238dae73e61662eb82b10cde38524b82 pmc_9103047.pdf c4e17900c7cabd0a5e08fd323f89f28731ab3b1f pmc_9103047.pdf b6fc25e42e19a5dd1c3b45624cfcff7b2c5ac4d112e0efa174c23a75820251e3 pmc_9103047.pdf Title: Low n-6/n-3 Gestation and Lactation Diets Influence Early Performance, Muscle and Adipose Polyunsaturated Fatty Acid Content and Deposition, and Relative Abundance of Proteins in Suckling Piglets Subject: Elevated omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) ratios in swine diets can potentially impose a higher risk of inflammatory and metabolic diseases in swine. A low ratio between the two omega PUFAs has beneficial effects on sows’ and piglets’ production performance and immunity status. At present, there are few studies on how sow nutrition directly affects the protein and fat deposition in suckling piglets. Two groups of sows were fed diets with high or low n-6/n-3 polyunsaturated ratios of 13:1 (SOY) and 4:1 (LIN), respectively, during gestation and lactation. Longissimus dorsi muscle and adipose tissue from newborn piglets, nourished only with sow’s milk, were subjected to fatty acid profiling by gas chromatography–mass spectrometry (GC-MS) and to proteomics assays based on nano-liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-HRMS). Fatty acid profiles on both muscle and adipose tissues resembled the magnitude of the differences between fatty acid across diets. Proteomic analysis revealed overabundance of 4 muscle and 11 adipose tissue proteins in SOY compared to LIN in both piglet tissues. The detected overabundance of haptoglobin, an acute-phase protein, and the stimulation of protein-coding genes and proteins related to the innate immune response and acute inflammatory response could be associated with the pro-inflammatory role of n-6 PUFAs. Keywords: piglets; longissimus dorsi; adipose tissue; PUFA; proteomics; omega-6; omega-3; inflammation; fat deposition Author: Yron Joseph Yabut Manaig, Silvia Sandrini, Sara Panseri, Gabriella Tedeschi, Josep M. Folch, Armand Sánchez, Giovanni Savoini and Alessandro Agazzi Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed May 4 08:52:42 2022 CEST ModDate: Wed May 4 08:57:00 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 746820 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KORLVE+VnURWPalladioL Type 1 Custom yes yes no 10 0 XIBHWZ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 LCTZRM+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 KWFAIW+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 AIQCCL+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 PUSVWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 XEYJZU+CMSY10 Type 1 Builtin yes yes no 68 0 NVJQPH+EURM10 Type 1 Builtin yes yes no 72 0 JFVOWE+URWPalladioL-BoldItal Type 1 Custom yes yes no 79 0 IDEOCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 95 0 IDEODE+PalatinoLinotype CID TrueType Identity-H yes yes yes 98 0 IDEODF+PalatinoLinotype TrueType WinAnsi yes yes no 104 0 IDEODH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 107 0 IDEOCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 122 0 IDEODE+PalatinoLinotype CID TrueType Identity-H yes yes yes 125 0 IDEODF+PalatinoLinotype TrueType WinAnsi yes yes no 131 0 IDEODH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 134 0 IDEPIB+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 137 0 IDFANK+CambriaMath CID TrueType Identity-H yes yes yes 140 0 ROWTUD+PazoMath Type 1 Builtin yes yes no 154 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-10-01 02:37:07 CEST RepresentationInformation: pmc_9103047.