype: Link Rect: 338, 301, 375, 312 Annotation: Subtype: Link Rect: 458, 275, 495, 286 Annotation: Subtype: Link Rect: 309, 249, 347, 260 Annotation: Subtype: Link Rect: 163, 212, 201, 222 Annotation: Subtype: Link Rect: 424, 185, 461, 196 Annotation: Subtype: Link Rect: 411, 159, 449, 170 Annotation: Subtype: Link Rect: 264, 134, 301, 144 Annotation: Subtype: Link Rect: 495, 120, 519, 131 Annotation: Subtype: Link Rect: 97, 107, 519, 118 Annotation: Subtype: Link Rect: 97, 94, 405, 105 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 141, 689, 178, 699 Annotation: Subtype: Link Rect: 344, 650, 519, 661 Annotation: Subtype: Link Rect: 97, 637, 182, 648 Annotation: Subtype: Link Rect: 332, 611, 369, 622 Annotation: Subtype: Link Rect: 191, 585, 519, 596 Annotation: Subtype: Link Rect: 97, 572, 259, 583 Annotation: Subtype: Link Rect: 449, 546, 519, 557 Annotation: Subtype: Link Rect: 97, 533, 438, 544 Annotation: Subtype: Link Rect: 150, 496, 187, 506 Annotation: Subtype: Link Rect: 416, 443, 519, 454 Annotation: Subtype: Link Rect: 97, 430, 364, 441 Annotation: Subtype: Link Rect: 410, 417, 519, 428 Annotation: Subtype: Link Rect: 97, 404, 305, 415 Annotation: Subtype: Link Rect: 127, 378, 519, 389 Annotation: Subtype: Link Rect: 97, 366, 222, 376 Annotation: Subtype: Link Rect: 100, 328, 137, 338 Annotation: Subtype: Link Rect: 379, 301, 519, 312 Annotation: Subtype: Link Rect: 97, 288, 519, 299 Annotation: Subtype: Link Rect: 97, 275, 114, 286 Annotation: Subtype: Link Rect: 329, 236, 366, 247 Annotation: Subtype: Link Rect: 177, 199, 214, 209 Annotation: Subtype: Link Rect: 359, 173, 396, 183 Annotation: Subtype: Link Rect: 145, 134, 182, 144 Annotation: Subtype: Link Rect: 283, 94, 320, 105 Annotation: Subtype: Link Rect: 271, 69, 308, 80 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 239, 728, 277, 738 Annotation: Subtype: Link Rect: 348, 701, 385, 712 Annotation: Subtype: Link Rect: 392, 701, 428, 712 Annotation: Subtype: Link Rect: 253, 612, 291, 622 Annotation: Subtype: Link Rect: 167, 586, 205, 596 Annotation: Subtype: Link Rect: 479, 559, 516, 570 Annotation: Subtype: Link Rect: 269, 521, 306, 531 Annotation: Subtype: Link Rect: 145, 483, 182, 493 Annotation: Subtype: Link Rect: 286, 444, 323, 454 Annotation: Subtype: Link Rect: 272, 418, 309, 428 Annotation: Subtype: Link Rect: 315, 418, 351, 428 Annotation: Subtype: Link Rect: 473, 353, 510, 364 Annotation: Subtype: Link Rect: 76, 281, 166, 312 Annotation: Subtype: Link Rect: 234, 275, 407, 286 Checksum: 286a0b96 Type: CRC32 Checksum: a0634328e8d85a85467c8b2b63d4b931 Type: MD5 Checksum: 03f7f4ff6a8f104ac970264b5eb50cfd444e3ebc Type: SHA-1 Checksum: 75a92dd557e49531da39e9e5100eae0684598367a5bc5f2b7faa75a24ea1b423 Type: SHA-256