22acee5419131d249dc4606c8063c396 pmc_34438659.pdf 3b5d13882f1bd819db6546f29f48d5fb496f630c pmc_34438659.pdf c00c47195992fd2549f84ccc4f2ea1c11fa602cac42b69c538f030d817fdc428 pmc_34438659.pdf Title: Hair Cortisol, Testosterone, Dehydroepiandrosterone Sulfate and Their Ratios in Stallions as a Retrospective Measure of Hypothalamic–Pituitary–Adrenal and Hypothalamic–Pituitary– Gonadal Axes Activity: Exploring the Influence of Seasonality Subject: The monitoring of stress physiology includes studying a wide range of endocrinological mechanisms, which can be assessed using multiple tissue samples. This study aimed to evaluate the seasonal variations of hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axis activity. Ten pure-breed Menorca stallions were included in the study. The hair samples were collected approximately every two months following the shave-reshave method caudally to the sternum. After a methanol-based extraction, samples were analyzed by enzyme immunoassay for cortisol, testosterone, and dehydroepiandrosterone sulphate. Following our findings, we detected that cortisol, testosterone and dehydroepiandrosterone sulphate were significantly affected by seasonality, with the highest values of cortisol during summer and the lowest values of testosterone during spring. Dehydroepiandrosterone sulphate concentrations were increased in autumn compared to the other studied periods. Additionally, the studied hormone ratios showed variations between seasons. To conclude, season should, therefore, be considered when assessing sexual and stress hormones in stallion hair, since this variable can be a potential influencing factor and led to misinterpretations. Keywords: hair; testosterone; cortisol; dehydroepiandrosterone sulphate; ratio; season; horse; stallion; non-invasive; hypothalamic–pituitary–adrenal axis; hypothalamic–pituitary–gonadal axis Author: Sergi Olvera-Maneu, Anaïs Carbajal, Jaume Gardela and Manel Lopez-Bejar Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Jul 25 12:07:36 2021 CEST ModDate: Sun Jul 25 12:11:40 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 10 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 859970 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DQHEJR+VnURWPalladioL Type 1 Custom yes yes no 10 0 XQPMLP+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 VLKQRP+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 RMKWIY+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 SZMRXJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 54 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 58 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 62 0 XOMUNQ+CMSY10 Type 1 Builtin yes yes no 77 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 84 0 LQLLFI+MSAM10 Type 1 Builtin yes yes no 91 0 RAMSXI+wasy9 Type 1 Builtin yes yes no 95 0 MPAGEJ+Wingdings CID TrueType Identity-H yes yes yes 102 0 MPAEAP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 108 0 MPAEBA+PalatinoLinotype CID TrueType Identity-H yes yes yes 111 0 MPAEBB+PalatinoLinotype TrueType WinAnsi yes yes no 117 0 MPAEBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 120 0 MPAEJC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 123 0 MPAGEI+MSGothic CID TrueType Identity-H yes yes yes 126 0 MPAGEJ+Wingdings CID TrueType Identity-H yes yes yes 139 0 MPAEAP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 145 0 MPAEBB+PalatinoLinotype TrueType WinAnsi yes yes no 148 0 MPAEBD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 151 0 MPAEJC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 154 0 MPAGEI+MSGothic CID TrueType Identity-H yes yes yes 157 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:06:26 CEST RepresentationInformation: pmc_34438659.