? ? ? Defining the Porcine Colostral Proteome: Changes in the Array of Proteins from Colostrum to Mature Milk by Alejandro J. Silva A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Masters of Science Auburn, Alabama December 12 th , 2011 Keywords: Pig, Uterus, Milk, Proteome Approved by Frank F. Bartol, Chair, Alumni Professor of Anatomy, Physiology and Pharmacology Terry D. Brandebourg, Assistant Professor of Animal Science Douglas C. Goodwin, Associate Professor of Chemistry and Biochemistry Dwight F. Wolfe, Professor of Clinical Sciences ? ? ? ii Abstract The?importance?of?colostrum?(first?milk)?for?immunological?and? nutritional?support?of?newborn?mammals?is?well?established.?Many?bioactive? peptides?are?present?in?milk?at?higher?concentrations?than?in?maternal? circulation.??Transmission?of?such?factors?from?mother?to?offspring?as?a?specific? consequence?of?nursing?is?characterized?as?lactocrine?communication.??Because? milk?borne,?lactocrine?acting?factors?affect?patterns?of?gene?expression?in? neonatal?somatic?tissues,?including?the?reproductive?tract?and?heart,?it?is? important?to?understand?the?biochemical?nature?of?colostrum/milk.??Data?for? relaxin,?a?prototypical?lactocrine?acting?factor?in?porcine?colostrum,?indicate?that? transmission?of?such?factors?is?significant?prior?to?gut?closure?in?the?neonatal?pig.? Additionally,?amino?acid?sequences?encoding?potentially?bioactive?peptides?are? encrypted?within?porcine?milk?proteins,?raising?the?possibility?that?proteolytic? cleavage?in?the?gut?could?release?such?factors?into?circulation?after?consumption.? The?array?of?proteins/peptides?constituting?the?porcine?colostral?proteome?has? not?been?defined.??Objectives?of?this?study?were?to?employ?two?dimensional?gel? electrophoresis?(2DE)?and?image?analysis?to:?(1)?define?the?porcine?colostral? proteome?on?lactation?day?(LD)?0;?and?(2)?determine?if?and?how?this?proteome? changed?from?LD?0?to?LD?6.?Colostrum?(LD?0)?and?milk?(LD?6)?samples?were? obtained?from?six?lactating?sows.??Protein?was?extracted?from?individual?samples? ? iii and?total?protein?concentrations?were?determined.??Extracted?proteins?were? prepared?for?and?subjected?to?both?standard?SDS?PAGE?(10%?total?monomer)? and?2DE.??For?2DE,?first?dimension?separations?were?carried?out?using?pH?3?10? immobilized?pH?gradient?strips?followed?by?SDS?PAGE?using?gradient? polyacrylamide?gels?(10?20%?total?monomer).?Individual?samples?were?run?on? duplicate?2DE?gels?and?stained?with?Sypro?RUBY.??Digital?images?of?individual? gels?were?obtained?and?analyzed?using?a?Typhoon?9400?digital?scanner?and? PDQuest?2?D?Analysis?Software.?Total?protein?concentrations?for?colostrum?(LD? 0)?and?milk?(LD?6)?were?8.5?mg/ml?and?8.3?mg/ml.?Standard?SDS?PAGE?analyses? revealed?distinct?differences?in?the?distribution?of?protein?bands?between?LD?0? and?LD?6.??PDQuest?analyses?identified?consistent?qualitative?and?quantitative? differences?between?colostrum?from?LD?0?and?milk?from?LD?6.??Systematic? analyses?of?the?primary?amino?acid?sequences?of?porcine?milk?proteins?using?the? BIOPEP?program?and?related?database? (www.uwm.edu.pl/biochemia/index_en.php) revealed that potentially bioactive peptides are encrypted within porcine milk proteins. Thus,?newborn?pigs?that?nurse? obtain?a?complex?mixture?of?proteins?and?peptides?from?birth?that?changes?with? time?during?a?period?of?neonatal?life?recognized?to?be?critical?for?female? reproductive?tract?development.??Gilts?deprived?of?colostrum?for?two?days?from? birth?exhibit?altered?gene?expression?patterns?essential?for?normal?development? of?female?reproductive?tract?tissues.?The?presence?of?encrypted?peptides?in? porcine?colostrum?increases?the?complexity?of?the?porcine?milk?proteome.??These? studies?provide?a?framework?for?future?efforts?to?be?aimed?at?identification?of? ? iv colostral?proteins?and?peptides?that?affect?lactocrine?programming?of?neonatal? development.? ? v Acknowledgements I would like to thank my friends and family for their support in the past three years. I would also like to thank and acknowledge my coworkers in the Bartol/Bagnell research labs including Dori Miller, Meghan Davolt, Elizabeth Talley, Dr. Amy Frankshun, Dr. Joseph Chen, and Anne Wiley. Also I extend special thanks and appreciation to Dr. Carol Bagnell and my mentor, Dr. Frank Bartol, not only for their help and support with this project, but also in helping me develop analytical and intellectual skills that I will use in my next endeavors. Finally I would like to thank Dr. Terry Brandebourg, Dr. Dwight Wolfe, and Dr. Douglas Goodman for their valuable feedback. ? vi Table of Contents ? Abstract.........................................................................................................................ii Acknowledgements........................................................................................................v List of Tables...............................................................................................................ix List of Figures................................................................................................................x List of Abbreviations..................................................................................................xii Chapter 1: Introduction .................................................................................................1 Chapter 2: Literature Review.........................................................................................3 1. Evolution of Lactation ......................................................................................... 1.1 Origins of Lactation, Mammary Glands, and Suckling ............................4 1.2 Conservation of Milk and Mammary Genes .............................................6 2. Lactation Functions .............................................................................................8 2.1 Ontogeny of the Mammary Gland ........................................................... 2.2 Lactogenesis and Regulation of Milk Secretion ......................................9 2.3 Absorption of Intact Macromolecules ...................................................10 3. Composition of Porcine Colostrum/Milk .........................................................13 3.1 Proteins .................................................................................................. 3.1.1 Anti-infection Agents...................................................................14 3.1.2 Growth Factors and Peptide Hormones .....................................17 3.2 Carbohydrates and Lipids ........................................................................20 ? vii 3.3 MicroRNAs ..............................................................................................22 3.4 Steroids ....................................................................................................23 3.4.1 Endocrine Disruptors ..................................................................25 4. Bioactive Encrypted Peptides ............................................................................27 5. Colostrum/Milk as a Conduit for Developmental Signals..................................31 5.1 Lactocrine Regulation of Porcine FRT Development .............................33 5.2 Evidence for Lactocrine Regulation of Marsupial Development ............35 6. Breastfeeding and Long-Term Effects ...............................................................39 7. Summary and Implications ................................................................................41 Chapter 3: Defining the Porcine Colostral Proteome: ................................................43 1. Abstract .............................................................................................................. 2. Introduction ........................................................................................................45 3. Materials and Methods .......................................................................................47 4. Results ................................................................................................................51 5. Discussion ..........................................................................................................70 References ...................................................................................................................74 Appendix A. Decrease Two-Fold from LD 0 .............................................................83 Appendix B. Increase Two- ...............................................................85 C. Milk Collection ......................................................................................88 Appendix D. Milk Preparation: Lipid Removal and Protein Solubilization ...............90 E. RCDC Assay ..........................................................................................91 Appendix F. Sample Preparation: Sequential Extraction 3 ........................................93 G. Sample Preparation: 2D Clean-Up Kit ..................................................94 ? viii Appendix H. Isoelectric Focusing ...............................................................................96 I. Equilibration and 2D-SDS-PAGE ..........................................................98 Appendix J. 2-D Gel Imaging ...................................................................................102 Appendix K. Gel Analysis: PDQuest ........................................................................103 L. Spot Identification Using Bioinformatic Methods ..............................108 Appendix M. Encrypted Peptides Identification .......................................................119 N. Raw 2D Gel Images.............................................................................111 ? ix List of Tables Table 1. Preliminary identification of spots unique to LD 0 using ExPASy TagIdent tool...............................................................................................................................59 Table 2. Preliminary identification of spots unique to LD 6 using ExPASy TagIdent tool...............................................................................................................................60 Table 3. BIOPEP analysis of common porcine milk proteins and the potential number of bioactive encrypted peptides that can be found within one sequence of the parent protein..........................................................................................................................64 Table 4. BIOPEP analysis showing the potential number of bioactive peptides excised using common proteolytic enzymes.............................................................................66 Table 5. BIOPEP analysis showing the potential number of bioactive peptides excised using combinations of common proteolytic enzymes..................................................68 Table 6. Preliminary spot identification of 53 spots downregulated at least 2-fold from LD 0....................................................................................................................84 Table 7. Preliminary spot identification of 105 spots downregulated at least 2-fold from LD 0....................................................................................................................86 ? x ? ? List of Figures Figure 1. Visual comparison of 2DE master images of porcine colostrum/milk at LD0 and LD 6 for representative sow..................................................................................52 Figure.2. Comparison of 2DE master images of colostrum at LD 0 and milk at LD 6 combining milk samples from all sows on respective days.........................................53 Figure 3. Overall master image showing consensus spots from all gels in both groups (LD 0 and LD 6) ........................................................................................................54 Figure 4. Master image with colored overlays showing qualitative changes between LD 0 and LD 6.............................................................................................................55 Figure 5. Master image with colored overlays showing quantitative changes between LD 0 and LD 6.............................................................................................................56 Figure 6. Master image with qualitative overlays and additional SSP numbers in purple...........................................................................................................................58 Figure 7: Comparison of parent protein size (by number of amino acids) and incidence of unique encrypted peptides occurring exactly three times within one parent protein...............................................................................................................61 Figure 8: Comparison of parent protein size (by number of amino acids) and incidence of unique encrypted peptides occurring greater than 4 times within one parent protein...............................................................................................................62 ? xi ? ? List of Abbreviations AA Arachidonic Acid ACE Angiotensin-Converting Enzyme BAP Bioactive Peptides BSA Bovine Serum Albumin CCP Casein Phosphopeptide DHA Docohexaenoic Acid ESR1 Estrogen Receptor-? EV Estradiol Valerate FITC-D Fluorescub Isothiocyanate Labeled Dextran HMG-CoA Hydroxylmethylglutaryl Coenzyme A HSA Human Serum Albumin IgA Immunoglobulin alpha chain IgG Immunoglobulin gamma chain IGF Insulin-like Growth Factor IGFBP Insulin-like Growth Factor Binding Protein LCPUFA Long-Chain Polyunsaturated Fatty Acid MbFs Milk-borne Factors miRNA micro-RNA MMP Matrix Metalloprotease ? xii Mya Million years ago PND Post-Natal Day RLX Relaxin RXFP-1 Relaxin Receptor SSP Standard Spot Numbers ? 1 CHAPTER 1 INTRODUCTION Lactation is a ubiquitous and critical part of the mammalian reproductive strategy. Milk produced by the mammary gland is generally rich in fats, proteins, water and other nutrients critical for the survival of the newborn [1]. These milk- borne factors are not only nutritionally beneficial; they can also modulate numerous aspects of neonatal development [2, 3]. Additionally, the development of agriculture has enabled humans to consume milk throughout their life span. Thus it becomes necessary to understand the characteristics of lactation and milk, as they are essential parts of the mammalian reproductive strategy and human nutrition. Much of the current research involving milk has focused on the immunological and developmental effects it may have on the neonate, including transfer of antibodies for passive immunity, modulation of gastrointestinal (GI) tract function and other effects on systemic tissues. Neonatal pigs are immunologically naive and their survival rate increases if they are able to nurse and receive critical antibodies to defend against pathogens [4]. Colostrum, or first milk, is particularly high in immunoglobulins and other bioactive proteins produced by the maternal system when the neonatal small intestine is capable of transferring intact macromolecules into circulation. Data for the prototypical milk-borne peptide hormone relaxin indicate that such bioactive factors can affect gene expression in the neonatal porcine uterus, cervix, and other tissues via ? 2 a lactocrine mechanism soon after birth [5]. Delay or disruption of this mechanism leads to altered patterns of gene expression with potential for long lasting consequences. Additionally, bioactive peptides encrypted within the primary sequence of bovine milk proteins have been identified and can affect a variety of organ systems once released into circulation [2]. Milk is also a vector for environmental endocrine disruptors entering neonatal circulation, suggesting that lactocrine signaling can also be affected by environmental conditions [6]. The conserved nature of milk and mammary genes suggests that lactocrine signaling is a conserved mechanism that remains incompletely understood. Research described in this thesis was designed to investigate the nature of the porcine colostral proteome and to determine if potentially bioactive peptides are encrypted within porcine milk proteins. ? 3 CHAPTER 2 LITERATURE REVIEW Lactation is a hallmark of mammalian reproductive physiology. Carl Linnaeus referred directly to the mammary gland in the naming of the class ?Mammalia? as the dominant characteristic by which mammals can be identified despite other major characteristics such as thermoregulation and hair growth [7]. Considerable progress has been made in understanding the physiological and biochemical mechanisms required to support lactation. Following is a review of mammary gland structure and function and a description of the effects of nursing on neonatal development. 