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-09-16 11:04:40 CEST Size: 746820 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 425 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4923ab24 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Children: Item: Title: Sow Reproductive Performance Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Growth Performance of Litter and Pre-Weaning Piglets Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Fatty Acid Composition of Muscle and Adipose Tissue Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Proteins Identification and Relative Abundance Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Children: Item: Title: From Muscle Tissues Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: From Adipose Tissue Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Gene Ontologies and Biological Pathways Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Experimental Design, Animals, and Housing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Experimental Diets Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Recording and Sampling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Muscle and Adipose Tissue Samples Fatty Acid Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Proteomics Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Protein Extraction, Processing, and Digestion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Nano-LC-MS/MS Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Proteomics Data Processing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Low n-6/n-3 Gestation and Lactation Diets Influence Early Performance, Muscle and Adipose Polyunsaturated Fatty Acid Content and Deposition, and Relative Abundance of Proteins in Suckling Piglets Author: Yron Joseph Yabut Manaig, Silvia Sandrini, Sara Panseri, Gabriella Tedeschi, Josep M. Folch, Armand Sánchez, Giovanni Savoini and Alessandro Agazzi Subject: Elevated omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) ratios in swine diets can potentially impose a higher risk of inflammatory and metabolic diseases in swine. A low ratio between the two omega PUFAs has beneficial effects on sows’ and piglets’ production performance and immunity status. At present, there are few studies on how sow nutrition directly affects the protein and fat deposition in suckling piglets. Two groups of sows were fed diets with high or low n-6/n-3 polyunsaturated ratios of 13:1 (SOY) and 4:1 (LIN), respectively, during gestation and lactation. Longissimus dorsi muscle and adipose tissue from newborn piglets, nourished only with sow’s milk, were subjected to fatty acid profiling by gas chromatography mass spectrometry (GC-MS) and to proteomics assays based on nano-liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-HRMS). Fatty acid profiles on both muscle and adipose tissues resembled the magnitude of the differences between fatty acid across diets. Proteomic analysis revealed overabundance of 4 muscle and 11 adipose tissue proteins in SOY compared to LIN in both piglet tissues. The detected overabundance of haptoglobin, an acute-phase protein, and the stimulation of protein-coding genes and proteins related to the innate immune response and acute inflammatory response could be associated with the pro-inflammatory role of n-6 PUFAs. Keywords: piglets; longissimus dorsi; adipose tissue; PUFA; proteomics; omega-6; omega-3; inflammation; fat deposition Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed May 04 08:52:42 CEST 2022 ModDate: Wed May 04 08:57:00 CEST 2022 ID: 0x6c7cd8b94a3960c162ed333877ea6d06, 0x6c7cd8b94a3960c162ed333877ea6d06 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LCTZRM+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 248 FontDescriptor: FontName: LCTZRM+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XEYJZU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: XEYJZU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: KWFAIW+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: KWFAIW+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: NVJQPH+EURM10 FontSubset: true FirstChar: 11 LastChar: 33 FontDescriptor: FontName: NVJQPH+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: KORLVE+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: KORLVE+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ROWTUD+PazoMath FontSubset: true FirstChar: 68 LastChar: 87 FontDescriptor: FontName: ROWTUD+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: AIQCCL+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 161 FontDescriptor: FontName: AIQCCL+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XIBHWZ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: XIBHWZ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JFVOWE+URWPalladioL-BoldItal FontSubset: true FirstChar: 80 LastChar: 112 FontDescriptor: FontName: JFVOWE+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 54, 335, 152, 344 Annotation: Subtype: Link Rect: 34, 325, 95, 332 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, 179, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 183, 623, 194, 636 Annotation: Subtype: Link Rect: 272, 623, 283, 636 Annotation: Subtype: Link Rect: 349, 623, 360, 636 Annotation: Subtype: Link Rect: 332, 610, 343, 624 Annotation: Subtype: Link Rect: 291, 135, 298, 144 ActionDest: 180 Annotation: Subtype: Link Rect: 290, 97, 297, 106 ActionDest: 180 Annotation: Subtype: Link Rect: 121, 17, 281, 28 Annotation: Subtype: Link Rect: 401, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 456, 87, 475 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 469, 723, 476, 732 ActionDest: 180 Annotation: Subtype: Link Rect: 477, 723, 484, 732 ActionDest: 180 Annotation: Subtype: Link Rect: 541, 685, 548, 694 ActionDest: 180 Annotation: Subtype: Link Rect: 549, 685, 556, 694 ActionDest: 180 Annotation: Subtype: Link Rect: 190, 648, 197, 657 ActionDest: 180 Annotation: Subtype: Link Rect: 511, 622, 518, 631 ActionDest: 180 Annotation: Subtype: Link Rect: 521, 623, 533, 631 ActionDest: 180 Annotation: Subtype: Link Rect: 530, 572, 542, 581 ActionDest: 180 Annotation: Subtype: Link Rect: 372, 522, 384, 531 ActionDest: 180 Annotation: Subtype: Link Rect: 384, 522, 396, 531 ActionDest: 180 Annotation: Subtype: Link Rect: 237, 484, 249, 493 ActionDest: 183 Annotation: Subtype: Link Rect: 320, 434, 332, 443 ActionDest: 183 Annotation: Subtype: Link Rect: 333, 434, 345, 443 ActionDest: 183 Annotation: Subtype: Link Rect: 262, 280, 269, 292 ActionDest: 76 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 399, 470, 406, 482 ActionDest: 76 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 383, 667, 390, 679 ActionDest: 112 Annotation: Subtype: Link Rect: 491, 617, 498, 629 ActionDest: 112 Annotation: Subtype: Link Rect: 481, 604, 488, 616 ActionDest: 112 Annotation: Subtype: Link Rect: 234, 592, 241, 604 ActionDest: 112 Annotation: Subtype: Link Rect: 427, 468, 439, 477 ActionDest: 183 Annotation: Subtype: Link Rect: 487, 455, 499, 464 ActionDest: 183 Annotation: Subtype: Link Rect: 378, 388, 385, 402 ActionDest: 112 Page: Sequence: 5 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 361, 708, 368, 720 ActionDest: 148 Annotation: Subtype: Link Rect: 454, 115, 466, 124 ActionDest: 183 Annotation: Subtype: Link Rect: 366, 77, 378, 86 ActionDest: 183 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 544, 736, 556, 744 ActionDest: 183 Annotation: Subtype: Link Rect: 302, 635, 314, 644 ActionDest: 183 Annotation: Subtype: Link Rect: 314, 635, 326, 644 ActionDest: 183 Annotation: Subtype: Link Rect: 327, 635, 339, 644 ActionDest: 183 Annotation: Subtype: Link Rect: 353, 597, 365, 606 ActionDest: 183 Annotation: Subtype: Link Rect: 370, 484, 382, 493 ActionDest: 183 Annotation: Subtype: Link Rect: 383, 484, 395, 493 ActionDest: 183 Annotation: Subtype: Link Rect: 312, 409, 324, 418 ActionDest: 183 Annotation: Subtype: Link Rect: 531, 346, 543, 355 ActionDest: 183 Annotation: Subtype: Link Rect: 544, 346, 556, 355 ActionDest: 183 Annotation: Subtype: Link Rect: 200, 309, 212, 318 ActionDest: 183 Annotation: Subtype: Link Rect: 321, 284, 328, 293 ActionDest: 180 Annotation: Subtype: Link Rect: 442, 170, 454, 180 ActionDest: 183 Annotation: Subtype: Link Rect: 457, 170, 469, 180 ActionDest: 183 Annotation: Subtype: Link Rect: 509, 70, 521, 79 ActionDest: 183 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 544, 647, 556, 657 ActionDest: 183 Annotation: Subtype: Link Rect: 544, 635, 556, 644 ActionDest: 183 Annotation: Subtype: Link Rect: 443, 597, 455, 606 ActionDest: 183 Annotation: Subtype: Link Rect: 417, 547, 429, 556 ActionDest: 183 Annotation: Subtype: Link Rect: 222, 422, 234, 431 ActionDest: 183 Annotation: Subtype: Link Rect: 442, 371, 449, 380 