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:14:53 CEST Size: 859970 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 381 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: Horses, Housing, Diet, Handling, and Ethical Considerations Destination: subsection.2.1 Item: Title: Hair Samples Destination: subsection.2.2 Item: Title: Hair Steroid Extraction Destination: subsection.2.3 Item: Title: Hormone Detection and Biochemical Validation Tests Destination: subsection.2.4 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Results Destination: section.3 Children: Item: Title: Biochemical Validation of the Enzyme Immunoassay Destination: subsection.3.1 Item: Title: Seasonal Effect on C, T and DHEA-S Concentrations Destination: subsection.3.2 Item: Title: Seasonal Variations of C/DHEA and T/C Ratios Destination: subsection.3.3 Item: Title: Hormone Correlations Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Hair Cortisol, Testosterone, Dehydroepiandrosterone Sulfate and Their Ratios in Stallions as a Retrospective Measure of Hypothalamic Pituitary Adrenal and Hypothalamic Pituitary  Gonadal Axes Activity: Exploring the Influence of Seasonality Author: Sergi Olvera-Maneu, Anaïs Carbajal, Jaume Gardela and Manel Lopez-Bejar Subject: The monitoring of stress physiology includes studying a wide range of endocrinological mechanisms, which can be assessed using multiple tissue samples. This study aimed to evaluate the seasonal variations of hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic pituitary adrenal and hypothalamic pituitary gonadal axis activity. Ten pure-breed Menorca stallions were included in the study. The hair samples were collected approximately every two months following the shave-reshave method caudally to the sternum. After a methanol-based extraction, samples were analyzed by enzyme immunoassay for cortisol, testosterone, and dehydroepiandrosterone sulphate. Following our findings, we detected that cortisol, testosterone and dehydroepiandrosterone sulphate were significantly affected by seasonality, with the highest values of cortisol during summer and the lowest values of testosterone during spring. Dehydroepiandrosterone sulphate concentrations were increased in autumn compared to the other studied periods. Additionally, the studied hormone ratios showed variations between seasons. To conclude, season should, therefore, be considered when assessing sexual and stress hormones in stallion hair, since this variable can be a potential influencing factor and led to misinterpretations. Keywords: hair; testosterone; cortisol; dehydroepiandrosterone sulphate; ratio; season; horse; stallion; non-invasive; hypothalamic pituitary adrenal axis; hypothalamic pituitary gonadal axis Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Jul 25 12:07:36 CEST 2021 ModDate: Sun Jul 25 12:11:40 CEST 2021 ID: 0xd03aafeaf481a9e885bc624fefc7f18c, 0xd03aafeaf481a9e885bc624fefc7f18c Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VLKQRP+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 239 FontDescriptor: FontName: VLKQRP+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: RMKWIY+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: RMKWIY+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DQHEJR+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: DQHEJR+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LQLLFI+MSAM10 FontSubset: true FirstChar: 3 LastChar: 7 FontDescriptor: FontName: LQLLFI+MSAM10 Flags: Symbolic FontBBox: 8, -463, 1331, 1003 FontFile: true Font: BaseFont: XOMUNQ+CMSY10 FontSubset: true FirstChar: 0 LastChar: 52 FontDescriptor: FontName: XOMUNQ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: XQPMLP+URWPalladioL-Ital FontSubset: true FirstChar: 44 LastChar: 122 FontDescriptor: FontName: XQPMLP+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RAMSXI+wasy9 FontSubset: true FirstChar: 35 LastChar: 35 FontDescriptor: FontName: RAMSXI+wasy9 Flags: Symbolic FontBBox: -21, -1667, 1768, 753 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 165, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 141, 622, 152, 635 Annotation: Subtype: Link Rect: 229, 622, 240, 635 Annotation: Subtype: Link Rect: 318, 622, 329, 635 Annotation: Subtype: Link Rect: 65, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 336, 74, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 339, 80, 346, 89 ActionDest: 170 Annotation: Subtype: Link Rect: 524, 68, 531, 76 ActionDest: 170 Annotation: Subtype: Link Rect: 116, 17, 251, 28 Annotation: Subtype: Link Rect: 407, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 482, 87, 501 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 379, 699, 386, 708 ActionDest: 170 Annotation: Subtype: Link Rect: 387, 699, 394, 708 ActionDest: 170 Annotation: Subtype: Link Rect: 406, 686, 413, 695 ActionDest: 170 Annotation: Subtype: Link Rect: 414, 686, 421, 695 ActionDest: 170 Annotation: Subtype: Link Rect: 244, 649, 251, 658 ActionDest: 170 Annotation: Subtype: Link Rect: 252, 649, 259, 658 ActionDest: 170 Annotation: Subtype: Link Rect: 229, 624, 236, 633 ActionDest: 170 Annotation: Subtype: Link Rect: 393, 598, 400, 608 ActionDest: 170 Annotation: Subtype: Link Rect: 256, 561, 263, 570 ActionDest: 170 Annotation: Subtype: Link Rect: 386, 548, 393, 557 ActionDest: 170 Annotation: Subtype: Link Rect: 373, 523, 380, 532 ActionDest: 170 Annotation: Subtype: Link Rect: 475, 498, 482, 507 ActionDest: 170 Annotation: Subtype: Link Rect: 483, 498, 490, 507 ActionDest: 170 Annotation: Subtype: Link Rect: 492, 498, 504, 507 ActionDest: 170 Annotation: Subtype: Link Rect: 265, 460, 272, 469 ActionDest: 170 Annotation: Subtype: Link Rect: 272, 461, 284, 469 ActionDest: 170 Annotation: Subtype: Link Rect: 285, 461, 297, 469 ActionDest: 170 Annotation: Subtype: Link Rect: 383, 448, 395, 457 ActionDest: 170 Annotation: Subtype: Link Rect: 510, 423, 517, 432 ActionDest: 170 Annotation: Subtype: Link Rect: 427, 385, 439, 394 ActionDest: 170 Annotation: Subtype: Link Rect: 439, 385, 451, 394 ActionDest: 170 Annotation: Subtype: Link Rect: 213, 360, 225, 369 ActionDest: 170 Annotation: Subtype: Link Rect: 226, 360, 238, 369 ActionDest: 170 Annotation: Subtype: Link Rect: 193, 347, 200, 356 ActionDest: 170 Annotation: Subtype: Link Rect: 482, 347, 494, 356 ActionDest: 170 Annotation: Subtype: Link Rect: 495, 347, 507, 356 ActionDest: 170 Annotation: Subtype: Link Rect: 410, 322, 422, 331 ActionDest: 170 Annotation: Subtype: Link Rect: 422, 322, 434, 331 ActionDest: 170 Annotation: Subtype: Link Rect: 308, 297, 320, 306 ActionDest: 170 Annotation: Subtype: Link Rect: 199, 285, 211, 294 ActionDest: 170 Annotation: Subtype: Link Rect: 302, 285, 314, 294 ActionDest: 170 Annotation: Subtype: Link Rect: 365, 285, 377, 294 ActionDest: 170 Annotation: Subtype: Link Rect: 377, 285, 389, 294 ActionDest: 170 Annotation: Subtype: Link Rect: 212, 272, 224, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 225, 272, 237, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 265, 272, 276, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 277, 272, 289, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 400, 272, 412, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 412, 272, 424, 281 ActionDest: 170 Annotation: Subtype: Link Rect: 226, 259, 238, 269 ActionDest: 170 Annotation: Subtype: Link Rect: 238, 260, 250, 269 ActionDest: 173 Annotation: Subtype: Link Rect: 484, 222, 496, 231 ActionDest: 170 Annotation: Subtype: Link Rect: 496, 222, 508, 231 ActionDest: 170 Annotation: Subtype: Link Rect: 509, 222, 521, 231 ActionDest: 173 Annotation: Subtype: Link Rect: 521, 222, 533, 231 ActionDest: 173 Annotation: Subtype: Link Rect: 333, 209, 345, 218 ActionDest: 170 Annotation: Subtype: Link Rect: 345, 209, 357, 218 