1. EVOLUTION OF LACTATION The pathways leading to the establishment of modern lactation systems are uncertain due to the lack of direct fossil evidence for mammary gland development and reliable living counterparts of early mammals. However, a combination of fossil and molecular evidence indicates that mammals first appeared toward the end of the Triassic period approximately 220 Mya [8] and complex lactation systems were already established in the last common ancestor of all modern mammals in the late Triassic period [9]. This implies that lactation evolved in mammalian precursors of the therapsid and cyanodont lineages concurrently with other hallmarks of mammalian physiology such as endothermy and fur growth. Modern mammals evolved to have unique reproductive strategies that are reflected in their approach to ? 4 lactation regarding milk composition and lactation cycles [7, 9]. Nevertheless, genomic analyses revealed the close relationship of mammary and lactation genes between even the most phylogenetically distant mammals, some of which have origins dating as far back as 240 Mya [9, 10] 1.1 ORIGINS OF LACTATION, MAMMARY GLANDS, AND SUCKLING ?Lactation?involves?the?coordination?of?complex?physiological,?ecological,? morphological,?and?biochemical?factors?[7].?Modern?mammalian?neonates?are? instinctually?driven?to?nurse?soon?after?birth.??The?mammary?gland?most?likely? evolved?alongside?other?hallmarks?of?mammalian?physiology?in?the?therapsid,? cyanodont,?and?mammaliforme?lineages?of?the?Triassic?period?[11].?Histological? similarities?between?mammary?glands?and?skin?glands?led?to?several?hypotheses? postulating?that?apocrine?and?sebaceous?glands?were?the?precursors?to? mammary?gland?development?[9,?11].?Blackburn?and?colleagues?suggested?the? predominant?theory?that?mammary?gland?formation?predated?full?differentiation? of?cutaneous?glands?and?may?have?evolved?as?a??neomorphic?mosaic??involving? both?cutaneous?and?apocrine?glands?[11].?This?combination?of?skin?glands?may? have?formed?the?first?primitive?mammary?gland?able?to?secrete?small?amounts?of? organic?substances?similar?to?its?precursors.?Another?theory?points?to?evidence? that?the?mammary?gland?may?have?evolved?from?an?association?between? apocrine?glands?and?hair?follicles,?the?remnants?of?which?can?be?seen?in?some? modern?day?mammals?[9].?In?monotremes?the?mammary?gland?lacks?a?nipple? and?opens?directly?into?the?areola?in?association?with?a?hair?follicle?[12].?This? ? 5 relationship?is?also?observed?in?the?ontogeny?of?the?mammary?gland?of? marsupials,?where?a?transient?association?is?observed?between?the?mammary? duct?and?hair?follicles.?Direct?association?between?mammary?gland?formation? and?hair?follicles?has?not?been?detected?in?eutherian?mammals,?but?there?is?active? repression?of?hair?follicle?formation?in?the?region?of?the?mammary?anlagen?[13].? Though?the?exact?mechanism?of?mammary?gland?evolution?is?unclear,?it?likely? formed?through?cooption?of?existing?structures?and?related?biochemical? pathways.?This?theory?would?explain?the?heavily?conserved?nature?of?milk?and? mammary?genes,?reviewed?in?the?next?section.? Numerous?scenarios?have?been?offered?to?explain?the?origins?of?lactation? and?suckling?behavior.?One?of?the?earliest?to?propose?a?mechanism?for?the?origin? of?lactation?was?Charles?Darwin,?who?proposed?that?brood?pouches?used?by? certain?fish?to?nourish?their?young?might?be?the?intermediate?form?of?a? mammary?gland?and?lactation?[14].?However,?it?is?now?clear?that?the?mammary? gland?did?not?evolve?from?a?piscine?brood?pouch?format.??Prevention?of?egg? desiccation?has?been?proposed?as?a?theory,?but?observations?on?monotreme? lactation?produced?conflicting?evidence?on?whether?mammary?secretions?are? necessary?to?protect?eggs?from?desiccation?[7,?15].??Others?proposed?that? apocrine?secretions?helped?egg?survivability?through?thermoregulation?by? evaporative?cooling?and?heat?transfer?[16].?Graves?and?Duval?[17]?suggested?that? pheromones?of?reptilian?like?mammals?millions?of?years?ago?may?have?had?a?role? in?attracting?offspring?to?the?mother?in?order?to?enhance?survival?and?that?these? or?other?secretions?could?have?evolved?to?have?a?nutritional?component.??The? ? 6 presence?of?antimicrobial?peptides?and?other?immuno?enhancing?proteins?in? milk?led?some?to?hypothesize?that?early?lacteal?secretions?were?for?the? immunological?benefit?of?the?neonate?[11],??similar?to?amphibian?secretion?of? antimicrobial?and?antitumor?peptides?on?their?skin?[18].??Antimicrobial?factors? may?have?incorporated?lysozyme,???lactalbumin,?and?other?iron?binding?peptides? that?are?nearly?universal?in?the?milk?of?modern?day?mammals?[19].?These?factors? were?shown?to?predate?the?emergence?of?lactose?as?the?primary?disaccharide?in? milk,?since???lactalbumin?is?one?of?the?functional?subunits?of?lactose?synthase? [19].?Thus?lactose?production?in?proto?mammals?could?only?have?begun?once? high?levels?of???lactalbumin?were?present?in?lacteal?secretions.?Though?the? origins?of?lactation?are?unclear,?it?is?clear?that?the?first?mammal?had?a?complex? lactation?system?and?differentiated?mammary?glands?approximately?220?Mya? [7].?? ? 1.2?CONSERVATION?OF?MILK?AND?MAMMARY?GENES? ?The?development?of?gene?sequencing?technology?allowed?for?comparison? of?milk?and?mammary?genes?from?various?species?[10].?Analysis?of?bovine?milk? genes?revealed?that?milk?genes?coding?for?immunoglobulin,?casein,?fibrinogen,? and?milk?fat?membrane?proteins?(MFMPs)?tend?to?be?clustered?with?mammary? genes?[10].??Co?expressed?genes?and?those?that?are?more?phylogenetically? conserved?are?found?in?paired?or?triplet?clusters?across?the?genome,?indicating? that?they?are?evolutionarily?related??[20].?Furthermore,?a?higher?number?of? bovine?milk?protein?orthologs?were?found?in?other?mammals?than?would? ? 7 normally?be?expected?compared?to?gene?orthologs?not?involved?in?lactation.?Most? of?the?highly?conserved?proteins?were?found?in?the?milk?fat?membrane?proteome,? which?generally?does?not?contribute?more?than?5%?of?total?protein?in?milk?[10,? 21].?Milk?protein?gene?loss?is?minimal?compared?to?the?rest?of?the?genome,?but? there?are?considerable?gene?copy?variations?that?may?contribute?to?the?diversity? of?milk?composition?across?species?[10].? ?Caseins,?a?class?of?major?milk?proteins,?are?universally?expressed?in? mammalian?milk.?Pigs?express?four?different?casein?genes,?including?three? calcium?sensitive?caseins?and?one???like?casein?to?aid?in?the?solubilization?of? other?caseins?[22].??The?ubiquity?of?casein?suggests?that?the?ancestral?precursor? to?casein?existed?before?mammalian?radiation?over?300?Mya.?The?predominant? theory?on?the?evolution?of?caseins?suggests?that?it?developed?alongside?other? ancient?antimicrobial?milk?proteins?such?a?lactoferrin,?lysozyme,?and? immunoglobulins?as?a?method?for?protecting?the?neonate?against?microbial? threats?[11].??An?evolutionary?relationship?between?caseins?was?found?in?the? casein?gene?cluster?region,?where?other?genes?involved?in?mineral?homeostasis? and?host?defense?are?also?found.?These?genes?are?expressed?in?both?mammary? and?salivary?glands,?which?share?developmental?and?morphological?properties? [23].?Genomic?sequencing?of?casein?genes?from?various?eutherian?and?non? eutherian?mammals?revealed?a?high?degree?of?divergence?in?coding?[10,?22].? Caseins?are?the?most?divergent?milk?borne?proteins?with?an?average?pairwise? percent?identity?(calculated?from?the?human?casein?orthologue?against?7? mammalian?species)?of?<54%?[10].?Nevertheless,?the?conserved?function?of? ? 8 casein?genes?across?mammals?suggests?that?coding?differences?in?their?amino? acid?sequences?does?not?impair?their?nutritional?and?immunological?functions.?? Further?analysis?of?the?casein?gene?clusters?showed?that?the?organization?and? orientation?of?these?genes?is?similar?in?placental?mammals?[22].? ? 2.?LACTATION?FUNCTIONS? ? 2.1?ONTOGENY?OF?THE?MAMMARY?GLAND? ?Porcine?mammary?glands?undergo?a?coordinated?morphological? progression?very?similar?to?human?mammary?gland?development?[24].? Mammary?glands?arise?from?two?lateral?lines?of?thickened?epidermal?tissue? known?as?the?mammary?ridge?on?the?developing?embryo?[25].?Cells?of?the? mammary?ridge?begin?to?push?inwards?forming?the?primary?mammary?bud.?As? the?embryo?develops,?the?primary?mammary?bud?lengthens?and?branches?out? away?from?the?epithelium.?Towards?the?end?of?gestation?these?branches?canalize? to?form?the?lactiferous?ducts,?which?eventually?empty?to?the?exterior?of?the? mammary?gland?[25].?The?mammary?gland?differentiates?further?during?puberty? under?the?influence?of?estrogen,?progesterone,?prolactin,?and?growth?hormone? [25].??These?hormones?are?responsible?for?the?increased?radial?growth?and? branching?of?the?mammary?ducts?in?this?period.?The?basic?unit?of?the?mammary? gland?is?the?sphere?shaped?alveolus,?composed?of?a?central?lumen?surrounded?by? a?single?layer?of?secretory?epithelial?cells?and?another?layer?of?myoepithelial?cells? and?capillaries.?Alveolar?formation?at?the?terminal?portion?of?each?duct?is? ? 9 upregulated?by?progesterone?produced?during?the?luteal?phase?of?estrous?cycles.? Complete?mammary?gland?development?takes?place?in?the?last?trimester?of? pregnancy,?specifically?between?the?75 th ?and?90 th ?day?of?gestation?in?gilts?[26],? characterized?by?a?decrease?in?alveolar?epithelial?tissue?combined?with?a? proportional?reduction?in?adipose?and?connective?tissues.?? ? 2.2?LACTOGENESIS?AND?REGULATION?OF?MILK?SECRETION? Towards?the?end?of?pregnancy,??alveoli?are?separated?into?lobes?that? eventually?empty?into?the?mammary?ducts?[25].?The?secretory?epithelial?cells?are? positioned?such?that?the?apical?end?is?positioned?next?to?the?lumen?and?the?basal? end?is?separated?from?blood?and?lymph?by?the?basement?membrane?[1].?These? cells?synthesize?milk?with?components?entering?from?the?blood?through?the? basement?membrane,?packaged?by?the?Golgi?apparatus,?and?excreted?via? exocytosis?through?apical?membrane?into?the?lumen.?Prolactin?secretion?from? the?anterior?pituitary?plays?a?major?role?in?the?onset?and?maintenance?of? lactation?in?the?sow?[1].?Prolactin?concentration?in?the?blood?of?pregnant?sows? increases?from?~25?ng/ml?three?days?before?parturition?up?to?~?150?ng/ml?on? the?day?of?farrowing?[27].?When?prolactin?secretion?is?inhibited?by? bromocryptine?during?late?pregnancy,?plasma?prolactin?concentrations?fall? below?2?ng/ml?and?the?onset?of?lactogenesis?is?delayed?[28].?Lactogenesis?is? characterized?by?two?phases?in?the?pig.?The?first?stage?is?the?gradual? accumulation?of?colostrum?between?the?90 th ?and?105 th ?day?of?gestation?[26].?The? second?stage?is?characterized?by?copious?milk?secretion?associated?with? ? 10 increasing?lactose?synthesis?by?the?mammary?gland.??This?results?in?an?increased? lactose?concentration?in?milk?and?maternal?blood?which?peaks?during?the?hours? prior?to?parturition?[29].? ?Milk?ejection?or??milk?letdown??is?primarily?a?neuroendocrine?reflex? involving?both?sensory?neurons?in?the?teats?as?wells?as?nontactile?stimulation? such?as?the?sight?and?sound?of?the?neonate.??Teat?stimulation?causes?a?nerve? impulse?to?travel?from?the?nipple?to?the?hypothalamus?where?it?stimulates? oxytocin?synthesizing?neurons?of?the?paraventricular?and?supraoptic?nuclei.? Oxytocin?is?released?into?the?bloodstream?where?it?can?bind?to?oxytocin? receptors?on?the?surface?of?myoepithelial?cells?surrounding?the?alveoli.?These? cells?contract?and?force?the?milk?stored?in?the?lumen?of?the?alveoli?into?the?larger? ducts?closer?to?the?teat?where?a?suckling?piglet?has?access?[1,?25].? ? 2.3.?ABSORPTION?OF?INTACT?MACROMOLECULES? ?The?ability?of?neonatal?intestinal?cells?to?absorb?whole?macromolecules? and??transport?them?intact?across?the?epithelium?into?circulation?is?a?unique? characteristic?of?intestinal?development?in?farm?animals,?including?the?neonatal? pig?[30].?Though?the?exact?time?the?porcine?gut?is?open?to?macromolecule? absorption?varies,?intestinal?closure?begins?6?12?hours?after?colostrum?ingestion? and?progresses?to?completion?by?24?36?hours[25,?30].?Such?transfer?of? macromolecules?facilitates?the?uptake?of?proteins?such?as?immunoglobulins,? growth?factors,?and?bioactive?compounds?found?in?milk?necessary?for?proper? neonatal?development.??Transport?involves?enterocytes?and?M?cells?involved?in? ? 11 non?receptor?passage?of?intact?macromolecules?[31].??More?broadly,?the?transfer? process?can?follow?two?pathways:?1)?specific?receptor?mediated?transocytosis? and?2)?nonspecific?transocytosis?mediated?by?nonselective?vesicular?transport? [31].??Non?specific?macromolecule?absorption?is?significant?for?the?first?two?days? in?neonatal?ungulates,?where?immunoglobulins?compete?with?other?proteins?for? absorption.?This?mechanism?is?facilitated?by?decreased?proteolytic?degradation? due?to?the?presence?of?colostral?protease?inhibitors?as?well?as?low?pancreatic?and? intestinal?enzyme?activities?[31,?32].?Gut?closure?occurs?when?the?intestinal? epithelium?matures?in?conjunction?with?increased?proteolytic?degradation? within?the?intestinal?lumen.? ?There?are?several?factors?influencing?the?length?of?time?that?the?porcine? intestinal?epithelium?remains?open?to?macromolecule?absorption.?Until?recently,? the?predominant?view?was?that?intestinal?uptake?capacity?is?due?primarily?to?an? immature??leaky?gut???that?allows?transport?of?all?molecules?[33].??More?recent? evidence?suggests?that?signals?inducing?gut?closure?involve?colostral?and? systemic?factors?that?influence?the?rate?of?maturation?of?intestinal?epithelium? [34].??Additionally,?diet?and?conditions?of?parturition?have?been?inplicated?as? modulators?of?gut?closure.?Multiple?studies?showed?that?artificial?rearing?of? newborn?pigs?with?hormone?free?milk?replacer?decreased?capacity?for?whole? macromolecule?absorption?[34,?35].?A?study?conducted?by?Jensen?and?colleagues? [35]?showed??that?neonatal?pigs?have?a?reduced?capicity?to?absorb?bovine??and? human?serum?albumin?and?IgG?if?they?are?fed?milk?replacer?or?bovine?colostrum? [35].?Piglets?maintained?on?mik?replacer?also?exhibited?delayed?gut?closure.?? ? 12 Thus,?milk?replacer?is?adequate?to?induce?normal?intestinal?growth?[31,?36].?? Collectively,?data?support?the?idea?that?there?are?factors?in?colostrum?necessary? to?support?normal?patterns?of?macromolecule?absorption?across?the?neonatal? gut.? ?Several?systemic?factors?have?been?implicated?in?regulation?of?gut?closure.? Cortisol?was?shown?to?stimulate?macromolecule?transport?across?the?newborn? small?intestine?in?farm?animals?[34].?Interestingly?the?neonatal?porcine?ileum? expresses?high?numbers?of?glucocorticoid?receptors?during?the?suckling?period? that?decrease?to?normal?adult?levels?during?weaning?[37].?The?presence?of? glucocorticoid?receptors?in?the?immature?small?intestine?may?have?regulatory? effects?on?intestinal?transport?of?ions,?amino?acids,?carbohydrates,?lipids,?and? proteins?[31].?Additionally,??intestinal?closure?at?18?hours?after?birth?is? associated?with?an?increase?in?serum?immunoreactive?insulin?levels,?an?effect? absent?in?fasted?piglets.?Exogenous?insulin?injections?decreased?bovine?serum? albumin?(BSA)?and?fluorescein?isothiocyanate?labeled?dextran?70,000?absorption? at?12?after?birth?[38].??Since?insulin?is?produced?in?response?to?nursing,? colostrum?may?be?at?least?partly?responsible?for?initiating?events?leading?to?gut? closure.??? ? ? ? ? ? ? 13 3.?COMPOSITION?OF?PORCINE?COLOSTRUM/MILK? ?? The?first?studies?on?porcine?milk?composition?were?published?in?1865?by? von?Gohren?[3].??Porcine?milk?is?a?complex?fluid?composed?of?numerous? constituents?including?proteins,?carbohydrates,?lipids,?steroids,?vitamins,? minerals,?nitrogen?compounds,?and?cells?[1].?Characteristics?of?mammalian?milk,? depend?on?the?stage?of?lactation?from?which?is?it?is?obtained.??Production?of? colostrum,?or?first?milk,?occurs?during?the?periparturient?period?and?ceases? between?24?and?48?hours?after?parturition?in?sows?[3,?25].?Mature?milk?is? synthesized?thereafter?and?production?continues?until?weaning.?Porcine? colostrum?differs?in?protein,?carbohydrate,?and?lipid?composition,?though?the? most?significant?difference?occurs?in?total?protein?and?protein?composition?[1,?3,? 39].?? ? 3.1?PROTEINS? Total?protein?content?varies?between?breeds,?but?porcine?colostrum?can? contain?up?to?three?times?more?proteins?than?mature?milk?[40].?Total?protein? declines?rapidly?after?parturition,?with?protein?content?decreasing?by?50%?in?the? first?12?hours?after?the?first?piglet?is?born?[3].?Major?proteins?in?colostrum?and? milk?are?classified?as?either?caseins?or?whey?proteins,?depending?on?their? physical?and?chemical?properties.??Caseins?make?up?about?8.8%?of?nitrogen? containing?compounds?in?colostrum,?but?increase?to?47.3%?of?the?nitrogen? containing?compounds?in?mature?milk.?Whey?proteins?make?up?91.0?%?of? ? 14 nitrogen?containing?compounds?in?colostrum,?and?about?52.6?%?of?these? compounds?in?mature?milk?[3].?Caseins,?such?as???,??,???,?and????variants?are? suspended?in?milk?in?the?form?of?micelles.?They?are?an?important?source?of? amino?acids?and?calcium?for?suckling?piglets,?though?they?also?have?other? bioactivities.??Additionally,?bioactive?peptides?encoded?within?these?