ActionDest: 180 Annotation: Subtype: Link Rect: 449, 371, 456, 380 ActionDest: 180 Annotation: Subtype: Link Rect: 277, 321, 289, 330 ActionDest: 183 Annotation: Subtype: Link Rect: 389, 296, 401, 305 ActionDest: 183 Annotation: Subtype: Link Rect: 404, 296, 416, 305 ActionDest: 186 Annotation: Subtype: Link Rect: 350, 146, 361, 154 ActionDest: 186 Annotation: Subtype: Link Rect: 200, 95, 211, 104 ActionDest: 186 Annotation: Subtype: Link Rect: 214, 95, 226, 104 ActionDest: 186 Annotation: Subtype: Link Rect: 544, 83, 556, 92 ActionDest: 186 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 273, 735, 285, 744 ActionDest: 186 Annotation: Subtype: Link Rect: 404, 710, 415, 719 ActionDest: 186 Annotation: Subtype: Link Rect: 416, 710, 427, 719 ActionDest: 186 Annotation: Subtype: Link Rect: 369, 635, 381, 644 ActionDest: 186 Annotation: Subtype: Link Rect: 381, 635, 393, 644 ActionDest: 186 Annotation: Subtype: Link Rect: 480, 610, 492, 619 ActionDest: 186 Annotation: Subtype: Link Rect: 212, 560, 224, 569 ActionDest: 186 Annotation: Subtype: Link Rect: 224, 560, 236, 569 ActionDest: 186 Annotation: Subtype: Link Rect: 428, 509, 440, 519 ActionDest: 186 Annotation: Subtype: Link Rect: 209, 472, 220, 481 ActionDest: 186 Annotation: Subtype: Link Rect: 221, 472, 233, 481 ActionDest: 186 Annotation: Subtype: Link Rect: 417, 434, 429, 443 ActionDest: 186 Annotation: Subtype: Link Rect: 429, 434, 441, 443 ActionDest: 186 Annotation: Subtype: Link Rect: 270, 384, 282, 393 ActionDest: 186 Annotation: Subtype: Link Rect: 283, 384, 295, 393 ActionDest: 186 Annotation: Subtype: Link Rect: 192, 309, 204, 318 ActionDest: 186 Annotation: Subtype: Link Rect: 207, 309, 219, 318 ActionDest: 186 Annotation: Subtype: Link Rect: 246, 283, 258, 293 ActionDest: 186 Annotation: Subtype: Link Rect: 351, 246, 363, 255 ActionDest: 186 Annotation: Subtype: Link Rect: 363, 246, 375, 255 ActionDest: 186 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 395, 507, 402, 519 ActionDest: 167 Annotation: Subtype: Link Rect: 366, 459, 378, 468 ActionDest: 183 Annotation: Subtype: Link Rect: 320, 444, 327, 456 ActionDest: 171 Annotation: Subtype: Link Rect: 444, 434, 451, 443 ActionDest: 180 Page: Sequence: 11 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 356, 237, 368, 246 ActionDest: 186 Annotation: Subtype: Link Rect: 383, 99, 395, 108 ActionDest: 189 Annotation: Subtype: Link Rect: 396, 99, 408, 108 ActionDest: 189 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 262, 596, 274, 605 ActionDest: 189 Annotation: Subtype: Link Rect: 206, 496, 218, 505 ActionDest: 189 Annotation: Subtype: Link Rect: 204, 231, 216, 241 ActionDest: 183 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 296, 723, 308, 732 ActionDest: 189 Annotation: Subtype: Link Rect: 421, 657, 560, 669 Annotation: Subtype: Link Rect: 165, 645, 192, 657 Annotation: Subtype: Link Rect: 536, 156, 560, 167 Annotation: Subtype: Link Rect: 165, 144, 399, 155 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 339, 645, 421, 656 Annotation: Subtype: Link Rect: 149, 407, 187, 418 Annotation: Subtype: Link Rect: 193, 407, 229, 418 Annotation: Subtype: Link Rect: 365, 395, 403, 406 Annotation: Subtype: Link Rect: 128, 361, 165, 372 Annotation: Subtype: Link Rect: 171, 361, 208, 372 Annotation: Subtype: Link Rect: 250, 338, 287, 349 Annotation: Subtype: Link Rect: 519, 327, 557, 337 Annotation: Subtype: Link Rect: 59, 315, 95, 326 Annotation: Subtype: Link Rect: 59, 292, 96, 303 Annotation: Subtype: Link Rect: 270, 257, 307, 268 Annotation: Subtype: Link Rect: 201, 234, 238, 245 Annotation: Subtype: Link Rect: 151, 200, 188, 210 Annotation: Subtype: Link Rect: 511, 188, 549, 199 Annotation: Subtype: Link Rect: 237, 165, 275, 176 Annotation: Subtype: Link Rect: 443, 142, 481, 153 Annotation: Subtype: Link Rect: 59, 108, 96, 118 Annotation: Subtype: Link Rect: 102, 108, 138, 118 Annotation: Subtype: Link Rect: 59, 74, 96, 84 Annotation: Subtype: Link Rect: 102, 74, 138, 84 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 467, 722, 505, 733 Annotation: Subtype: Link Rect: 511, 722, 547, 733 Annotation: Subtype: Link Rect: 108, 688, 146, 698 Annotation: Subtype: Link Rect: 367, 664, 405, 675 Annotation: Subtype: Link Rect: 59, 642, 96, 652 Annotation: Subtype: Link Rect: 470, 618, 508, 629 Annotation: Subtype: Link Rect: 267, 596, 305, 606 Annotation: Subtype: Link Rect: 407, 572, 445, 583 Annotation: Subtype: Link Rect: 519, 549, 557, 560 Annotation: Subtype: Link Rect: 59, 538, 95, 548 Annotation: Subtype: Link Rect: 463, 526, 501, 537 Annotation: Subtype: Link Rect: 507, 526, 543, 537 Annotation: Subtype: Link Rect: 250, 503, 287, 514 Annotation: Subtype: Link Rect: 293, 503, 329, 514 Annotation: Subtype: Link Rect: 519, 480, 557, 491 Annotation: Subtype: Link Rect: 59, 469, 95, 479 Annotation: Subtype: Link Rect: 491, 445, 528, 456 Annotation: Subtype: Link Rect: 127, 412, 164, 422 Annotation: Subtype: Link Rect: 59, 389, 96, 399 Annotation: Subtype: Link Rect: 514, 376, 552, 387 Annotation: Subtype: Link Rect: 505, 365, 543, 376 Annotation: Subtype: Link Rect: 59, 343, 96, 353 Annotation: Subtype: Link Rect: 257, 319, 295, 330 Annotation: Subtype: Link Rect: 59, 285, 96, 295 Annotation: Subtype: Link Rect: 102, 285, 138, 295 Annotation: Subtype: Link Rect: 265, 261, 302, 272 Annotation: Subtype: Link Rect: 519, 238, 557, 249 Annotation: Subtype: Link Rect: 59, 228, 95, 238 Annotation: Subtype: Link Rect: 194, 205, 231, 215 Annotation: Subtype: Link Rect: 435, 181, 473, 192 Annotation: Subtype: Link Rect: 479, 181, 515, 192 Annotation: Subtype: Link Rect: 320, 158, 357, 169 Annotation: Subtype: Link Rect: 193, 124, 231, 134 Annotation: Subtype: Link Rect: 323, 100, 361, 111 Annotation: Subtype: Link Rect: 384, 77, 422, 88 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 448, 722, 485, 733 Annotation: Subtype: Link Rect: 391, 687, 429, 698 Annotation: Subtype: Link Rect: 479, 676, 515, 687 Annotation: Subtype: Link Rect: 521, 676, 557, 687 Annotation: Subtype: Link Rect: 148, 653, 184, 664 Annotation: Subtype: Link Rect: 246, 630, 284, 640 Annotation: Subtype: Link Rect: 59, 607, 96, 617 Annotation: Subtype: Link Rect: 457, 583, 494, 594 Annotation: Subtype: Link Rect: 501, 583, 537, 594 Annotation: Subtype: Link Rect: 273, 560, 311, 571 Annotation: Subtype: Link Rect: 317, 560, 353, 571 Annotation: Subtype: Link Rect: 95, 538, 133, 548 Annotation: Subtype: Link Rect: 149, 515, 187, 525 Annotation: Subtype: Link Rect: 264, 491, 301, 502 Annotation: Subtype: Link Rect: 262, 468, 300, 479 Annotation: Subtype: Link Rect: 394, 445, 431, 456 Annotation: Subtype: Link Rect: 437, 445, 473, 456 Annotation: Subtype: Link Rect: 240, 423, 277, 433 Annotation: Subtype: Link Rect: 284, 423, 320, 433 Annotation: Subtype: Link Rect: 502, 399, 540, 410 Annotation: Subtype: Link Rect: 428, 377, 465, 387 Annotation: Subtype: Link Rect: 472, 377, 508, 387 Annotation: Subtype: Link Rect: 224, 342, 261, 353 Annotation: Subtype: Link Rect: 155, 319, 193, 330 Annotation: Subtype: Link Rect: 388, 296, 425, 307 Annotation: Subtype: Link Rect: 240, 261, 278, 272 Annotation: Subtype: Link Rect: 452, 238, 490, 249 Annotation: Subtype: Link Rect: 495, 227, 533, 238 Annotation: Subtype: Link Rect: 430, 204, 467, 215 Annotation: Subtype: Link Rect: 473, 204, 509, 215 Annotation: Subtype: Link Rect: 404, 181, 442, 192 Annotation: Subtype: Link Rect: 448, 181, 484, 192 Annotation: Subtype: Link Rect: 368, 158, 405, 169 Annotation: Subtype: Link Rect: 411, 158, 447, 169 Annotation: Subtype: Link Rect: 440, 135, 477, 146 Annotation: Subtype: Link Rect: 483, 135, 519, 146 Annotation: Subtype: Link Rect: 372, 112, 409, 123 Annotation: Subtype: Link Rect: 415, 112, 451, 123 Annotation: Subtype: Link Rect: 346, 89, 383, 100 Annotation: Subtype: Link Rect: 182, 67, 219, 77 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 161, 722, 199, 733 Annotation: Subtype: Link Rect: 273, 699, 311, 710 Annotation: Subtype: Link Rect: 486, 676, 523, 687 Annotation: Subtype: Link Rect: 108, 642, 146, 652 Annotation: Subtype: Link Rect: 152, 642, 188, 652 Checksum: 88139f8f Type: CRC32 Checksum: 238dae73e61662eb82b10cde38524b82 Type: MD5 Checksum: c4e17900c7cabd0a5e08fd323f89f28731ab3b1f Type: SHA-1