ActionDest: 173 Annotation: Subtype: Link Rect: 360, 209, 372, 218 ActionDest: 173 Annotation: Subtype: Link Rect: 331, 172, 338, 181 ActionDest: 170 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 544, 656, 556, 665 ActionDest: 173 Annotation: Subtype: Link Rect: 475, 342, 487, 351 ActionDest: 173 Annotation: Subtype: Link Rect: 434, 241, 446, 250 ActionDest: 173 Annotation: Subtype: Link Rect: 261, 178, 273, 187 ActionDest: 170 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 533, 483, 545, 492 ActionDest: 173 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 497, 577, 504, 589 ActionDest: 88 Annotation: Subtype: Link Rect: 549, 93, 556, 105 ActionDest: 133 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 288, 271, 300, 280 ActionDest: 170 Annotation: Subtype: Link Rect: 482, 196, 494, 205 ActionDest: 170 Annotation: Subtype: Link Rect: 494, 196, 506, 205 ActionDest: 170 Annotation: Subtype: Link Rect: 218, 133, 230, 142 ActionDest: 170 Annotation: Subtype: Link Rect: 297, 133, 309, 142 ActionDest: 173 Annotation: Subtype: Link Rect: 249, 83, 261, 92 ActionDest: 170 Annotation: Subtype: Link Rect: 386, 70, 398, 79 ActionDest: 173 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 208, 697, 220, 706 ActionDest: 173 Annotation: Subtype: Link Rect: 297, 672, 309, 681 ActionDest: 173 Annotation: Subtype: Link Rect: 311, 672, 323, 681 ActionDest: 173 Annotation: Subtype: Link Rect: 340, 647, 351, 656 ActionDest: 170 Annotation: Subtype: Link Rect: 352, 647, 364, 656 ActionDest: 173 Annotation: Subtype: Link Rect: 331, 584, 343, 593 ActionDest: 170 Annotation: Subtype: Link Rect: 375, 546, 387, 556 ActionDest: 170 Annotation: Subtype: Link Rect: 387, 546, 399, 556 ActionDest: 170 Annotation: Subtype: Link Rect: 199, 521, 211, 530 ActionDest: 173 Annotation: Subtype: Link Rect: 220, 471, 232, 480 ActionDest: 173 Annotation: Subtype: Link Rect: 468, 471, 480, 480 ActionDest: 173 Annotation: Subtype: Link Rect: 198, 396, 210, 405 ActionDest: 173 Annotation: Subtype: Link Rect: 211, 396, 223, 405 ActionDest: 173 Annotation: Subtype: Link Rect: 283, 396, 295, 405 ActionDest: 170 Annotation: Subtype: Link Rect: 343, 371, 355, 380 ActionDest: 173 Annotation: Subtype: Link Rect: 356, 371, 368, 380 ActionDest: 173 Annotation: Subtype: Link Rect: 312, 321, 324, 330 ActionDest: 170 Annotation: Subtype: Link Rect: 325, 320, 337, 330 ActionDest: 173 Annotation: Subtype: Link Rect: 321, 295, 333, 304 ActionDest: 173 Annotation: Subtype: Link Rect: 333, 295, 345, 304 ActionDest: 173 Annotation: Subtype: Link Rect: 403, 233, 415, 242 ActionDest: 173 Annotation: Subtype: Link Rect: 529, 220, 541, 229 ActionDest: 173 Annotation: Subtype: Link Rect: 246, 182, 258, 192 ActionDest: 173 Annotation: Subtype: Link Rect: 259, 183, 271, 192 ActionDest: 173 Annotation: Subtype: Link Rect: 493, 170, 505, 179 ActionDest: 173 Annotation: Subtype: Link Rect: 210, 120, 217, 129 ActionDest: 170 Annotation: Subtype: Link Rect: 217, 120, 229, 129 ActionDest: 173 Annotation: Subtype: Link Rect: 430, 120, 437, 129 ActionDest: 170 Annotation: Subtype: Link Rect: 468, 120, 475, 129 ActionDest: 170 Annotation: Subtype: Link Rect: 528, 120, 540, 129 ActionDest: 170 Annotation: Subtype: Link Rect: 541, 120, 553, 129 ActionDest: 173 Annotation: Subtype: Link Rect: 373, 95, 379, 104 ActionDest: 170 Annotation: Subtype: Link Rect: 464, 82, 476, 91 ActionDest: 173 Annotation: Subtype: Link Rect: 479, 82, 491, 91 ActionDest: 173 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 214, 722, 221, 731 ActionDest: 170 Annotation: Subtype: Link Rect: 320, 672, 332, 681 ActionDest: 173 Annotation: Subtype: Link Rect: 499, 647, 511, 656 ActionDest: 173 Annotation: Subtype: Link Rect: 512, 647, 524, 656 ActionDest: 173 Annotation: Subtype: Link Rect: 256, 622, 268, 631 ActionDest: 173 Annotation: Subtype: Link Rect: 