(and?other)? milk?proteins?have?the?potential?to?influence?the?function?of?the?GI?tract?and? other?organ?systems?[1,?22].?Whey?proteins?include?serum?albumin,??? lactalbumin,???lactoglobulin,?immunoglobulins,?growth?factors?and?many?other? peptides?with?variable?bioactivities?[1].?Of?the?proteins?found?in?colostrum,?more? than?90%?are?immunoglobulins?that?provide?passive?immunity?for?newborn? piglets.?Immunoglobulin?concentrations?decrease?in?mature?milk,?though? secretory?IgA?levels?remain?high?relative?to?other?body?fluids?[3].? ? 3.1.1?ANTI?INFECTION?AGENTS?IN?PORCINE?MILK?? Porcine?colostrum?and?milk?contain?a?large?number?of?defense?factors? including?immunoglobulins,?lactoferrin,?lysozymes,?lactoperoxidases,?and? leukocytes.?These?factors?are?crucial?for?developing?the?passive?immunity?in? porcine?neonates.?Neonatal?vitality?has?a?positive?correlation?with?the?degree?of? passive?immunization?and?circulating?anti?infection?agents.?The?most?important? of?these?is?IgG,?which?constitutes?a?key?element?in?host?defense?against? pathogens?[34].?The?porcine?fetus?is?well?protected?from?antigens?by?the? placenta.?However,?the?neonatal?porcine?immune?system?is?immature?at?birth,? offering?little?resistance?to?pathogens.?Unlike?humans,?pigs?do?not?transfer? ? 15 immunoglobulins?through?the?placenta?prior?to?parturition,?thus?the?piglet?is? dependent?on?intestinal?absorption?of?immunoglobulins?and?other?immune? enhancing?factors?that?exist?in?colostrum?for?their?passive?immunity?[30].? There?are?three?types?of?immunoglobulins?in?porcine?colostrum;?IgG,?IgA,? and?IgM?[1,?39].??The?predominant?immunoglobulin?in?colostrum,?IgG?is?absorbed? into?the?neonatal?circulation?where?it?provides?passive?protection?against? immune?challenges.??Some?mammals?exhibit?intestinal?selectivity?to?IgG?because? of?the?similar?concentrations?of?IgG?in?colostrum?and?within?the?plasma?of?piglets? allowed?to?nurse.?[34].?The?intestinal?Fc?receptor?in?the?rodent?was?implicated?in? regulating?IgG?absorption?in?the?suckling?rat,?however?this?receptor?has?yet?to?be? identified?in?the?neonatal?pig?[34,?41].?By?the?third?day?of?lactation?IgA?becomes? the?predominant?immunoglobulin?in?colostrum/milk.?It?is?found?in?the?form?of? secretory?IgA?in?milk,?which?combines?IgA?with?a?glycoprotein.?Secretory?IgA? functions?primarily?in?the?intestinal?lumen?where?it?blocks?adhesion?of? pathogens?and?toxins?to?the?epithelial?surface.?The?largest?immunoglobulin,?IgM? is?detectable?in?low?concentrations?in?milk?throughout?lactation.?It?is?thought?to? serve?a?function?similar?to?that?of?IgA?in?protection?of?the?intestinal?lumen?[42].? Lactoferrin?is?an?iron?binding?glycoprotein?expressed?in?most?biological? fluids?and?is?an?important?part?of?the?immune?system.?Milk?borne?lactoferrin,? identified?in?1960,?is?synthesized?by?mammary?epithelial?cells?[43].?Lactoferrin? concentrations?are?higher?in?porcine?colostrum?than?in?milk?by?four?weeks?after? parturition?[44].?Lactoferrin?was?shown?to?protect?the?body?against?bacteria,? viruses,?fungi,?inflammation?and?even?cancer?[43].?This?milk?protein?functions?by? ? 16 binding?free?iron,?thereby?competing?with?pathogens?to?limit?their?growth.?It?also? binds?directly?to?and?destabilizes?bacterial?and?fungal?membranes?and?activates? the?host?immune?cells?through?nuclear?activation?pathways?[43,?45,?46].? Lactoferrin?contains?an?abundance?of?bioactive?peptides?within?its?primary? sequence?that?may?have?higher?antimicrobial?capabilities?than?the?parent?protein? [47].?These?bioactive?peptides?are?released?and?activated?after?proteolytic? degradation.? Lysozyme,?one?of?the?most?intensively?studied?antibacterial?milk?proteins,? is?also?found?in?mucus,?tears,?saliva,?and?cytoplasmic?granules?[44].?Found? ubiquitously,?it?has?a?close?structural?relationship?with???lactalbumin?[10].? Lysozyme?functions?by?binding?to?peptidoglycans?in?bacterial?cell?walls?and? hydrolyzing?the?glycosidic?bond?that?connects?N?acetylnuramic?acid?with?the? fourth?carbon?atom?of?N?acetylglucosamine?[48].?It?is?stable?in?acid?and?trypsin? solutions?and?is?considered?an?important?part?of?GI?defense?in?suckling?neonates.? However,?its?role?in?the?pig?has?yet?to?be?determined?as?porcine?milk?borne? lysozyme?has?not?been?identified[1].?Lactoperoxidase?is?another?common? bacteriostatic?factor?found?in?milk,?tears,?and?saliva.?It?catalyzes?the?oxidation?of? certain?molecules?with?hydrogen?peroxide?to?generate?reactive?products?with?a? wide?range?of?antimicrobial?activity?[49,?50].?Lactoperoxidase?activity?in?porcine? milk?was?detected?in?both?colostrum?and?milk?36?hours?after?parturition[49].? Though?the?importance?of?this?enzyme?in?neonatal?porcine?GI?defense?is?unclear,? lactoperoxidase?is?important?in?protecting?the?newborn?piglet?against?reactive? ? 17 oxygen?species?created?during?parturition?and?subsequent?adaptation?to?living? outside?the?womb?[50].? ? 3.1.2?GROWTH?FACTORS?AND?PEPTIDE?HORMONES? ?Growth?factors?are?a?heterogeneous?group?of?proteins?that?promote? cellular?growth,?differentiation,?and?may?act?in?a?similar?manner?to?classic? endocrine?hormones?in?circulation?[51].??The?ability?of?porcine?milk?and,?to?a? greater?extent,?colostrum?to?stimulate?gastrointestinal?DNA?and?protein? synthesis?in?the?neonate?is?recognized.?Colostrum?fed?piglets?have?greater? protein?synthesis?in?liver,?kidney,?spleen?and?skeletal?muscle?compared?to?those? fed?mature?milk.??This?indicates?that?colostral?factors?affect?the?growth?of? systemic?tissues?[52].?Porcine?colostrum?and?milk?contains?epidermal?growth? factor?(EGF),?insulin,?insulin?like?growth?factor?I?(IGF?I),?insulin?like?growth? factor?II?(IGF?II),?IGF?binding?proteins,?transforming?growth?factor???(TFG??),? transforming?growth?factor???1?and?2?(?TGF?B1?&?B2),?relaxin,?growth?hormone,? prolactin,?and?numerous?other?peptide?growth?factors?[51,?53].??Colostrum? contains?a?greater?concentration?of?these?growth?factors?compared?to?mature? milk?and?was?shown?to?play?an?important?role?in?support?of?growth?and? development?of?the?neonatal?piglet?[53].?? ?The?EGF?family?of?growth?factors?includes?EGF,?TGF??,?and?TGF???as?the? three?major?proteins?having?a?role?in?development?of?neonatal?mammals?[54].? EGF?is?a?single?chain?polypeptide?of?approximately?6?kDa.??Porcine?colostrum? was?reported?to?contain?up?to?1500?ng/ml?of?EGF,?with?concentrations?declining? ? 18 to?150?250?ng/ml?nine?days?after?parturition?[1,?53].??Additionally,?EGF?receptors? were?identified?in?the?villi?and?crypts?of?the?neonatal?porcine?small?intestine? [53].?Data?for?mice?suggest?that?EGF?has?a?role?in?the?regulation?of?GI?tract? development.?The?influence?of?EGF?on?porcine?systemic?tissues?has?yet?to?be? defined.?Data?for?the?mouse?indicate?that?EGF?conveys?important?regulatory? signals?affecting?development?of?the?liver,?pancreas?and?pulmonary?system?[54].? TGF???is?a?dimeric?protein?of?25?kDa.?Three?isoforms?were?identified?in?porcine? colostrum?and?milk,?with?concentrations?in?colostrum?being?higher?than?those? observed?for?milk?[1].?Recent?studies?in?mice?suggest?that?TGF???is?also?involved? in?intestinal,?hepatic,?cardiac,?and?pulmonary?system?development[1,?54].? However,?the?physiological?function?of?milk?borne?TGF???is?not?well?understood? in?pigs.?TGF???is?found?in?low?levels?in?both?porcine?colostrum?and?milk?and?can? stimulate?mitotic?events?through?the?EGF?receptor?[51].?The?most?likely?role?for? TGF???in?milk?is?as?a?local?regulator?for?GI?function?and?repair?[51].? ?The?insulin?like?family?of?peptides?is?composed?of?four?members?(insulin,? relaxin,?IGF?I,?and?IGF?II).?These?peptides?are?very?closely?related,?with?the?IGFs? sharing?70%?amino?acid?homology?to?proinsulin?and?40%?to?insulin?[1,?51].?The? primary?structures?of?these?proteins?are?heavily?conserved?across?species;?IGF?I? and?IGF?II?are?identical?in?humans,?pigs,?and?cattle?[1].?Porcine?colostrum? contains?all?members?in?high?concentration?with?comparatively?reduced?levels? found?in?mature?milk?[1,?51,?53].?Insulin,?IGF?I,?and?IGF?II?receptors?were? identified?in?the?small?intestine.?Though?relaxin?is?primarily?known?for?its?role?in? ? 19 cervical?softening?prior?to?parturition,?it?also?promotes?mammary?gland? development?and?is?involved?in?uterine?morphogenesis?in?the?pig?[5,?51,?55].? ?Milk?borne?insulin?is?absorbed?from?the?neonatal?GI?tract?in?a?biologically? active?form.?Oral?administration?of?pharmacological?levels?of?insulin?to?the? suckling?rat?and?pig?results?in?hypoglycemia,?indicating?that?insulin?is?absorbed? intact?and?retains?its?biological?activity?[51].?However,?studies?in?pigs?showed? that?insulin?can?also?survive?in?the?lumen?of?the?GI?tract?and?subsequently?act?on? receptors?there?[56].?Newborn?piglets?bottle?fed?formula?containing?60?unit/l?of? insulin?had?higher?levels?of?brush?border?enzyme?activities?than?those?fed? formula?alone?[57].?Svendsen?and?colleagues?[38]?determined?that?milk?borne? insulin?may?be?a?player?in?the?timing?of?gut?closure?of?neonatal?piglets,?as?those? piglets?given?exogenous?insulin?had?a?70%?reduction?of?macromolecular? transport.?They?hypothesized?that?insulin?changes?enterocyte?basement? membrane?proteoglycan?synthesis,?which?enhances?gut?closure.?? ?IGF?I?and?IGF?II?are?similar?peptides?with?a?molecular?weight?of? approximately?7.5?kDa?[1].?They?exist?in?milk?in?association?with?specific?high? molecular?weight?binding?proteins?called?IGF?binding?proteins?(IGFBPs).?IGFBPs? modulate?the?ability?of?IGFs?to?interact?with?target?tissues?and?provide?the?IGFs? with?protection?from?proteolytic?degradation[53].?Porcine?colostrum?can?contain? 500?fold?higher?levels?of?IGF?I?and?IGF?II?compared?to?mature?milk.?[58].?IGFs? and?IGFBPs?are?very?stable?in?acidic?environments.??Also,?type?I?and?II?receptors? were?identified?in?both?mucosal?and?serosal?surfaces?of?the?small?intestine?where? they?stimulate?GI?cell?proliferation.?There?is?relatively?low?absorption?of?ingested? ? 20 IGF?I?or?Long?R 3 IGF?I?in?piglets?[53,?58],?though?several?investigators?dispute? this?assessment?[56,?59].?A?study?conducted?by?Xu?et?al?[59]?indicated?that? labeled?IGF?I?administered?via?orogastric?tube?represents?20%?of?serum?IGF?I?in? newborn?piglets?and?10%?in?three?day?old?piglets,?implying?that?IGF?I?absorption? can?occur?after?gut?closure.?? ?Relaxin?(RLX)?is?a?6?kDa?peptide?hormone?similar?in?structure?to?insulin? and?has?been?detected?in?human,?canine,?rat,?bovine,?and?porcine?milk?[51,?55].? The?concentration?of?RLX?in?milk?over?time?is?species?specific.?Milk?borne?human? RLX?concentrations?are?low?early?in?lactation,?increasing?as?milk?matures?[60].? Conversely,?RLX?concentrations?in?porcine?colostrum?are?high?but?drop?after?the? second?day?of?lactation?[61].?The?role?of?RLX?and?other?milk?borne?bioactive? factors?(MbFs)?on?neonatal?development?has?only?recently?been?explored?in?the? pig.??Ingested?RLX?is?absorbed?into?the?neonatal?circulation?of?dogs?and?pigs? where?it?can?affect?downstream?targets?[61,?62].?Evidence?for?the?pig?shows?that? the?neonatal?uterus?is?RLX?receptor?(RXFP1)?positive?at?birth?and?that?RLX?is important?for?the?proper?development?of?the?porcine?female?reproductive?tract? (FRT)?[5,?63].?A?more?expansive?review?of?porcine?MbFs?and?their?effects?on? mammalian?neonates?is?presented?below.? ? 3.2?CARBOHYDRATES?&?LIPIDS? ?Several?reviews?described?the?relative?nutrient?composition?of?porcine? colostrum?and?milk?[1,?3,?64].?Colostrum?has?a?high?concentration?of?total?solids? including?proteins?and?a?low?concentration?of?carbohydrates,?lipids,?and?ash.?The? ? 21 transition?from?colostrum?to?milk?is?marked?by?a?decline?in?total?protein?(15.7?? 6.4%)?with?a?simultaneous?increase?in?lactose?(3.1?5.5%)?and?fat?(5.0?13.0%)? [3].?Lactose?is?the?predominant?carbohydrate?in?porcine?colostrum?and?milk?[1].? It?is?produced?by?mammary?epithelial?cells?and?secreted?actively?into?the? alveolar?lumen.?Lactose?is?the?major?carbohydrate?in?milk?and?is?one?of?the? factors?that?determines?milk?volume?[64].?Lactase?readily?hydrolyzes?lactose?in? the?neonatal?porcine?small?intestine.?Additionally,?milk?contains?smaller? quantities?of?nucleotide?sugars,?glycolipids,?glycoproteins,?oligosaccharides,?and? monosaccharides?[1].??Some?milk?borne?oligosaccharides?have?protective?effects? against?pathogenic?bacteria?in?the?intestinal?lumen?while?promoting?the?growth? of?beneficial?bacteria.? ?Porcine?milk?fat?is?mainly?composed?of?triglycerides,?though?there?are? smaller?quantities?of?phospholipids,?glycolipids,?cholesterols,?fat?soluble? vitamins,?and?free?fatty?acids?[1].??The?newborn?piglet?does?not?metabolize?free? fatty?acids?very?well?on?its?own?because?tissue?and?liver?concentrations?of? carnitine?are?low.?Carnitine?is?a?compound?that?is?responsible?for?transferring? free?fatty?acids?into?the?mitochondrial?membrane,?thus?playing?an?important?role? in?fat?metabolism.?However,?Kerner?et?al?[65]?showed?that?colostrum?contains?a? high?concentration?(370?nmol/ml)?of?carnitine?while?serum?levels?of?the? compound?increase?dramatically?after?two?days?of?suckling.??They?concluded?that? colostrum?is?the?primary?source?for?carnitine,?suggesting?lactocrine?regulation?of? neonatal?fat?metabolism.? ? ? 22 3.3?microRNAs? ?The?first?microRNA?(miRNA)?was?discovered?in?1993?in?nematode?worms? [66].?The?recent?development?of?deep?sequencing?technologies?accelerated?the? number?of?miRNAs?discovered?in?multiple?species.??These?small,?bioactive?RNA? molecules?display?regulatory?functions?including?cell?differentiation,? developmental?timing,?apoptosis,?cell?proliferation,?metabolism,?transposon? silencing,?and?immunity?[66].?miRNAs?are?20?30?nucleotides?in?length?that?target? mRNA,?thus?they?are?post?transcriptional?regulators.??They?function?as?guide? molecules?for?mRNA?by?binding?to?untranslated?3??region?of?target?RNAs,?which? typically?leads?to?regression?and?exonucleic?mRNA?decay.??Other?types?of? regulation?such?as?transcriptional?activation?and?heterochromatin?formation? may?also?be?important?[66].?Like?most?other?types?of?RNA,?miRNAs?are? transcribed?by?RNA?polymerase?II,?though?a?smaller?subclass?of?miRNAs?are? transcribed?exclusively?by?RNA?polymerase?III?[67].?Although?individual?miRNAs? repress?their?targets?only?moderately,?miRNAs?can?have?broad?effects?because? each?can?have?multiple?targets?[66].? ?A?recent?study?identified?miRNAs?in?human?breast?milk?[68].?The?highest? concentrations?of?miRNAs?were?found?between?days?4?and?240?of?lactation?with? most?relating?to?immune?function.??In?vitro?observations?of?milk?borne?miRNAs? indicate?that?these?molecules?are?resistant?to?RNases?,?may?be?encapsulated?in? microvesicles?and?are?resistant?to?harsh?conditions?[68].??This?suggests?that?they? can?survive?in?the?neonatal?intestinal?lumen?and?could?be?absorbed?into? circulation,?affecting?downstream?targets.?miRNAs?in?humans?were?shown?to? ? 23 increase?T?cell?numbers?and?B?cell?differentiation.?miRNAs?may?also?serve?as?a? vehicle?for?transferring?genetic?material?from?mother?to?offspring.?It?was? estimated?that?1.3?X?10 7 ?copies/liter/day?of?mR181a,?an?immunomodulating? miRNA,?are?transferred?from?mother?to?infant?[68].?There?were?also?hundreds?of? miRNAs?found?in?breast?milk?with?undetermined?function.?While?the?jury?is?still? out?on?this?new?class?of?potential?MbFs,?evidence?of?the?presence?of?miRNAs?in? milk?opens?the?possibility?that?milk?borne?miRNAs?may?have?significant?impact? on?the?growth?and?development?of?the?neonate.? ? 3.4?STEROID?HORMONES? ?Several?different?steroid?hormones?are?present?in?the?milk?and?colostrum? of?a?wide?variety?of?mammals.?Bovine?milk?contains?testosterone,?progesterone,? cortisol,?and?several?different?types?of?estrogens?including?17???estradiol?[69].? All?were?shown?to?consist?of?58?92%?conjugated?(inactive)?forms,?which?render? them?more?easily?water?soluble?for?excretion.?Conjugated?steroids?can?become? activated?upon?exposure?to?bacterial?sulfatases?or?glucouronidases?in?the?GI?tract? [53].??Data?for?steroids?in?porcine?milk?is?less?abundant.?Farmer?et?al?[70]? reported?that?porcine?colostrum?contains?almost?15?ng/ml?of?estrone,?decreasing? to?~2?ng/ml?by?30?hours?after?parturition.?Since?non?conjugated?steroids?can? pass?through?biological?membranes,?it?is?reasonable?to?assume?that?estrogens?in? milk?arise?from?the?ovary.?However,?a?recent?study?showed?that?ovariectomized? sows?can?secrete?17???estradiol?into?circulation?from?their?mammary?glands? [71].??It?has?not?been?determined?whether?17???estradiol?produced?by?the? ? 24 porcine?mammary?gland?is?secreted?into?milk,?though?the?diffusible?nature?of? steroids?makes?this?scenario?likely.?? ?The?roles?of?milk?borne?estrogens?have?yet?to?be?fully?understood.??A? positive?correlation?exists?between?estrogen?levels?in?neonatal?circulation?and? survival?[70].?Estrogen?receptor?(ER)?expression?and?activation?in?the?neonatal? porcine?uterus?is?required?for?adenogenesis?and?uterine?maturation?[72].? Administration?of?17???estradiol?valerate?(EV)?to?neonatal?gilts?promoted? uterine?gland?genesis?by?postnatal?day?(PND)?14?while?administration?of?the? speci?c?antiestrogen?ICI?182,780?retarded?uterine?wall?development?and? inhibited?gland?formation?[72].?Uterine?ER?is?activated?by?MbFs?[55],?a? mechanism?that?will?be?reviewed?in?the?next?section.?Nevertheless,?uterotrophic? effects?of?EV?on?the?neonatal?uterus?were?ultimately?detrimental?to?uterine? development?and?functional?uterine?capacity.?Adult,?primiparous?gilts?exposed?to? EV?for?two?weeks?from?birth?exhibited?abnormal?responses?to?signals?associated? with?the?periattachment?phase?of?early?pregnancy?and?displayed:?1)?reduced? uterine?fluid?protein?content;?2)?abnormal?uterine?growth?response?to?early? pregnancy;?and?3)?altered?endometrial?gene?expression?patterns?compared?to? unexposed?controls?[73,?74].?In?humans,?estrogenic?endocrine?disruptors?found? in?the?environment?can?be?detrimental?to?the?health?and?development?of?the? infant,?and?evidence?indicates?that?these?disruptors?can?enter?neonatal? circulation?by?ingestion?of?compromised?milk?from?the?mother?[75]? ? ? ? 25 3.4.1?ENDOCRINE?DISRUPTORS? ?In?recent?years?there?has?been?considerable?interest?on?environmental? endocrine?disruptors?(EDs)?and?their?effects?on?humans,?domestic?animals,?and? wildlife?[75].?The?Environmental?Protection?Agency?defines?EDs?as??exogenous? agents?that?interfere?with?the?production,?release,?transport,?metabolism,? binding,?action,?or?elimination?of?natural?hormones?in?the?body?responsible?for? the?maintenance?of?homeostasis?and?the?regulation?of?developmental?processes?? [6].?These?compounds?are?diverse?in?their?structure?and?can?be?found?in?water,? soil,?air,?food,?household?products,?and?packing?material?[75].?Common? endocrine?disruptors?include?bisphenol?A?(BPA)?and?other?phthalates,?paraben? compounds,?polychlorinated?biphenols,?pesticides,?herbicides,?heavy?metals,?and? many?others?[6].?Particular?attention?has?been?paid?to?estrogenic?EDs?that?mimic? the?effects?of?endogenous?estrogen?once?they?are?in?circulation.?Biologically? active?xenoestrogens?can?freely?cross?cellular?plasma?membranes?and?target? cytoplasmic?estrogen?receptors,?ER??(ESR1)?and?ER??(ESR2),?where?they?can? induce?transcription?[75].?Exposure?to?these?xenoestrogens?may?have?significant? consequences?in?reproductive?growth?and?sexual?differentiation.? ?Milk?iswell?recognized?as?a?potential?sink?for?toxic?substances?and? endocrine?disruptors?[75].??Adipose?tissue?is?a?reservoir?for?lipophilic?EDs?such? as?xenoestrogens?and?fat?deposits?in?breast?tissue?were?used?to?monitor?ED? levels?in?peripheral?adipose?tissue?during?pregnancy?and?lactation.??Not? surprisingly,?multiple?xenoestrogenic?compounds?are?found?in?milk?since?these? compounds?are?able?to?diffuse?into?the?alveolar?lumen?from?circulation?[75].? ? 26 ?Zearalenone?(ZEA),?an?estrogenic?mycotoxin?produced?predominantly?by? fungi?in?the?genus?Fusarium,?is?the?most?common?ED?affecting?swine?[76].?Fungi? producing?ZEA?contaminate?maize?and?other?cereals?in?the?field,?but?it?can?also? grow?after?harvesting?if?the?cereals?are?not?handled?correctly.?Pigs?can?absorb? 80?85%?of?an?oral?ZEA?dose?and?are?very?efficient?in?internalizing?ZEA?from?the? GI?tract?[77].??Once?ingested,?intestinal?epithelial?cells?degrade?ZEA?into?its? metabolites???zearalenol?(??ZEA)?and???zearalenol?(??ZEA).??Malekinejad?et?al? [69]?showed?that?pigs?produce?mainly???ZEA?upon?oral?ingestion?of?ZEA.? Reduced?forms?of?ZEA?display?increased?estrogenic?activity?compared?to?the? parent?compound,?since?they?can?bind?to?both?ESR1?and?ESR2?competitively? [75].??ZEA?and?its?metabolites?were?reported?in?porcine?urine,?plasma,?and?milk,? though?concentrations?in?porcine?milk?were?much?lower?when?compared?to? intrauterine?concentrations?of?ZEA?[78,?79].?Therefore,?signs?of? hyperestrogenism?associated?with?ZEA?exposure?in?the?neonatal?pig?were?mainly? attributed?to?prenatal?rather?than?postnatal?exposure?through?milk.??? ?Pigs?and?sheep?are?very?sensitive?to?the?reproductive?and?developmental? effects?of?ZEA.?It?reduces?embryonic?survival,?fetal?weight,?and?is?known?to? decrease?luteinizing?hormone?(LH)?and?progesterone?secretion?as?well?as? altering?uterine?morphology?[76].??Chen?et?al?[80]?reported?that?prenatal?and? postnatal?ZEA?exposure?affected?uterine?gene?expression?in?the?neonatal?pig? [80].??Pregnant?sows?were?fed?1500??g?ZEA/kg?of?feed/day?for?14?days?prior?to? parturition?and?21?days?afterwards.?Gilts?were?cross?fostered?to?obtain?four? groups;?unexposed?controls,?prenatal?exposure,?postnatal?exposure,?and? ? 27 continuous?exposure.?Results?indicated?that?continuous?ZEA?exposure?decreased? RXFP1,?RXFP2,?ER??,?Wnt7a,?and?Hoxa10?uterine?transcripts?while?postnatal? exposure?only?decreased?RXFP1?transcripts?in?the?piglets[80].?Thus,?neonatal? exposure?to?estrogen?or?estrogen?like?compounds?can?alter?the?uterine? developmental?program?[5,?81].??? ? 4.?ENCRYPTED?BIOACTIVE?PEPTIDES? ? ?Milk?exhibits?a?wide?range?of?biological?activities?that?can?influence? neonatal?digestion,?metabolism,?immunity,?and?development.?Most?of?this? activity?is?due?to?the?hormones,?proteins?and?peptides?synthesized?by?the? mother,?deposited?in?colostrum/milk?and?consumed?by?the?newborn.?However,? it?is?possible?that?some?bioactive?peptides?communicated?via?a?lactocrine? mechanism?from?mother?to?offspring?in?milk?have?latent?activity,?becoming? active?only?after?proteolytic?digestion?of?the?parent?protein?while?in?the?neonatal? GI?tract?or?circulation?[82].??Peptides?in?this?category?are?likely?to?be??encrypted?? within?the?primary?amino?acid?sequence?of?parent?proteins.??Such?encrypted? peptides?can?be?multifunctional,?meaning?that?specific?peptide?sequences?have? two?or?more?biological?activities?with?their?expression?depending?on?degree?of? proteolysis?[82,?83].??Logically,?the?two?biggest?contributors?to?milk?proteins,? caseins?and?whey?proteins,?would?be?expected?to?contain?the?highest?number?of? encrypted,?potentially?bioactive?peptides.? ?Proteolytic?activation?of?encrypted?peptides?can?be?achieved?through?two? ? 28 mechanisms.?Most?of?the?known?milk?borne?bioactive?peptides?were?identified? by?hydrolyzing?the?parent?protein?with?pancreatic?enzymes?such?as?trypsin,? chymotrypsin,?carboxypeptidases,?and?aminopeptidases?[83].?Encrypted? peptides?have?also?been?released?using?pepsin,?thermolysin,?and?proline?specific? peptidases?[84].?However,?the?neonatal?pig?does?not?have?high?concentrations?of? proteolytic?enzymes?due?to?the?immaturity?of?the?GI?tract?and?pancreas.??This? suggests?the?likelihood?of?encrypted?peptide?release?in?the?GI?tract?to?be?low? [35].?However,?naturally?occurring?bacteria?in?the?small?intestine?can?produce? bioactive?peptides?from?milk?borne?proteins.?Yamamoto?et?al?[85]?found?that? Lactobacillus?helveticus?produces?a?serine?type?proteinase?that?can?bind?to??? casein?and?produce?several?casokinins?[85].?Casokinins?are?ACE?inhibitory? peptides?derived?from????and???caseins?[2,?85].?Many?different?bacterial? proteinases?with?specificities?to?the?different?caseins?found?in?milk?are?known.? Not?surprisingly,?specific?cleavage?cites?to?bacterial?proteinases?have?been? identified?in??S1?casein,??S2?casein,???casein,?and???caseins?[86].? ?Milk?borne?encrypted?peptides?identified?in?bovine?and?human?milk?can?be? divided?into?four?groups,?including?those?affecting?gastrointestinal?function,? modulation?of?postprandial?metabolism,?antimicrobial?defense,?and? immunoregulation?[2].?These?peptides?can?interact?with?target?receptors?in?the? intestinal?lumen?or?may?be?absorbed?and?potentially?reach?target?sites?via?the? circulation?[2].?Among?the?most?abundant?of?milk?peptides?with?latent?activity? are?the?opioid?peptides?and?their?antagonists,?both?mainly?affecting?GI?function? in?the?neonate.?Opioid?agonists?are?called?casomorphins?and?exorphins?for?their? ? 29 ability?to?bind?to?opioid?receptors.??These?peptides?are?encrypted?within??S1? casein,???casein,?and?most?of?the?major?whey?proteins?[87].?In?contrast,?opioid? antagonists,?known?as?casoxins?and?lactoferroxins,?are?derived?from???casein? and?lactoferrin?respectively?[84].?Casomorphins/exorphins?bind?to?opioid? receptors?and?decrease?intestinal?mobility?while?increasing?amino?acid?and? electrolyte?uptake?in?the?calf,?while?casoxins/lactoferroxins?have?the?opposite? effect?[84,?87].??Presence?of?bioactive?peptides?encrypted?within?milk?proteins? has?enormous?implications?concerning?the?extent?of?influence?that?milk?borne? bioactive?peptides?have?on?intestinal?and?systemic?health.?Colostrum?has?higher? concentrations?of??s1?casein?and???casein?compared?to???casein?and?lactoferrin? [87],?implying?that?the?number?of?opioid?agonist?peptides?out?numbers?opioid? antagonists?.?This?may?be?important,?as?the?decrease?in?intestinal?mobility? combined?with?higher?amino?acid?uptake?that?such?signaling?might?effect?would? give?more?time?for?colostral?peptides?and?growth?factors?to?influence?GI? development?and?get?absorbed?into?circulation.?Opioid?agonists?can?influence? postprandial?metabolism?in?canines?by?stimulating?the?secretion?of?insulin?and? somatostatin?once?inside?circulation?[88,?89].?Antimicrobial?bioactive?peptides? can?be?found?within?whey?proteins?casein?micelles?[47].?The?most?common?is? lactoferricin,?found?after?hydrolyzing?lactoferrin?with?pepsin,?though?others? were?identified?after?hydrolysis?with?different?GI?enzymes.?Lactoferricin?has? activity?against?a?broad?spectrum?of?Gram?positive?and?negative?bacteria.?[47].? Antimicrobial?peptide?fragments?can?also?come?from?all?the?different?caseins,? though??S1?casein?is?the?primary?source?[90].?Immunomodulatory?peptides?can? ? 30 enhance?the?immune?response?by?stimulating?lymphocyte?proliferation?and? interleukin?production?[91].?? ?The?mechanism?by?which?these?peptides?exert?their?effects?is?undefined,? though?it?was?hypothesized?that?they?act?through?opiate?receptors?found?on?the? surface?of?lymphocytes?and?macrophages?[2].?Although?direct?studies?aimed?at? identifying?encrypted?peptides?in?porcine?milk?are?lacking,?the?idea?that? encrypted?peptides?affect?the?nursing?piglet?is?believable?considering?the? conserved?nature?of?milk?and?mammary?genes.??? ?Some?milk?borne?proteins?contain?angiotensin?I?converting?enzyme?(ACE)? inhibitory?peptides?within?their?sequence?[2,?83,?84].?ACE?is?predominantly? found?in?membrane?bound?vascular?endothelial?cells,?neuroepithelial?cells,?and? solubilized?in?the?blood.?Its?main?function?is?to?cleave?the?C?terminal?dipeptide? from?angiotensin?I?to?ultimately?form?angiotensin?II,?a?powerful?vasoconstrictor? [92].?ACE?inhibitory?peptides?in?milk?are?called?casokinins?and?lactokinins? depending?on?whether?the?parent?proteins?are??/??caseins?or???lactalbumin/?? lactoglobulin?respectively?[2].?These?peptides?lower?blood?pressure?by? competing?with?angiotensin?I?for?the?active?site?on?the?enzyme.?Over?50?different? ACE?inhibitory?peptides?were?identified?in?vitro?by?hydrolysis?of?milk?proteins? using?proteases?and?bacterial?fermentation?[92].?However,?some?of?the?larger? peptides?have?no?effect?in?vivo,?most?likely?because?they?are?hydrolyzed?to? smaller?inactive?fragments?in?the?intestine?before?they?are?able?to?pass?into? circulation.? ?Mineral?binding?properties?of?casein?are?well?described.??The?high? ? 31 concentration?of?calcium?in?milk?has?long?been?credited?to?the?presence?of? phosphorylated?serine?and?glutamyl?residue?clusters?in?casein?that?are?able?to? bind?other?minerals?present?in?milk?such?as?zinc?and?iron?[2].?Tryptic?digestion?of? caseins?yields?caseinphosphopeptides?(CCPs)?containing?clusters?of? phosporylated?serine?and?glutamyl?residues,?thus?retaining?the?mineral?binding? capabilities?of?the?intact?casein?molecule?[2,?90].?Studies?showed?that?CCPs?in? milk?can?increase?the?availability?of?calcium,?iron,?magnesium?and?zinc?to?the? neonate?[93,?94].?Though?milk?borne?zinc?concentrations?are?rarely?deficient?in? porcine?milk?[1],?zinc?levels?in?breast?milk?can?vary?greatly?and?low? concentrations?of?milk?borne?zinc?in?humans?can?lead?to?a?severe?zinc?deficiency? in?nursing?infants?which?has?behavioral,?neurological,?and?immunological? consequences?[95].?However,?there?are?conflicting?reports?on?whether?or?not? increasing?the?availability?of?minerals?using?CCPs?leads?to?an?increase?in?passive? mineral?absorption,?particularly?calcium?[90].?? ?? 5.?COLOSTRUM/MILK?AS?A?CONDUIT?FOR?DEVELOPMENTAL?SIGNALS? ?? ?The?previous?section?focused?on?describing?some?of?the?most?important? components?of?colostrum?and?milk.?Colostrum?is?known?to?contain?significant? amounts?of?immunoglobulins?and?other?anti?infection?agents?that?protect?against? pathogens?in?the?neonatal?GI?tract?and?enhance?passive?immunity?within? circulation?[4].??Colostrum?and?milk?are?both?rich?in?growth?factors?that?function? as?mediators?of?neonatal?development.?The?EGF?and?IGF?families?of?proteins?in? ? 32 milk?affect?multiple?aspects?of?neonatal?development,?from?GI?maturation?to? reproductive?tract?development?[51,?53].?Bioactive?peptides?encrypted?in?milk? proteins?~2?20?residues?are?also?important?as?they?have?latent?activity?in?the?GI? tract?and?circulation?following?hydrolysis?of?the?parent?protein.?These?small? peptides?can?modulate?neonatal?immunity,?blood?pressure,?GI?development,?and? systemic?targets?[90].?Milk?borne?steroids?have?also?been?described?in?the?milk? of?several?organisms,?including?the?pig,?though?their?role?has?yet?to?be?fully? determined?[53].??The?diversity?of?growth?factors?and?hormones?in? colostrum/milk?requires?that?the?role?of?this?maternal?lacteal?secretion?be? extended?beyond?nutritional?and?immunological?borders?to?include? consideration?of??colostrum/milk?as?a?conduit?for?lactocrine?transmission?of? developmental?signals?from?mother?to?offspring?[96].? ?The?term?lactocrine?was?coined?to?describe?a?mechanism?by?which?milk? borne?factors?are?delivered?from?mother?to?offspring?as?a?specific?consequence?of? nursing?[5].??In?pigs,?lactocrine?acting?factors?are?present?predominantly?in? colostrum?synthesized?within?the?first?two?days?of?lactation,?a?timing?that? coincides?with?the?period?of?gut?closure?to?macromolecule?absorption.?Studies? reviewed?in?the?next?section?were?designed?to?determine?how?colostrum? consumption?during?this?period?affects?development?of?the?female?reproductive? tract?(FRT),?male?reproductive?tract,?and?other?tissues?in?the?neonatal?pig?[55,? 61,?63,?97?99].? Studies?of?marsupial?species?support?the?idea?that?lactocrine?regulation?of? development?is?not?unique?to?the?pig?[100].?Additionally,?mammotroph? ? 33 differentiation?in?the?rodent?is?dependent?on?milk?borne?factors?shortly?after? birth?[96].??In?rats,?colostrum?consumption?in?the?first?two?days?is?necessary?to? induce?the?appearance?of?prolactin?releasing?cells?within?the?anterior?pituitary? gland?and?delays?development?of?mammotrophs?in?the?neonate.?Milk?deprived? rats?had?altered?mammotroph?function?as?adults?compared?to?their?siblings? allowed?to?nurse?normally?[96].?This?suggests?that?there?are?milk?borne?factors? in?rat?colostrum?necessary?for?proper?differentiation?and?long?term?function?of? mammotrophs.??Finally,?the?World?Health?Organization?recommends?that? mothers?nurse?their?infants?for?at?least?six?months?after?birth,?citing?multiple? studies?describing?the?short??and?long?term?benefits?of?breastfeeding?[101].?The? fact?that?all?newborn?mammals?consume?milk?containing?bioactive?proteins?and? peptides?combined?with?the?ability?of?the?neonatal?gut?to?absorb?these?molecules? intact?makes?the?lactocrine?hypothesis?[5]?for?maternal?programming?of? mammalian?neonatal?development?compelling.? 5.1 LACTOCRINE REGULATION OF PORCINE FRT DEVELOPMENT In the pig, FRT development begins prenatally and is completed postnatally [81, 102]. Throughout this period, there are systematic cellular interactions involving epithelial and stromal cells of the FRT, that support both structural and functional patterning of the oviduct, uterus, cervix, and vagina [102, 103]. One of the most important events during this period is uterine adenogenesis, or budding and differentiation of glandular epithelium from luminal epithelium. Genesis of uterine endometrial glands in the neonatal pig requires both the expression and activation of ? 