271, 622, 283, 631 ActionDest: 173 Annotation: Subtype: Link Rect: 494, 546, 501, 556 ActionDest: 170 Annotation: Subtype: Link Rect: 549, 534, 556, 543 ActionDest: 170 Annotation: Subtype: Link Rect: 512, 521, 523, 530 ActionDest: 170 Annotation: Subtype: Link Rect: 524, 521, 536, 530 ActionDest: 173 Annotation: Subtype: Link Rect: 199, 509, 206, 518 ActionDest: 170 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 100, 632, 137, 642 Annotation: Subtype: Link Rect: 143, 632, 179, 642 Annotation: Subtype: Link Rect: 494, 620, 531, 631 Annotation: Subtype: Link Rect: 223, 598, 261, 608 Annotation: Subtype: Link Rect: 131, 574, 169, 585 Annotation: Subtype: Link Rect: 175, 574, 211, 585 Annotation: Subtype: Link Rect: 239, 551, 277, 562 Annotation: Subtype: Link Rect: 283, 551, 319, 562 Annotation: Subtype: Link Rect: 439, 540, 477, 550 Annotation: Subtype: Link Rect: 375, 516, 412, 527 Annotation: Subtype: Link Rect: 419, 516, 455, 527 Annotation: Subtype: Link Rect: 109, 482, 146, 493 Annotation: Subtype: Link Rect: 418, 459, 456, 470 Annotation: Subtype: Link Rect: 370, 436, 407, 447 Annotation: Subtype: Link Rect: 384, 413, 421, 424 Annotation: Subtype: Link Rect: 427, 413, 463, 424 Annotation: Subtype: Link Rect: 153, 390, 190, 401 Annotation: Subtype: Link Rect: 104, 367, 142, 378 Annotation: Subtype: Link Rect: 148, 367, 184, 378 Annotation: Subtype: Link Rect: 255, 344, 293, 354 Annotation: Subtype: Link Rect: 118, 321, 155, 331 Annotation: Subtype: Link Rect: 161, 321, 197, 331 Annotation: Subtype: Link Rect: 347, 297, 385, 308 Annotation: Subtype: Link Rect: 422, 274, 460, 285 Annotation: Subtype: Link Rect: 249, 251, 286, 262 Annotation: Subtype: Link Rect: 293, 251, 329, 262 Annotation: Subtype: Link Rect: 434, 195, 471, 205 Annotation: Subtype: Link Rect: 437, 171, 474, 182 Annotation: Subtype: Link Rect: 305, 148, 342, 159 Annotation: Subtype: Link Rect: 458, 125, 495, 136 Annotation: Subtype: Link Rect: 362, 102, 400, 113 Annotation: Subtype: Link Rect: 520, 79, 557, 90 Annotation: Subtype: Link Rect: 283, 57, 320, 67 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 113, 710, 151, 720 Annotation: Subtype: Link Rect: 157, 710, 193, 720 Annotation: Subtype: Link Rect: 288, 686, 325, 697 Annotation: Subtype: Link Rect: 408, 652, 445, 663 Annotation: Subtype: Link Rect: 451, 652, 487, 663 Annotation: Subtype: Link Rect: 359, 630, 396, 640 Annotation: Subtype: Link Rect: 402, 630, 438, 640 Annotation: Subtype: Link Rect: 519, 617, 557, 628 Annotation: Subtype: Link Rect: 131, 595, 169, 605 Annotation: Subtype: Link Rect: 422, 571, 460, 582 Annotation: Subtype: Link Rect: 192, 548, 229, 559 Annotation: Subtype: Link Rect: 106, 526, 144, 536 Annotation: Subtype: Link Rect: 59, 492, 97, 502 Annotation: Subtype: Link Rect: 129, 457, 167, 467 Annotation: Subtype: Link Rect: 136, 423, 173, 433 Annotation: Subtype: Link Rect: 179, 423, 216, 433 Annotation: Subtype: Link Rect: 109, 400, 146, 410 Annotation: Subtype: Link Rect: 441, 387, 479, 398 Annotation: Subtype: Link Rect: 59, 354, 97, 364 Annotation: Subtype: Link Rect: 409, 330, 447, 341 Annotation: Subtype: Link Rect: 430, 307, 467, 318 Annotation: Subtype: Link Rect: 148, 285, 186, 295 Annotation: Subtype: Link Rect: 192, 285, 228, 295 Annotation: Subtype: Link Rect: 252, 261, 290, 272 Annotation: Subtype: Link Rect: 376, 238, 413, 249 Annotation: Subtype: Link Rect: 420, 238, 456, 249 Annotation: Subtype: Link Rect: 157, 215, 195, 226 Annotation: Subtype: Link Rect: 201, 215, 237, 226 Annotation: Subtype: Link Rect: 104, 192, 142, 203 Annotation: Subtype: Link Rect: 214, 169, 252, 180 Checksum: 09af5712 Type: CRC32 Checksum: 22acee5419131d249dc4606c8063c396 Type: MD5 Checksum: 3b5d13882f1bd819db6546f29f48d5fb496f630c Type: SHA-1 Checksum: c00c47195992fd2549f84ccc4f2ea1c11fa602cac42b69c538f030d817fdc428 Type: SHA-256