34 uterine ESR1 [102] .Development of the neonatal porcine uterus is both estrogen sensitive and ESR1 dependent [74]. Patterns of expression and activation of the uterine ESR1 system can define the developmental program and determine the developmental trajectory porcine FRT tissues, including the uterus [5]. Using relaxin as a prototypical milk-borne morphoregulatory factor, a series of experiments was conducted to determine the extent to which lactocrine mechanisms might support development of the neonatal porcine uterus. Bioactive prorelaxin was detected in porcine milk, with highest concentrations found in colostrum at birth [99]. Immunoreactive RLX was only detectable in serum of neonatal piglets allowed to nurse [63], indicating lactocrine transmission of this bioactive morphoregulatory peptide. The neonatal uterus is RLX receptor (RXFP1) - positive and ESR1-negative at birth [5]. Administration of exogenous RLX upregulated uterine ESR1 expression and had trophic effects on uterine growth similar to estrogen that could be inhibited by co-administration of the ESR1 antagonist ICI 182,780 [55, 63, 104]. Evidence supporting lactocrine transmission of RLX to the neonatal circulation [55] indicated that the window for delivery of such MbFs is open for approximately 48 h from birth. This is approximately the same time when the gut closes and mammary secretions characteristic of colostrum cease [55]. It is important to note that, as reviewed above, RLX is only one of many potentially lactocrine-active factors. Piglets maintained in a lactocrine null state (deprived of colostrum) for the first 48 hours from birth displayed dramatically altered uterine expression patterns for ESR1, vascular endothelial growth factor (VEGFA) and matrix metalloproteinase 9 ? 35 (MMP9) on PND 2 as compared to nursed controls [105]. While no effects on patterns of growth as reflected by body weight were noted, uterine ESR1, VEGFA and MMP9 protein levels were uniformly below assay sensitivity in lactocrine null gilts at PND 2. Results provided the first, unequivocal evidence of a requirement for lactocrine support of gene expression events necessary to establish an optimal uterine developmental program [105]. Similar effects of lactocrine signaling were also seen in the cervix [106], male reproductive tissues [97] and the heart [98]. Generally, the fact that trophic effects of exogenous RLX were more pronounced in nursing gilts than in lactocrine null gilts indicated that factors other than RLX in colostrum/milk cooperate to support RLX-dependent effects [98, 105]. 5.2 EVIDENCE FOR LACTOCRINE REGULATION OF MARSUPIAL DEVELOPMENT Given the conserved nature of milk and mammary genes (see above) it is reasonable to theorize that lactocrine regulation of development occurs across a wide range of mammalian species. Direct studies on lactocrine signaling in mammals excluding the pig are scarce. However, studies focusing on marsupial lactation strategies provide evidence that lactocrine signaling supports development in the metatherians. The tammar wallaby is a member of the kangaroo family indigenous to Australia. As a marsupial their lactation strategy differs from that of eutherian mammals in that they have long lactation periods relative to gestation, where the inverse relationship is true for eutherians [100]. ? 36 In contrast to eutherian mammals that display one major change in milk composition, the transition from colostrum to mature milk [107], marsupial (wallaby) lactation involves multiple changes in milk-protein composition which occur through a series of lactational phases matched to the extrauterine development of offspring. Phase 1 is the preparatory phase beginning 26 days before parturition; Phase II begins when, following birth, the neonate climbs into the pouch and attaches to a teat secreting Phase II milk for ~200 days. Phase II includes two sub-phases: Phase IIA ? when milk contains high levels of ?-lactalbumin, ?-lactoglobulin, ?-casein, and ?- casein; and Phase IIB ? involving concurrent changes in whey protein composition - when pouch young (PY) cease to be permanently attached to the teat [108]. Phase III occurs as the PY begin consumption of herbage. This phase of wallaby lactation is characterized by high milk volume coupled with an increase in milk protein and fat content[108]. Key stages of PY development are correlated with changes in lactation patterns of the mother [109]. Joss et al [109] found that all eight milk samples of specific time points tested between lactation day (LD) 0 and LD 250 contained unique proteins. Proteins that changed in abundance at least 3-fold between time points consisted of up to 80% of the total proteins within that stage [109]. Clearly, given the extreme, altricial state of development of metatherian PY at the time they emerge from the uterus, lactocrine signaling is central to developmental success in these species. Kwek et al [100] provided further evidence that lactocrine regulation of development functions the tammar wallaby. They determined that disruption of the lactational/developmental relationship between mother and joey results in altered ? 37 patterns of gene expression in the forestomach [100]. In that study, PY at 120 days of age were cross-fostered to mothers at 170 days of lactation for 60 days. Thus, the developmental age of the joey and lactation stage of the mother was asynchronous. Joeys around 120 days of age exhibit significant changes in different regions of their stomach and the effect of later stage milk on fore-stomach maturation was unknown. They found that gross morphology and cell proliferation were not altered, however there was increased apoptosis and decreased parietal cell numbers in the fore-stomach of fostered joeys at 180 days of age [100]. Parietal cells are normally abundant in 180 day-old joey fore-stomachs but disappear by day 230. Not surprisingly, the parietal cell marker ATP4A was reduced in the forestomach of the fostered group. Other gastric glandular cell markers, such as GHRL and GKN2, were also downregulated. These observations suggested that milk consumed by the fostered PY from a later stage in lactation contained factors that retarded development of gastric glandular cell types of the forestomach and increased related apoptosis [100]. Moreover, fostered PY were over twice as heavy and had a head length 11 mm longer than non-fostered PY [100]. While the advanced rate of growth could be attributed to higher protein and lipid content of later stage milk consumed by the fostered group, it is also possible that the different proteins or other factors consumed by the fostered joeys affected their development. A similar study by Menzies et al [110] showed how cross-fostering between wallaby species can alter growth and development of the fostered group [110]. In this case tammar joeys were fostered to parma wallaby mothers and parma joeys were fostered to tammar wallaby mothers at the same stage of lactation and development. ? 38 Parma and tammar wallabies were chosen for their similarities in size and lactation length [110]. Joeys were fostered at 15 days and 30 days and timing of developmental milestones were recorded. Fostered tammar joeys grew at similar rates to the controls and there was no change in the timing of developmental milestones such as appearance of hair, pigmentation, and opening of the eyes. In contrast, parma joeys did not survive when fostered at 15 days and struggled when fostered at 30 days of age. The surviving joeys had retarded growth rates and delayed appearance of developmental milestones. The difference in size between fostered joeys is not dependent on nutritional factors because fostered tammar joeys grew at a similar rate to control tammar and parma joeys, demonstrating that the nutritional milk components of each species are similar. It has long been known that an intrinsic maternal program unaffected by suckling patterns of the neonate regulates changes in expression of tammar milk proteins [111]. The authors suggested that differences in key milk-proteins consumed by fostered parma joeys produced an immature endocrine growth axis and retarded development. They pointed to early lactation protein (ELP), whey acidic protein (WAP) and IGF-1 as possible candidates affecting development of the parma endocrine growth axis [110]. Concentrations of these candidate proteins peak earlier in parma lactation compared to tammar lactation, suggesting that growth defects observed in fostered parmas were caussed by delayed consumption of ELP and WAP [110]. The fact that no parma joeys fostered before PND 30 survived supports this theory, as the concentration of these proteins in tammar milk would have increased enough to support the needs of fostered parma joeys. Authors concluded that maternal milk influenced aspects of joey development ? 39 and that altering the period when each joey receives critical milk components can have profound developmental consequences [110]. While mechanistic details remain to be delineated, developmental differences observed between these groups makes a convincing argument that maternal factors in wallaby milk may be affecting gene expression events critical to the success of neonatal development in this metatherian species. 6. BREASTFEEDING AND LONG-TERM EFFECTS Short-term benefits of breastfeeding are clear. Numerous studies show that nursed infants have reduced risk of mortality from infectious diseases and decreased morbidity from GI and allergic ailments [112]. Long-term benefits of breastfeeding are less clear and more difficult to describe considering the limitations inherent in human studies. However, in a review published by the World Health Organization several correlations between breastfed children and long-term health such as risks for hypertension, high cholesterol, obesity, type II diabetes, and intelligence were described [101]. There is still controversy regarding the effects of breastfeeding beyond infancy since numerous studies suggest opposite conclusions (101) Nevertheless, infant formula typically does not contain the variety of milk-borne factors that the infant would ingest with breast milk. Meta-analytical studies and observations of the impact of imposition of a lactocrine null state on human development are imperfect. However, such studies may provide some insight into the role of lactocrine signaling in postanatal development of our species. ? 40 In humans, neonatal diet may influence risk of hypertension and heart disease in adulthood [113]. On average, the risk of hypertension in adults that were breastfed as infants was lower than those that were not [101]. The leading theory explaining this finding involves the presence of long-chain polyunsaturated fatty acids (LCPUFA) in breast milk that are not found in most brands of milk formula [101]. These fatty acids are incorporated into tissue membrane systems, including neural membranes and vascular epithelia [114]. Supplementation of formula with LCPUFAs results in lower blood pressure by age 6 compared to those fed with standard formula and comparable to infants that were breastfed [115]. After correcting for confounding factors and publication bias, meta-analyses of several studies concluded that both systolic and diastolic blood pressure are reduced in adults breastfed as infants [101]. Higher LCPUFA concentrations in breast milk were suggested as the reason for higher performance on intelligence tests applied to breastfed infants compared to infants that were fed formula. The major lipid components of neural membranes are docosahexaenoic acid (DHA) and arachidonic acid (AA), both of which are necessary for cortical and retinal brain development [101, 116]. DHA and AA levels decrease soon after parturition, but was also shown that DHA and AA levels are higher in breastfed infants, suggesting that the milk-borne LCPUFAs are replenishing DHA and AA levels. However, breastfeeding also enhances bonding between mother and child, which may contribute to intellectual development [117]. These factors are impossible to separate in observational studies and the extent to which breastfed infants are cognitively superior to those fed formula exclusively remains unclear [118]. ? 41 Breastfeeding was associated with lower cholesterol levels in adults [101]. This may be due to the ability of milk to downregulate hepatic hydroxylmethylglutaryl coenzyme (HMG-CoA), as observed in piglets nursed normally compared to those fed with milk replacer (101) Similarly, piglets fed a high cholesterol diet had lower cholesterol levels as adults [119]. HMG-CoA is the rate limiting enzyme in the conversion of acetate to cholesterol [101]. Breast milk has significantly higher cholesterol compared to standard formulas, suggesting that high cholesterol levels have a long-term programming effect on cholesterol synthesis [101]. In contrast to the analysis of hypertension, protective effects of milk against high cholesterol were observed exclusively in adults (101) Risks of obesity and type 2 diabetes in adults were also linked to breastfeeding [101]. A possible explanation is that formula fed infants express higher levels of insulin, motilin, enteroglucagon, neurotensin, and pancreatic polypeptide compared to breastfed counterparts [120]. Higher insulin results in increased fat deposition and adipocyte numbers, leading to a higher risk for obesity and insulin resistance in the future [101]. Studies (101) showed that breastfeeding has a small protective effect against obesity and development of type 2 diabetes, though the issue still remains controversial. 7. SUMMARY AND IMPLICATIONS The studies discussed in this literature review conveyed the importance of colostrum and milk in enhancing the health and development of the neonate. Milk is a ? 42 complex fluid consisting of a vast array of substances suited for the survival of the neonate. The fact that all mammals nurse their young, the highly conserved nature of milk and mammary genes, and the changes observed in milk composition across several different species during developmentally critical periods suggests the value of milk to the neonate exceeds pure nutritional and immunological needs. Data for the pig demonstrate the effects of nursing within the first 48 hours of life on development of FRT and other somatic tissues in both male and female neonates. The first objective of research described here was to determine if and how the array of proteins/peptides that define the porcine colostral proteome changes in the transition from colostrum to milk by determining qualitative and quantitative differences between protein profiles characteristic of colostrum at PND 0 with milk obtained at PND 6 using two-dimensional gel electrophoresis. The second objective was to confirm the hypothesis, as established for other species, that potentially bioactive peptides are encrypted within porcine milk proteins. Results will serve as a reference point for future studies aimed at identifying novel lactocrine acting factors in the porcine colostral proteome that may be contributing to FRT programming. ? 43 CHAPTER 3 DEFINING THE PORCINE COLOSTRAL PROTEOME ABSTRACT Colostrum (first milk) provides nutritional and immunological support for newborn mammals. In addition, colostrum contains a variety of bioactive peptides and growth factors, some of which are present in higher concentrations than in maternal circulation. Milk-borne, lactocrine acting factors affect patterns of gene expression in neonatal somatic tissues such as the reproductive tract and heart. Data for relaxin, a prototypical lactocrine-acting factor in porcine colostrum, indicate that its transmission of such factors from mother to offspring is significant prior to gut closure in the neonate. Additionally, amino acid sequences encoding potentially bioactive peptides may be encrypted within porcine milk proteins. These encrypted peptides become activated after proteolytic cleavage in the intestinal lumen or within the circulation. Thus it is important to understand the biochemical nature of colostrum/milk. The array of proteins/peptides constituting the porcine colostral proteome has not been defined. Objectives of this study were to employ two- dimensional gel electrophoresis (2DE) and image analysis to: (1) define the porcine colostral proteome on lactation day (LD) 0; (2) determine if and how this proteome changes from LD 0 to LD 6; and to (3) determine if potentially bioactive peptides are encrypted within porcine milk proteins. Colostrum (LD 0) and milk (LD 6) samples ? 44 were obtained from six lactating sows. Protein was extracted from individual samples and total protein concentrations were determined. For 2DE, first dimension separations were carried out using pH 3-10 immobilized pH gradient strips followed by SDS-PAGE using gradient polyacrylamide gels (10-20% total monomer). Individual samples were run on duplicate 2DE gels and stained with Sypro RUBY. Digital images of individual gels were obtained and analyzed using a Typhoon 9400 digital scanner and PDQuest 2-D Analysis Software. Total protein concentrations for colostrum (LD 0) and milk (LD 6) were 8.5 mg/ml and 8.3 mg/ml. PDQuest analysis identified 304 spots on 2DE gels that defined the colostral/milk proteome. Of these, 25 were unique to LD 0 and 15 were unique to LD 6. Differences in relative spot intensity between groups were also identified. There were 158 spots common to LD 0 and LD 6 that changed quantitatively at least 2-fold between days. Of these 158 spots, 105 increased in abundance while 53 decreased in abundance from LD 0 to LD 6. Data indicate that the colostral proteome (LD 0) is distinct from that identified for milk (LD 6). A large number of encrypted peptides were found within the sequences of common porcine milk-borne proteins. Thus, newborn pigs that nurse obtain a complex mixture of proteins and peptides from birth, which changes with time during a critical developmental period. This idea is supported by the fact that gilts deprived of colostrum for only two days from birth exhibit altered gene expression patterns associated with development of reproductive tract tissues and which persist when colostrum-deprived neonates are returned to milk feeding immediately thereafter. Though this study is largely predictive, it serves as a starting point in identifying ? 45 potential bioactive peptides in colostrum that act in a lactocrine faction and are necessary for the normal programming of the neonate. INTRODUCTION Milk is the main source of nutrients for the neonatal pig early in development. [3]. Colostrum is produced exclusively during the late stages of pregnancy and early lactation. It is high in protein and is classically known for boosting passive immunity in the neonate [1]. Fifty to eighty percent of the crude protein in porcine colostrum consists of immunoglobulins that are necessary in protecting the piglet from infection [4, 121]. However, colostrum also contains a large number of bioactive compounds, including growth factors, hydrolytic enzymes, hormones, and anti-infection agents that have local and systemic targets [1, 121]. Additionally, many milk proteins contain potentially bioactive peptides encrypted within their primary sequences that may become active after proteolysis. Effects of milk-borne factors on the health and development of the neonatal gastrointestinal (GI) tract are well documented [32]. The porcine GI tract develops rapidly during the perinatal period after colostrum consumption and intestinal villi are temporarily damaged after milk withdrawal [57]. Newborn piglets fed colostrum for 24 hours had greater intestinal villous height and crypt depth compared to those that were fed mature milk or milk replacer as well as greater internalization of transforming growth factor-? receptors of the intestinal epithelium [39]. Bioactive factors can also affect systemic targets before the porcine GI tract closes to absorption of whole macromolecules at 24-48 hours after birth [32] This process is facilitated by ? 46 protease inhibitors present in colostrum as well as the undeveloped production of gastric acids and pancreatic enzymes [32, 96]. Collectively this arrangement provides an effective method for milk-borne bioactive factors (Mbfs) to enter systemic circulation. Evidence suggests that colostrum serves as conduit for developmental signals required for the normal growth of the neonatal mammal. Prolactin secreting cells of the anterior pituitary in the rat proliferate quickly immediately after birth [96]. A study where neonatal rats were immediately fostered to mothers at day 4 of lactation showed that fostered pups had a decreased percentage of prolactin-secreting cells compared to nursed controls [122]. Similar studies done with tammar wallabies where fostered joeys nursing from mothers at 60 day advanced lactation phase exhibited altered patterns of gastrointestinal and somatic growth [100]. Analysis of wallaby milk revealed that changes in milk proteins throughout lactation correlated with the changing needs of the joey during development [109]. Using a porcine model the term ?lactocrine? was coined to describe a mechanism through which milk-borne bioactive factors enter circulation as a specific consequence of nursing [5]. The prototypical milk-borne bioactive factor relaxin (RLX) is present in porcine colostrum and only detectable in neonatal circulation if piglets are allowed to nurse [55, 61]. Subsequent studies showed that maternally driven relaxin signaling is required for estrogen receptor-? (ESR1) and vascular endothelial growth factor (VEGFA) expression in the porcine uterus by postnatal day 2. The lactocrine null state describes neonates deprived of colostrum and instead fed a hormone free milk replacer. Piglets maintained in this state for 48h from birth exhibit abnormal, ? 47 developmentally critical gene expression patterns the heart and both male/female reproductive tract tissues. [97, 98]. Relaxin is only one of many Mbfs present in porcine milk with potential to affect somatic development in the neonate. However, the milk proteome is poorly characterized in pigs and indeed in most mammals. In this study two dimensional polyacrylamide gene electrophoresis (2DE), image analysis and bioinformatic methods were used to (1) define the porcine colostral proteome on lactation day (LD) 0; (2) determine if and how this proteome changes from LD 0 to LD 6; and to (3) determine if potentially bioactive peptides are encrypted within porcine milk proteins. MATERIALS AND METHODS PORCINE MILK COLLECTION AND WHEY PROTEIN EXTRACTION Purebred Yorkshire sows were milked manually immediately after parturition (lactation day 0) for colostrum and on lactation day 6 for mature milk (n = 6 sows/day). Teats were washed with warm water and massaged to stimulate milk letdown for both days. Composite samples were obtained on ice from multiple teats from each sow and frozen at -4?C. To isolate the whey protein fraction individual milk samples were diluted 1:1 with ethylenediamine tetra-acetic acid (EDTA; pH 7.0) and centrifuged twice for 15 minutes at 4000 x g at 4?C [61, 123]. Excess fat was removed with a spatula after each centrifugation. All samples were stored at -20?C. SAMPLE PREPARATION AND DETERMINATION OF TOTAL PROTEIN ? 48 Samples were not pooled. Individual samples were prepared for 2DE. Total protein concentration was determined with the RCDC assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard. For each sample, 500?g of protein was subjected to third level sequential extraction (Bio-Rad) and further processed using the ReadyPrep 2-D Clean-Up Kit (Bio-Rad) to improve spot quality. Protein pellets were rehydrated with 50?l of ReadyPrep 2-D Starter Kit Rehydration/Sample Buffer (Bio-Rad). ISOELECTRIC FOCUSING (IEF) AND 2DE For each sample, 75?g of protein were loaded unto 11cm pH 3-10 ReadyStrip IPG Strips (Bio-Rad) following the protocol from the manufacturer. Strips were loaded on to a Protean IEF Cell (Bio-Rad) and focused for 20 hours per the protocol established by Boehmer et al in 2008: 500V for 1h, 1000V for 1h, 2000V for 2h, 4000V for 4h, 8000V for 12h[124]. Focused IPG strips were equilibrated in Buffer I (6 M Urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% Glycerol, and 2% wt/vol dithiotrheitol) for 30 minutes and Buffer II (6 M Urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% Glycerol, and 2.5% wt/vol iodoacetamide) for an additional 30 minutes (Bio-Rad). In every case, strips representing each animal and lactation day were run together to avoid procedural confounding. Equilibrated IPG strips were run in duplicate in the second dimension on 10- 20% Tris-HCl polyacrylamide gels (Bio-Rad) in 1X Tris-glycine-SDS running buffer at 200V for 1 hour with a Criterion Dodeca Cell (Bio-Rad). Since 12 second dimension gels can be run simultaneously on this device, care was taken to insure that ? 49 samples representing each animal and lactation day were run together to avoid procedural confounding. Second dimension gels were removed from casings and fixed in 40% methanol, 10 % acetic acid solution for 1 hour and stained with SYPRO Ruby overnight. 2D gels were destained with 10% methanol, 7% acetic acid solution for 2 hours. Each gel was scanned using a Typhoon 9410 Variable Mode Imager (GE Healthcare Bio-Sciences). 2D GEL IMAGE ANALYSIS Digital images of each gel were analyzed using PDQuest Advanced 2-D Analysis software version 8.0.1 (Bio-Rad). Gel images were set up such that the pI range (3-10) was oriented left-toright and the molecular weight range (M r x 10 -3 , 250-to10) was oriented top-tobottom. All images were cropped to standardize their size and minimize false spots detected around gel edges. Individual spots were detected using the automatic spot detection wizard along with manual modifications to spot detection parameters. The speckle filter was turned to a sensitivity of 200 and images were corrected for horizontal and vertical streaks. Gel images were matched automatically to create a ?master ?gel, or a digital image combining all spots identified in each raw image for a given animal and day. Three master gels were created; LD 0 master, LD 6 master and the combined master which included data from all gels in both groups. Matched spots were inspected visually using the Spot Review Tool to verify accurate spot matching and identification. Some spots were matched or unmatched manually based on visual inspection. The Multi-Channel ? 50 Viewer was used to manually overlay gel images stained digitally with different colors (pesudocolored) to aid in manual spot matching. Analysis sets were then created to determine qualitative and quantitative differences between groups, which were identified using PDQuest software. SPOT IDENTIFICATION Spots indicative of proteins and peptides separated by 2DE that changed qualitatively and up to two-fold quantitatively from LD 0 to LD 6 were identified preliminarily using the ExPASy TagIdent tool (http://expasy. org/tools/tagident. html). To do this, estimated molecular weight and isoelectric point values were obtained for each targeted spot. These values were entered into the ExPASy TagIdent database obtain a preliminary identification. The error ranges for molecular weight and isoelectric point were set at 10% and pH 0.2 respectively. The organism name was specified to Sus scrofa whenever possible. If no matches were found, a general search for mammalian proteins in the approximated pI x molecular weight range was conducted. Molecular weight and pI values with multiple matches and no consensus between matches were labeled ?No Consensus? in Tables 1 and 2. ENCRYPTED PEPTIDE IDENTIFICATION Primary amino acid sequences of common porcine milk proteins were obtained from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/) and each was analyzed using the BIOPEP database (http://www.uwm.edu.pl/ biochemia/index _en.php). A correlation was obtained between the size of the protein ? 51 and the incidence of unique encrypted peptides occurring three times or > 4 times per sequence using SAS analytics software version 9.1 (SAS Institute, Cary, NC). In silico analyses were performed to determine projected effects of proteolytic enzyme action on targeted porcine milk proteins. The enzymes chosen for these studies included pepsin, trypsin, chymotrypsin, elastase I, and elastase II. These enzymes were selected as they are known to be expressed by or to be present in the porcine GI tract during early postnatal development (35). RESULTS Relative qualitative and quantitative changes in the colostral/milk proteome observed between LD 0 and LD 6 of lactation are illustrated in Figure 1. Proteins and peptides are separated by pI (3-10, left to right) and molecular weight (250-10 x 10 -3 , top to bottom). In this example, illustrating results for a single animal, details within outset boxes (Figure 1, A, B and C) show how the same areas in LD 0 and LD 6 gels differ in both spot number and relative spot density between days as assessed visually. ? 52 Figure 1: Visual comparison of 2DE master images of porcine colostrum/milk at LD0 and LD 6 for representative sow. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. Boxes A, B, and C show areas where quantitative and qualitative changes were observed in the porcine milk proteome between LD 0 and LD 6. ? 53 Figure 2 depicts master gels for LD 0 (top) and LD 6 (bottom). Qualitative and quantitative differences in the proteomes are shown. Figure 3 is the master image including all components from both LD 0 and LD 6. Differences between the images in Figures 2 and 3 can be explained by the higher number of gels that were matched for Figure 3. Digital matching procedures resulted in fewer spots matched at 70% in all gels. Figure 2: Comparison of 2DE master images for colostrum at LD 0 and milk at LD 6 combining milk samples from all sows on respective days. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. ? 54 Figure 3: Overall master image showing consensus spots from all gels in both groups (LD 0 and LD 6). PDQuest identified 304 common spots representative of the porcine milk proteome at LD0 and LD 6. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. ? 55 Figures 4 and 5 are the master gel images showing qualitative and two-fold or greater quantitative changes between LD 0 and LD 6. ? Figure 4: Master image with colored overlays showing qualitative changes between LD 0 and LD 6. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. Green X?s represent the 25 spots unique to LD 0 while Red X?s represent the 15 spots unique to LD 6. These spots are described in Tables 1 and 2. ? 56 Figure 5: Master image with colored overlays showing quantitative changes between LD 0 and LD 6. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. Red squares highlight the 53 spots that decrease at least two-fold from LD0 while green squares show the 105 spots that increase two-fold from LD 0. ? 57 With spot identification parameters established using both automated and visual inspection protocols, PDQuest identified 304 spots defined here to represent the LD 0-6 porcine milk proteome. Twenty-five of these spots were unique to LD 0 and 15 were unique to LD 6. There were 158 spots common to LD 0 and LD 6 that changed quantitatively at least 2-fold between days. Of these 158 spots, 105 increased in relative abundance while 53 decreased in relative abundance from LD 0 to LD 6. Figure 6 shows the location of spots and Standard Spot Numbers (SSP) at each location on the master gel image. The SSP numbers were used as a reference for identification of proteins/peptides. Results of preliminary TagIdent identification of spots unique to LD 0 and LD 6 are listed in Tables 1 and 2. ? 58 Figure 6: Master image with qualitative overlays and additional SSP numbers in purple. SSP numbers arbitrarily identify the identification of all spots in the master image. Additional information on SSP identified spots and can be tracked to Tables 1 and 2. Molecular weight range (250-10 X 10 -3 ) and pI (3-10) markers are shown. ? 59 SSPMr x10 -3 pI TagIdent IDProtein Name SP 046 4.63.0Ukow Mtchs 272 49 RSCA1_Pig RegulatrySlu Cri Protein 85 1. .2n o ates SP307546 6 F2i Cltin Fctr XI 2 8.04.71GH_PgGrwth Hrm Rptor 573 8 C1Si omple1Subnet SP 360 .15.990Aat ock Prtei 90 5026 5 7L_Pig h71 21 24.36.2OMAplirtin M SP63050 87 FCN1i Fcol 6.5.0PL_PgPlasmige 1834 69 TREi Sertnfri SP 604 .27.8 lsio 72356 2 Unkow No Cesu 31.89. IBP2_igInsuli-keGrwth Factor BP 2 SP040 743 TGFmuse Trafrming o1 735 2. . nko Mtes 6318 9 Uw No Cnu SP 0 6.07.1FIBA_vinFibrngeAlpha Cin 787 6 nko ss 23 .29.5 onu SP198 82 Uw N Cses 860 70. .1nko 413 934 o nsensus SP 5 .9.5 nseus Table 1: Preliminary identification of spots unique to LD0 using ExPASy TagIdent tool. SSP numbers can be tracked to their location on the image in Figure 6. The table shows TagIdent IDs and protein names based on molecular weight and pI. ? 60 SSP Mr pI TagIdent IDProtein Name SP10315.404.10HRB_Pig hrbi 2 2378 378 Ukow atces 546. .6n no SP 708 95 45 T may atches 3221. .03CATB_PigCathepsiBHvCain 1 68 7 ENL Endolasi SP407.45.2GK3i Gtrkne3 26 2130 6 POR_g Apliti R 5.9.30S10Ai Prote S10-A1 SP 10 46 1 TFB3 Transfmingrwth factor Beta 3 313.286.7G_Pigr G 54 3 SODC SupeoxideDisutase (Cn, Z) SP106.5.20TFB3iTransfrng rohfctorBeta3 2 196 61 RIH_Pg FeitHeavy ai 7. 7.52MGBa Micrlubln Table 2: Preliminary identification of spots unique to LD 6 using ExPASy TagIdent tool. SSP numbers can be tracked to their location on the image in Figure 6. The table shows TagIdent IDs and protein names based on molecular weight and pI. ? 61 Figure 7 illustrates the regression line (p < 0.0001) with incidence of unique peptides occurring three times per sequence as the dependent variable. Figure 8 illustrates the regression line (p < 0.0001) with incidence of unique peptides occurring > 4 times per sequence. Figure 7: Comparison of parent protein size (by number of amino acids) and incidence of unique encrypted peptides occurring exactly three times within one parent protein. The equation of the line appears above the graph. Number of proteins (N), R 2 , Adjusted R 2 , and root-mean square error (RMSE) is shown to the right of the graph. ? 62 Figure 8: Comparison of parent protein size (by number of amino acids) and incidence of unique encrypted peptides occurring greater than 4 times within one parent protein. The equation of the line appears above the graph. Number of proteins (N), R 2 , Adjusted R 2 , and root-mean square error (RMSE) is shown to the right of the graph. Not surprisingly, the number of potentially bioactive peptides likely to be encrypted repeatedly (three or more times) within parent proteins was determined to be related positively to the size, as defined by number of amino acids, of each parent molecule (Figures 7 and 8). ? 63 BIOPEP analysis of common milk-borne proteins revealed the presence of potentially bioactive encrypted peptides (BAPs) in all proteins tested (Table 3). Many of these encrypted peptides occurred over 100 times in a single parent molecule. Tables 4 and 5 show projected effects of proteolytic enzyme action on porcine milk proteins as reflected by the number of potential BAPs that could be excised. As expected, the decrease in number of potential BAPs identified through this in silico procedure likely reflects the limited number of enzyme-specific excision sites in the parent molecule. ? 64 Milk Proteins # As Potenial BAP BAP Milk Org cur >3 Ac 2468986 mylase511 146 28 polirotin -1 26490614 Ceuplas 335 109 8 22 rti Kie 1024613 EGFPrecsor574 125 11 35 ndoplamin 80412870 Htgb347 103 4 9 eat Shck 27DA 1245033 o0 a643 134 10 50 Ig 33899821 541 126 40 IGF-11717564 2 181 65 10 9 IBP-II316113425 FIII 293 111 5 23 IgG4671331135 M 403 115 6 18 Insuli 1085645 Lactdhern 431 134 11 19 te Dyogase 332993 acoprxi 712 127 47 Letn 1676060 iriipase 492 150 9 30 Lymphocyte CtolcProtin 5101371144 Lszme 146 49 2 6 Min-4193720422137 Phospglycerat Kiase 417 112 5 35 rena XRptor330114615 Prolctin 229 75 4 Seum Abu 6071401244 rylid89 47 1 2 TGF? 391132929 ?2434 121 12 19 ?-lactlbumin 141517 ofer 703 155 8 68 s1-Casi 20110511 ?s2en235 8 11 7 ?-asi 232107813 lctoglbul 178 56 5 3 2-Mirin 1184241 ?Casei 188 82 16 4 ? 65 Table 3: BIOPEP analysis of common porcine milk proteins and the potential number of bioactive encrypted peptides that can be found within one sequence of the parent protein. The first two columns describe the protein names and their size based on amino acid number. The third column shows the number of bioactive peptides (BAPs) occurring in one protein. The fourth and fifth columns show the number of BAPs that occur exactly three times or greater than four times respectively in one protein. ? 66 MilkProteins Pepsin Trypsin Chymotrpsin Elaste I Elaste I Ac 1 3 3 11 2 mylase02230 polirotin -1 1 9 Ceuplas 2 5253 rti Kie 4 8 13 10 9 EGFPrecsor 133181 ndoplamin 8 5 11 22 Htgb24 213 eat Shck 27DA 1 0 1 3 1 o0 a3 540 Ig 4 14 3 629506 IGF3 1 8 -2 3391 IBPII 0 0 6 F-III 2 230 IgG5 1 7 31 5 M 0 12 Insuli 3 1 10 3 Lactdhern 534136 te Dyogase 8 5 Lacoprxi 5923 Letn 2 0 3 13 2 iriipase 568219 Lymphocyte CtolcProtin 7 5 15 szm 00020 Min-417 22 12 76 11 Phospglycerat Kiase 252124 rena XRptor 2 5 14 0 Prolctin 0452 Seum Abu 2 5 12 rylid01080 TGF? 8 0 13 24 9 ?26 12146 ?-lactlbumin 0 1 1 4 1 ?ofer 74 319 s1-Casi 2 3 6 3 ?s2en12232 ?-asi 3 1 9 4 lctoglbul 1111 2-Mirin 4 3 3 6 ?Casei 0 30 ? 67 Table 4: BIOPEP analysis showing the potential number of bioactive peptides excised using common proteolytic enzymes. The first column names the porcine milk proteins and the subsequent columns list the number of bioactive peptides excised using pepsin, trypsin, chymotrypsin, elastase I, and elastase II respectively. ? 68 Milk Proteins Pepsin +Trypsin + Chmot Elaste I+ Elaste I Ac 8 11 mylase 2442 polirotin -1 16 Ceuplas 1728 rti Kie 3 2 EGFPrecsor 9 26 ndoplamin 34 36 Htgb 2434 eat Shck 27DA 3 2 o0 a 1447 Ig 12 15 50 IGF 5 12 -2 109 IBPII 9 13 F-III 8 10 IgG 11 32 M 12 Insuli 3 9 Lactdhern 4 11 te Dyogase 18 acoprxi 2936 Letn 3 13 LireiLipase 1339 ymphocyt CtolcProtin 15 18 szme 3 4 Min-4 39 19 Phospglycerat Kiase 2021 rena XRptor 10 Prolctin 4 7 Seum Abu 20 23 rylid 3 9 TGF? 10 28 ?2 1419 ?-lactlbumin 2 7 ofer 2735 s1-Casi 3 9 ?s2en 8 5 ?-asi 6 12 ? 69 Table 5: BIOPEP analysis showing the potential number of bioactive peptides excised using combinations of common proteolytic enzymes. The first column names the porcine milk proteins and the subsequent columns list the number of bioactive peptides excised using combinations of pepsin, trypsin, chymotrypsin, elastase I, and elastase II respectively. ? 70 DISCUSSION Previous studies on proteins found in porcine milk focused on those necessary to maximize the health of the piglet. Transfer of milk-borne antibodies from mother to offspring is particularly important in pigs because the epitheliochorial nature of the placenta prevents transfer of immunoglobulins into neonatal circulation [4]. This, combined with an undeveloped immune system in neonatal pigs at birth, makes ingestion of colostrum crucial in the short period where the neonatal gut is open to absorption of whole macromolecules. Nevertheless, as can be inferred from studies of pigs [5] and metatherians [100, 108], lactocrine communication of bioactive factors from mother to offspring is likely to represent a conserved mechanism for maternal regulation of extrauterine development of mammalian neonates. Present results are consistent with observations indicating considerable changes in porcine milk quality during the transition from colostrum to mature milk [1, 3]. As expected, both qualitative and quantitative changes were documented for the array of proteins found on LD 0 as compared to that identified on LD 6. Of the 304 total spots defined as porcine colostral and milk proteomes, 25 were unique to colostrum and 15 were unique to mature milk (Figure 4). Quantitative changes were also observed; 53 spots decreased at least 2-fold while 105 spots increased at least 2- fold in abundance between days (Figure 5). Interestingly, nearly half of the spots downregulated at least 2-fold from LD 0 were those determined to be unique to colostrum (Figure 6, Table 1). Fairly liberal PDQuest settings were used to establish conditions for identification of these spots by their pI and molecular weight coordinates using TagIdent. This may explain why this approach to identification of ? 71 peptides and proteins in colostrum and milk proved to be less than accurate and of minimal objective value. Many of the spots matched using TagIdent had potentially hundreds of candidate protein identities matched to a specific pI and molecular weight range. As a consequence, no consensus sequence or related peptide/protein identify could be defined. However, some systematic patterns of change in colostral/milk proteomic patterns were observed between days. Spots unique to colostrum and those downregulated at least two-fold from LD 0 were observed, in general, to have higher molecular weights and pI estimates compared to those spots defned to be unique to LD 6 milk and those upregulated two- fold from LD 0. (Figures 4 and 5) Variation in proteomic profiles was also seen between individual sows of the same experimental group within lactation day. This could be explained by the natural variation that exists between individual dams. However, evidence that milk protein composition varies depending on mammary gland location was recently reported. Wu and colleagues [125] used 2DE procedures to identify proteomic differences in porcine colostrum and LD 14 milk from anterior and posterior mammary glands [125]. In the present study, care was taken to collect milk from multiple teats in different locations. Therefore, variation of the kind described by Wu et al [125] should have been masked. Careful visual comparisons of proteomic profiles between the present study and images published by Wu et al [125] revealed a number of similarities, including the location of several proteins identified in their study by mass spectrometry. A high degree of consensus was observed upon visual comparison of gel images presented by Wu et al [125] with LD 0 gel images generated in this study. Interestingly, some spots identified by Wu et al ? 72 [125] using mass spectroscopy were also identified here using TagIdent. For example, albumin (SSP 5502 in appendix Table 6) was found in the same area in both of these studies. Nevertheless, positive identification of these peptides and proteins will require more sophisticated analysis such as mass spectrometry. A hidden component of the porcine colostral/milk proteome, not visible in 2DE images, is the presence of potentially bioactive peptides encrypted within larger, parent proteins. These peptides are generally a few amino acids in length and require enzymatic release from their milk-protein precursors [2]. Meisel and colleagues [2, 83] studied the activities of encrypted peptides in bovine milk. This group determined that such encrypted peptides can be divided into four general categories including peptides that affect gastrointestinal function, those that modulate postprandial metabolism or those that function in antimicrobial defense or immunoregulation [2, 83]. Potentially bioactive peptides in similar categories were identified in porcine milk proteins. Though the porcine neonatal GI tract is immature, some proteolytic enzymes are present and active early in development. Although chymosin is the predominant enzyme in the stomach at birth, it has weak proteolytic activity [126]. Trypsin, chymotrypsin, pepsin and pancreatic elastases are present in variable levels in the young pig [126, 127]. Analysis of the primary sequences of common porcine milk proteins using BIOPEP, a database for identification of encrypted bioactive peptides, revealed the presence of potentially thousands of bioactive peptides present within milk-borne proteins (Table 3) and hundreds that may be released as a result of enzyme action (Table 4 and Table 5). ? 73 Results reported here not only illustrate the number of potentially bioactive peptides that can be identified within porcine milk proteins, they also emphasize the extreme complexity of the potential lactocrine signaling domain after degradation of MbFs. The positive correlation identified between the size of parent milk proteins and the incidence of specific encrypted peptides occurring three or more times per parent molecule (Figure 7 and 8) was expected. These relationships reinforce the idea that molar quantities of potentially bioactive peptides encrypted in colostral/milk proteins could be available to be unleashed in the neonate and affect development and growth. This dimension of lactocrine signaling remains to be explored systematically. Changes occurring in the porcine milk proteome may reflect changing needs of the neonatal piglet in a manner similar to that described for lactationally dependent tammar wallaby development [100]. Genomic studies analyzing the relationship between milk and mammary genes from marsupials, monotremes, and placental mammals revealed that milk and mammary genes are more likely to be present in all mammals and are highly conserved across Mammalia [10]. These relationships are likely to reflect the importance of such factors in the mammalian reproductive continuum. 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Mol?Recognit?1996;?9:?407?414.? 124.?Boehmer?JL,?Bannerman?DD,?Shefcheck?K,?Ward?JL.?Proteomic?analysis?of? differentially?expressed?proteins?in?bovine?milk?during?experimentally? induced?Escherichia?coli?mastitis.?J?Dairy?Sci?2008;?91:?4206?4218.? 125.?Wu?WZ,?Wang?XQ,?Wu?GY,?Kim?SW,?Chen?F,?Wang?JJ.?Differential? composition?of?proteomes?in?sow?colostrum?and?milk?from?anterior?and? posterior?mammary?glands.?In:?J?Anim?Sci,?vol.?88.?United?States;?2010:? 2657?2664.? 126.?Shen?WH,?Liechty?EA.?Digestion?and?Absorption.?In:?Xu?RJ,?Cranwel?PD? (eds.),?The?Neonatal?Pig.?Nottingham,?UK:?Nottingham?University?Press;? 2003:?157?184.? 127.?Gestin?M,?Le?Huerou?Luron?I,?Peiniau?J,?Le?Drean?G,?Rome?V,?Aumaitre?A,? Guilloteau?P.?Diet?modifies?elastase?I?and?II?activities?and?mRNA?levels? during?postnatal?development?and?weaning?in?piglets.?J?Nutr?1997;?127:? 2205?2211.? ? ? 83 Appendix A. TagIdent Identification of spots downregulated at least two-fold from LD 0 SSP MR x 10 -3 pI TagIdent IDProtein Name 406 44.16 3.3 Unkow o Matches 1305 30.78 4.18 N Cnu 1603 73.95 4.01 nko o atches 1604 74.47 4.22 CAD1_PIG drin 1 2203 25.89 4.5 Unkow No Cseus 2406 45.88 4.54 n 2606 71.02 4.49 RSCA1_PIG Regulatory Slute arie Protein 3005 10.54 5.17 ITL Cathlin 3207 25.46 4.96 F12_PI oagultio Fctor XI 3210 19.94 5.13 AG Alph 1cid Glyptein 3502 68 4.71 GHR_PI Growt Hormne Rctor 3505 67.34 4.88 C1S Cmplent C1S ubpnet 3611 86.1 5.23 AD5_PIG adherin 5 4208 26.14 5.8 Unkow No sus 4603 85.14 5.42 Cne 4606 86.49 5.68 nko o sus 5502 63.11 5.95 ALBU_PIG Serum Albin 6201 24.13 6.52 OM polirotei M 6305 36 6.87 nkow N Cnsus 6403 52.04 6.64 U o e 6603 74.51 6.92 nko nsus 7302 31.89 7.22 IBP2_IG Insulin-like Growth Factor BP 2 7304 35.6 7.43 Unkow N Cnseus 7305 28.65 7.72 o 7306 30.18 7.97 nko nseus 7307 37.82 7.74 Uw No C 7505 66.64 7.44 TPA_IG Tisue yp Plaminoge Activaor 7506 62.08 7.71 FIB Fibrnoge lpha Cin 7507 67 7.76 _PI i l i 7508 61.08 7.82 G6 Glucose 6Phospate Isomeras 7510 62.32 8.01 PI_ l t 7512 55.94 8.2 Unkow No Cnseus 7602 74.55 7.16 7605 80.29 7.64 nko o nseus 7606 80.49 7.77 Uw N C 7608 70.6 7.96 nko o nseus 7703 93.8 7.24 PLMN_PIG Plamiog 8106 15.31 8.96 Unkow No Cnseus 8308 28.91 9.19 8309 35.97 9.25 LEG4_PI Galcetin4 8404 46.58 9.17 Unkow No Consensus ? 84 SSP MR x 10 -3 pI TagIdent IDProtein Name 8501 67.38 8.2 Unkow o Cnseus 8504 68.44 8.73 SC5A_PIG Sodium/glc otraporte 5 8505 69.07 8.84 F Caltion Fat Vlight cain 8506 68.75 8.94 5_PI ogulti ctor lit i 8603 70.22 9.15 Unkow N Cnseus 8706 106.41 8.93 TLR9_PIG Tol Lik Rcptor 9 8707 108.22 9.15 nko onseus 8709 67.2 9.2 Uw N C 8711 90.7 8.78 nko o nseus 8717 45.6 9.2 8720 97.9 9.1 Unkow No Cnseus Table 6: Preliminary spot identification of 53 spots downregulated at least 2-fold from LD 0. For clarity, SSP numbers are not tracked to a table. Molecular weight and isoelectric point for each spot are shown. Tagident ID and preliminary protein identification are also listed. ? 85 Appendix B. Tagident ID of spots upregulated at least two-fold from LD 0 SSP Mr x10 -3 pI TagIdent IDProtein Name 403 43.9 3 Ukow atches 4041.1n no 407 0.5 PEA_IG Pepsi APrecusor 4094863.65C Gatin 1103 1. 41 THRBI rohmbi 1201237.78Unkown atces 1203 .95 39 12054.0 No Csenus 1306 37.26 2 CHYM_PIG hymi (ri) 1407409.16Unkow ses 1408 5. 4 D4N51IG Calrticuln 1504631. No ses 1701 90.4 385 nko 17038754.1Uw Cnseus 2108 13. 6 LAB_PIG Alpha-Lctalbmin 230120. nko No ses 2303 .5 45 Cnu 2305369.6Uw ses 2402 40.8 3 nko o 240374.D4N51_PIGCalreticuln 2604 . 2 N ss 2608832.5Unkowoe 2611 76.5 46 Cnsus 270191.2 e 2705 04. TSP4 Thrombspodin4 27075364.51 2708 98. 6 Unkow N Cnseus 28011.2 o 2806 .5 438 ses 3001234.1nko Cnu 3004 10 5 Uw No ses 310317.564.9LACB_PIGBetaLctgloblin 3104 4 06 u 31052.5.2I Alpha-ctalmi 3204 195 4.87 LCB_P Bet Logbln 3206.74.9AS1IGlaS1 Casei 3208 26 503 T Cathepsin Hvyhai 3209.01.8PO_I Aolrotein A-1 3211 75 2 CSB Beta Cs 330128.4.7Unkownmche 3401 493 81 o ats 34035.15.02 ce 3404 68 nko n aths ? 86 SSP MR x 10 -3 pI TagIdent IDProtein Name 3602 87.5 4.81 Ukow tchs 3603292n noae 3609 6.3 .97 mtces 3701843ENPL_IGEdplasin 4102 1. 5. CAS2 Alh S2Ce 4103346I aasi 4104 2.6 .7 YT1_P ystin A-1 42035851CSIGlpha SCasei 4206 1.3 .6 AOR Aoirot R 44024942Unkown maches 4406 5.67 5.9 CLS_PI Clustrin 45043 o aces 4505 . .7 nko nth 45075302581Uw maces 4605 86. .6 ot 4607717nko n aches 4703 9. 5.41 t 470406252Uwo maces 4705 . .7 nko nth 500419363S10A_PIGPrtei S10-A 5102 2.46 .01 TFB3 TransfogGrowt factor Beta 3 510387Unkown mahes 5104 16.64 6.3 SODC_PI SuperxideDisut (Cn, Z) 510612.52TGFB3Transfog rofactorBeta3 5201 96 .1 RIHI FeritnHevy hi 52043.63CASK_PKap Casn 5205 21 .2 I ei 52077.47Unkowno matchs 5402 53 6.01 e 54051. 24 atcs 5507 672 .35 nko nohe 56040.461Uw matcs 5606 83 .4 e 57019.610nko no atchs 5705 207 6.31 e 5708. 45Uw matcs 6203 146 .9 nko nohe 65022.06 N Csnus 6701 95 .57 e 6703.4Unkowo ss 6704 21 6. Cneu 67059. 8 N ss 7001 7.25 B2MG BetaMicroglblin 70021.5 u 7105 89 . IL10_PI Interlki 10 7702.721UnkowNo Csns 8006 17 8. I3IG Iterlui 3 80082.090 ss ? 87 SSP MR x 10 -3 pI TagIdent IDProtein Name 8010 .4 9.24 ELAF_PIG Elfi 810217583Unkowo Cnseus 8104 2. . QN3I CarbhydratBidgProtein 810786902TFB_PLmpx Alpha 8110 13.79 . IL4IG Interlukin4 8201287SAMSeru Ayod PComnet 8704 6.8 .6 Unkow N ses 900114954NFB_PI atritic ptiB 9104 5.2 .7 o Cnseus Table 7: Preliminary spot identification of 105 spots downregulated at least 2- fold from LD 0. For clarity, SSP numbers are not tracked to a table. Molecular weight and isoelectric point for each spot are shown. Tagident ID and preliminary protein identification are also listed ? 88 Appendix C. Milk Collection Materials: Latex gloves 15 ml Fisherbrand centrifuge tubes Rescue decks Cheese-cloth Procedure: 1. Sows were milked manually following the birth of the first piglet and milked again 6 days later. 2. Teats were milked directly into centrifuge tubes, with the cheese-cloth serving as a filter for foreign particles. 3. Piglets were temporarily separated from the mother for 1 hour in order to collect a significant volume of milk at LD 6. After 1 hour, 2-3 piglets were reintroduced to induce milk let down. This process was repeated several times in order to obtain a significant volume of milk. 4. Samples were put on ice and stored at -4.0?C Considerations: 1. Teats were cleaned with warm water before collection. 2. Milk was collected from as many teats as physically feasible from each sow. ? 89 3. Bloody or injured teats were not included in the study. ? 90 Appendix D Milk Preparation: Lipid Removal and Protein Solubilization Materials: 2.0 ml Eppendorf centrifuge tubes 0.2 M EDTA pH 7.0 10 M NaOH Stir bars pH sensor (Symphony) Centrifuge (Fisher Scientific Marathon 26KM) Procedure: 1. Create 0.2 M EDTA 37.22g EDTA in 500 ml distiller water. Add stir bar to bottle. 2. Use pH sensor and 10 M NaOH to adjust pH of stock EDTA to 8.0 to solubilize the EDTA. Add sequential drops of HCl to reduce pH to 7.0. 3. Add 0.8 ml EDTA to 0.8 ml thawed and vortexed milk samples 4. Centrifuge for 15 minutes at 6000 rpms. 5. Remove whey fraction with 1.0 ml pipette and transfer to new centrifuge tube. 6. Centrifuge for 15 minutes at 6000 rpms. 7. Remove whey fraction from any remaining lipids and transfer to new centrifuge tube. 8. Store at -20?C. ? 91 Appendix E RCDC Protein Assay Materials: Prepared milk/EDTA samples RCDC Protein Assay Kit (Bio-Rad) BSA standard 0.1 M TRIS 1.5 ml centrifuge tubes (Eppendorf) Vortex Genie Mixer 96 well plates (Bio-Rad) Densitometer (Molecular Devices) Centrifuge (Fisher Scientific Marathon 26KM) Procedure: 1. Create stock BSA solution. 1.25 mg/ml 2. Create 1-5 serial dilution for standards using 0.1 M TRIS as buffer. Make enough volume for triplicate wells. 3. Create 100X and 200X dilutions of all milk samples using 0.1 M TRIS. 5?l sample / 495?l TRIS for 100X 200?l 100X / 200 ?l TRIS for 200X 4. Add 25?l samples and standards to labeled 1.5 ml centrifuge tubes. ? 92 5. Add 125?l of RC Reagent I to each tube. Incubate for 1 minute. 6. Add 125?l of DC Reagent II to each tube. Centrifuge at 15,000 g for 4 minutes. 7. Discard supernatant. Remove remaining supernatant with a quick spin in the centrifuge and a gel loading pipette tip. 8. Create Reagent A? by adding 60.96?l of Reagent S and 2987?l of Reagent A. 9. Add 127?l of Reagent A? to each sample/standard. Vortex. Incubate at room temperature for 5 minutes. 10. Add 1ml of Reagent B to each sample/standard. Vortex. 11. Add 300?l of sample/standard to each well. Each sample is done in triplicate. 12. Incubate at room temperature for 15 minutes. 13. Insert well plate in densitometer for analysis. ? 93 Appendix F. Sample Preparation: Sequential Extraction 3 Materials: Sequential Extraction 3 Kit (Bio-Rad) Tributyl Phosphine (TBP) PMSF Vortex Genie Mixer Centrifuge (Fisher Scientific Marathon 26KM) Procedure: 1. Mix a 1:100 ratio of TBP and Reagent 3 to create extraction buffer in the hood. Vortex 2. Calculate 500?g of protein in a volume based on the RCDC protein assay and transfer to 1.5ml centrifuge tube. 3. Add 200?l of extraction buffer to each sample. 4. Add 8?l of PMSF to each sample. Incubate at room temperature for 15 minutes. 5. Vortex for 5 minutes. Centrifuge at 10,000 g for 5 minutes. 6. Recover supernatant and proceed to 2D Clean-Up Kit protocol. ? 94 Appendix G. Sample Preparation: 2D Clean-Up Kit Materials: Vortex Genie Mixer ReadyPrep 2D Clean-Up Kit (Bio-Rad) Centrifuge (Eppendorf 5417R) ReadyPrep 2-D Starter Kit Rehydration/Sample Buffer (Bio-Rad) Procedure: 1. Add 300?l of Precipitating Agent I to each sample. Incubate on ice for 15 minutes. 2. Add 300?l of Precipitating Agent II to each sample. Vortex. 3. Centrifuge at 15,000 g for 5 minutes. 4. Discard supernatant. 5. Add 40?l of Wash Reagent I. Centrifuge at 15,000 g for 5 minutes 6. Remove supernatant and transfer to new microfuge tube. 7. Add 25?l of nanodrop water to each sample. Vortex for 30 seconds. 8. Add 1ml of Wash Reagent II to each sample. Vortex for 1 minute. 9. Add 5?l of Wash Additive to each sample. Vortex. 10. Incubate at -20?C for 30 minutes. Every 10 minutes vortex for 30 seconds. ? 95 11. Centrifuge at 15,000 g for 5 minutes. Discard supernatant. Air-dry tubes for 5 minutes. 12. Re-suspend each sample with 50?l Rehydration Buffer. 13. Store at -20?C. ? 96 Appendix H. Isoelectric Focusing Materials: Protean IEF cell (Bio-Rad) ReadyStrip IPG Strip 11cm pH 3-10 (Bio-Rad) ReadyPrep 2-D Starter Kit Rehydration/Sample Buffer (Bio-Rad) Loading Trays (Bio-Rad) Mineral Oil (Bio- Paper Wicks Procedure: 1. Thaw IPG strips and prepared samples on ice. 2. Transfer 75?g of protein by volume from samples and add enough rehydration buffer to total 185?l. Vortex. 3. Transfer the 185?l of sample onto their respective loading trays evenly. 4. Remove plastic backing from 11cm IPG strips and load gel side down onto loading wells, taking care that there are no bubbles under the sample. Incubate for 1h at room temperature. 5. Cover each strip with mineral oil and incubate at room temperature overnight. 6. Cover IEF cell tray electrodes with paper wicks weighted down with nanodrop water. ? 97 7. Load IPG strips gel side up onto IEF cell tray and cover strips with mineral oil. 8. Load tray onto IEF cell focus strips for 20 hours per the following protocol: 500V for 1h, 1000V for 1h, 2000V for 2h, 4000V for 4h, 8000V for 12h. 9. Remove tray from IEF cell and transfer IPG strips gel side up to clean loading tray. Cover with plastic wrap and freeze immediately at -80?C. ? 98 Appendix I. Equilibration and 2D-SDS-PAGE Materials: SDS Urea Tris Glycerol Acetic Acid Methanol Iodoacetamide (Bio-Rad) Dithiothreitol (DTT) (Bio- Focused IPG strips (Bio-Rad) Bromophenol Blue Agarose (Bio-Rad) Precision Plus Unstained Protein Standard (Bio-Rad) Loading Trays (Bio-Rad) Orbital Shaker (Stovall) Power Pack 300 (Bio-Rad) Criterion Dodeca Cell (Bio- Water Circulator / Cooler (Brinkmann) Criterion Tris-HCl Gels 10-20% 11cm Sypro Ruby Protein Gel Stain ? 99 Buffers: 1. 60ml Equilibration Buffer I. 6M Urea, 0.375M Tris pH 8.8, 2% SDS, 20% Glycerol, 2% (w/v) DTT 2. 60ml Equilibration Buffer II 6M Urea, 0.375M Tris pH 8.8, 2% SDS, 20% Glycerol, 2.5% (w/v) Iodoacetamide 3. 10X and 1X tank buffers. To make 1L of 10X tank buffer: 30g Tris, 144g Glycine, 10g SDS, bring to volume using distilled water. 6L 1X tank buffer: Mix 600ml 10X tank buffer with 5.4L distilled water. 4. Fixative solution. 40% Methanol, 10 % acetic acid 5. De-stain solution. 10% Methanol, 7% Acetic acid Procedure: 1. Thaw focused IPG strips at room temperature until gel is pliable and translucent. 2. Cover each strip with 4ml of Equilibration Buffer I. Place tray in orbital shaker and incubate for 30 minutes. 3. Carefully decant buffer I from tray. Cover each strip with 4ml of Equilibration Buffer II. Place in orbital shaker and incubate for an additional 30 minutes. 4. Melt the agarose overlay during the incubation period. ? 100 5. Prepare the SDS-PAGE gels by removing combs and blotting away excess water from the IPG well. 6. Fill dodeca tank with approximately 5L of 1X tank buffer and connect to water circulator. Set temperature to 20?C. Place 2 stir bars at the bottom of the tank 7. Mount the tank on stir plate. 8. Remove IPG strips from buffer II and dip strips briefly in 1X tank buffer. 9. Lay strips gel side up on back casing of SDS-PAGE gel directly above the IPG well. 10. Use pipette to place overlay agarose solution into the IPG well of the gel. 11. Gently push IPG strip into the well using forceps or a spatula. 12. Place SDS-PAGE gels in slots of the dodeca tank. 13. Load 10?l of protein standard in standard well of the gel. 14. Fill reservoirs with 1X tank buffer. 15. Connect dodeca to powerpack and begin electrophoresis with running conditions of 200V for approximately 1 hour or until bromopehnol blue line is 1cm from bottom of the gel. 16. Remove gels from casing after electrophoresis is complete. Place gels in 100 ml of fixative solution and incubate for 1 hour on orbital shaker. 17. Remove fixative and add 60 ml Sypro RUBY stain to each gel. Cover containers in aluminum foil and incubate overnight on orbital shaker. 18. Remove stain and add 100 ml of destain solution to each gel. Incubate for 2 hours on orbital shaker. Minimize light exposure to gels. ? 101 19. Remove destain and wash gels in distilled water before imaging. Minimize light exposure to gels. ? 102 Appendix J. 2-D Gel Imaging Materials: Typhoon 9410 Variable Mode Imager PC with ImageQuant version 5.2 Squirt bottle with distilled water Procedure: 1. Open Typhoon Scanner Control on the desktop. 2. Change acquisition mode to fluorescence. 3. Click Set-Up button ? Emission Filter ? Sypro RUBY 4. Set up scan size to an H-6 grid 5. Open the scanner and carefully place a single gel on the bottom left corner. Use water to remove any air bubbles that appear under the gel. 6. Close the scanner and press the scan button on the PC. Save and name the gel image when prompted. Repeat for all gels. 7. Open ImageQuant on the desktop. 8. Open all gels from ImageQuant. Click Save As ? File Type TIFF file. Save. ? 103 Appendix K. Gel Analysis: PDQuest Materials: 2-D gel images PC with PDQuest Advanced 2-D Analysis Software version 8.0.1 Microsoft Excel Cropping Images: 1) Open PDQuest software and close the experiment wizard that appears. 2) Click File ?Open open collected 2-D gel images. 3) Click the ?Advanced Crop Tools? tab ? Crop select area of interest of gel. Crop an area that includes the molecular weight markers but avoids all four edges of the gel. 4) Select the ?Crop and Save? button that appears. Name and save the new gel images. 5) Click the ?Save Crop Settings? button under the ?Advanced Crop Tools? tab. Name the settings. 6) Click ?Load Saved Cropped Settings? under the ?Advanced Crop Tools? tab for each subsequent gel. Repeat step 4 for every gel. The crop size and gel size will be the same for each gel. New Experiment Wizard ? 104 1) Click ?New Experiment? in PDQuest Analysis Quick Guide. 2) Name the experiment and click ?Next?. 3) Click the ?Add? button and select all the images cropped using PDQuest. Click ?Next?. 4) Group replicate gel images. LD 0 and LD 6 gels were grouped separately. 5) Select the ?Use Spot Detection Wizard? when prompted. Select ?Warp Gel Images Before Matching? under Matching Options. Spot Detection Parameters Wizard 1) Click on the ?Advanced? tab on the top right to open up all the options. 2) Zoom in on an area of the gel with a low spot concentration. Select the ?Click on a faint spot? button and find the faintest spot on the gel. 3) Follow the same steps for the ?Click on a small button if different from the faint spot? button and ?Box the largest spot cluster?. The casein clusters around pI of 5.0 MW of 30 kD was chosen as the largest spot cluster. 4) Under ?Test Settings? check all three options. Change ?Sensitivity? to 8.00, ?Size Scale? to 5. Leave the ?Min Peak? unchanged. 5) Under ?Optional Controls?: a) Streaks: Check ?Vertical? and ?Horizontal? b) Background: Do not change settings c) Smoothing: Do not change settings d) Speckles: Check ? Apply speckle filter?. Change ?Sensitivity? to 200 ? 105 6) Click the ?Proceed? button. Click ?Next? until software begins to count the spots for each gel. Do not change any of the standard settings. When analysis is completed, select ?Open Spot Review Tool? when prompted. Spot Review: 1) This tool is used to make sure that the spots identified by the software are real and accurately matched to each other. Each histogram numbers each spot and represents the relative abundance of each matched spot per gel. 2) Open the ?Edit Spot Tools? tab. 3) Click on each histogram and inspect the spot matching accuracy in each gel. 4) If the spot is false use ?Cancel spot at cursor? under the ?Edit Spot Tools? tab and cancel the false spot in each gel. 5) If the software identifies a large saturated spot as multiple spots, click on ?Combine spots in a box? button under the ?Edit Spot Tools? tab and draw a square around the spots to combine them into a single spot. 6) Repeat steps 4 and 5 for every histogram to improve the accuracy of the matching software. 7) Make sure to cancel all the spots associated with the MW markers. Creating Analysis Set for Qualitative Changes: 1) Click on ?Analysis? and select ?Analysis Set Manager?. 2) Click on ?Create? and select ?Qualitative?. 3) Name the analysis set (On LD 0, Off LD 6). ? 106 4) Check the ?Repl. Gels? button under ?Compare? and select LD 0 for ?A? and LD 6 for ?B?. Check ?Estimate missing spots? button and ? Estimate saturated spots? button under ?Options?. 5) Spot count should appear on the lower left hand corner. Click on the ?Save? button. 6) Repeat steps 1-5 to analyze for spots unique to LD 6. For step 4 choose LD 6 for ?A? and LD 0 for ?B?. Creating Analysis Set for Quantitative Changes: 1) Click on ?Analysis? and select ?Analysis Set Manager?. 2) Click on ?Create? and select ?Quantitative?. 3) Name the analysis set (2-Fold Change from LD 0 to LD 6) 4) Check the ?Repl. Gels? button under ?Compare? and select LD 0 for ?A? and LD 6 for ?B?. Check ?Estimate missing spots?, ? Estimate saturated spots?, and ?Include qualitative changes? under ?Options?. 5) Check ?Outside limits? under ?Method? 6) Spot count should appear on the lower left hand corner. Click on the ?Save? button to accept changes. 7) To see spots that increase in expression, select ?Upper limit? under ?Method?. To see spots that decrease in expression, select ?Lower limit? under ?Method?. Determining MW and pI: ? 107 1) Click on the ?Mr, pI Standard Tools? tab and select ?Enter Mr, pI data for spot?. 2) Estimate the MW and pI for 5-7 spots on various locations of the gel, include areas around the 4 corners as well. 3) After estimating 5-7 spots, the software will be able to plot a MW and pI grid over the gel and will be able to estimate the MW and pI for the remaining spots. Exporting Spot Data and Gel Images 1) Click on ?File? ? Export Export (Text) Experiment. 2) Specify the analysis set to export and leave default settings unchanged. 3) Specify analysis set under ?Spots to export?. Check ?Clipboard? under ?Export to?. Click on the ?done? button 4) Open Microsoft Excel, Edit? Paste 5) To export gel images click on ?File? Export ? Export JPEG image. Click on the image to be exported. 6) Click ?Export?. Image will be saved on the desktop. ? 108 Appendix L. Spot Identification Using Bioinformatic Methods Materials: Spot MW and pI data ExPASy Proteomics Server (http://ca.expasy.org/tools/tagident.html) Procedure: 1) Obtain the MW and pI data for each spot. For the maximum and minimum pI range, enter the actual pI given by PDQuest + 0.01 respectively. 2) Enter the MW in the specified box. Reduce the MW range to 10% error. 3) Check the box with ?Check for protein sequences with cysteines in reduced form (-SH)? 4) Under ?Organism name or classification? input Sus scrofa. 5) Scroll down the screen and click on the ?Start Tagident? button. 6) If minimal or no results appear, input mammalia under ? Organism name or classification?. ? 109 Appendix M. Encrypted Peptide Identification Materials: BIOPEP database (http://www.uwm.edu.pl/biochemia/index_en.php) NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/) List of porcine colostral /milk proteins Finding Primary Amino Acid Sequences of Porcine Milk Proteins: 1) Access the NCBI protein database and type the name of the target protein in the ?Search? box. 2) Click on the protein name. Under ?Display Settings? select ?FASTA? and click ?Apply?. 3) Copy and paste the sequence to a text document for future reference. Determining Potential Biological Activity of Encrypted Peptides: 1) Access the BIOPEP database. Select the ?BIOPEP? tab and click on ?Bioactive Peptides?. 2) Select the ?Analysis? tab and click on ?Profiles of Potential Biological Activity?. Select the ?For Your Sequence? button. 3) Copy and paste the selected amino acid sequence onto the search box. Click ?Report? on the far right of the page. ? 110 4) The website will display all potential bioactive peptides for the inputted sequence. Determining Possible Encrypted Peptides After Enzymatic Cleavage: 1) Access the BIOPEP database. Select the ?BIOPEP? tab and click on ?Bioactive Peptides?. 2) Select the ?Analysis? tab and click on ?Enzymes Action?. Select the ?For Your Sequence? button. 3) Copy and paste the selected amino acid sequence onto the search box. Under ?Select enzymes? choose the enzymes required for analysis. Click on the ?Report of enzyme action? button. 4) Under results, click on the ?Search for active fragments? button. The database will search the cleaved protein for bioactive peptides within the BIOPEP database. ? 111 Appendix N. Raw 2D Gels Sow 129 LD 0 Gel Run Date: 5/28/09 Sow 129 LD 0 Gel Run Date: 6/11/09 ? 112 Sow 136 LD 0 Gel Run Date: 5/28/09 Sow 136 LD 0 Gel Run Date: 6/11/09 ? 113 Sow 192 LD 0 Run Date: 5/28/09 Sow 192 LD 0 Run Date: 6/11/09 ? 114 Sow 223 LD 0 Run Date: 5/29/09 Sow 223 LD 0 Run Date: 6/11/09 ? 115 Sow 126 LD 6 Run Date: 5/11/09 Sow 126 LD 6 Run Date: 6/11/09 ? 116 Sow 129 LD 6 Run Date: 5/28/09 Sow 129 LD 6 Run Date: 6/11/09 ? 117 Sow 188 LD 6 Run Date: 5/28/10 Sow 188 LD 6 Run Date: 6/11/10 ? 118 Sow 192 LD 6 Run Date: 5/28/09 Sow 192 LD 6 Run Date: 6/11/09 ? 119 Sow 223 LD 6 Run Date: 5/28/09 Sow 223 LD 6 Run Date: 6/11/09