Effect of Supplementing an Omega-3 Fatty Acid-Containing Product to Pig Diets on Growth and Immune Responses of Weanling Pigs


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Effect of Supplementing an Omega-3 Fatty Acid-Containing Product to Pig Diets on Growth and Immune Responses of Weanling Pigs
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Li, Qizhang
University of Florida
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Master's ( M.S.)
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University of Florida
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Animal Sciences
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growth -- immunity -- omega -- pig -- weanling
Animal Sciences -- Dissertations, Academic -- UF
Animal Sciences thesis, M.S.
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The recognition that omega-3 polyunsaturated fatty acids (n-3 PUFA) possess potent anti-inflammatory properties in human models has prompted studies investigating their efficacy for animal growth and immunity. The objective of this study was to examine the effect of feeding an n-3 PUFA-enriched diet on growth and immune response of weanling piglets. Newly weaned pigs (averaging 27 2 days of age and 8.1 0.7 kg of body weight were assigned randomly to receive a control (3% vegetable oil, n = 20) or n-3 PUFA-supplemented (3% Omega, n = 20) diet for 28 days after weaning. Diets were formulated to be isocaloric (3.3 Mcal / kg of diet) and isolysinic (14 g Lys / kg of diet). A diet gender week interaction was detected (P < 0.04) for body weight. Female pigs consuming the n-3 PUFA-enriched diet were lighter (P <0.01) at week 4 post-weaning than their counterparts fed the vegetable oil-supplemented diet. Newly weaned pigs gained more weight (P < 0.01), consumed more feed (P < 0.01) and had better feed efficiency (P < 0.01) between day 14 and 28 than between days 0 and 14 post-weaning. Peripheral IGF-1 concentration decreased (P < 0.01) between days 0 and 14 after weaning and then increased again by day 28 post-weaning. In piglets consuming the vegetable oil-enriched diet, plasma TNF concentration increased (P < 0.04) from 33.7 16.9 pg/mL to 92.7 14.1 pg/mL between days 0 and 14 post-weaning and remained elevated through out the remaining weeks of the study. The weaning-induced increase in serum TNF was completely abolished by adding n-3 PUFA to the piglet diet. With the exception of blood platelets and eosinophil cells, dietary n-3 PUFA had minimal effects on hematological characteristics. Fecal consistency scores improved (P < 0.01) with increasing weeks after weaning, but were similar between the two dietary treatments. Results of this study provided no evidence for n-3 PUFA modulation of fecal consistency score or hematological parameters of weanling piglets raised in the absence of significant immunological and environmental challenges. However, female pigs consuming the n-3 PUFA-supplemented diet were lighter at week 4 post-weaning compared with control group. Dietary n-3 PUFA may improve the immune status of weanling pigs, as reflected by considerably lower plasma TNF in pigs consuming n-3 PUFA than those fed a vegetable oil-enriched diet. Furthermore, the gradual increase of body weight, feed intake and feed efficiency following weaning may reflect a slow adaptation to post-weaning diets and a gradual improvement of the gastrointestinal microbiota.
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by Qizhang Li.
Thesis (M.S.)--University of Florida, 2014.

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2014 Qizhang Li


To all the people who ever helped me


4 ACKNOWLEDGMENTS I would like to express my greatest appr eciation to my major professor, Dr. Lokenga Badinga, for his pat ient guidance, support and assistance throughout my graduate life. I have been very fortunate to be his student, and his active spirit regarding teaching, research and life truly built up the model for the rest of my life. Sincere thanks are also expressed to my co mmittee members, Dr. J oel H. Brendemuhl, for his assistance with designing and conducting my experiment, and Dr. John P. Driver, for his suggestions about immunological interpre tation of my data. I also appreciate Dr. Adegbola Adesogan, Graduate C oordinator of Animal Scie nces, and Dr. Geoff Dahl, Chairman of the Department of Animal Sciences. I am gr ateful to Joe Spencer at JBS United, for providing omega -3 fat supplement fo r my research. Sincere thanks are extended to Tom Crawfo rd, the manager of t he University of Florida Swine Unit, for his direction and assist ance during the field phase of this work. I am grateful to Dustin Loche and Christy Odom student assistants at the Swine Unit, for their help with animal handling during feeding, weighing and blood sample collection. Appreciations are extended to all the fri ends and colleagues at the University of Florida: Mr. Yan Guo, Ms. Zhengxin Ma, Ms. Chengcheng Li, Mr. Junyi Tao, Ms. Fei Du, Dr. Sha Tao, Ms. Yun Jiang, Ms. Maria Ceva llos, Ms. Leticia Del-Penho Sinedino, Mr. Marcos G. Zenobi and any others for thei r help and friendship during my graduate studies at the University of Florida. I really apprecia te Ms. Bianca Libanori Artiaga and Paula Morelli Mercadante, for their support and help during the field phase of this study. My appreciations are extended to everybod y who helped me during these two years when I was far away from home.


5 Finally, I would like to express my deepest gratitude to my family for their financial support during my ent ire graduate studies and for giving me the opportunity to be in the position I am in today. My father Mr. Xiaosu Li, inspired my interest to the biology world and my mother, Ms. Minglang Xi a, encouraged me to pursue my dream of studying in the U.S.A. Thanks to my sister, Xia Pu, for her suggestions about choosing my major and career path. Fi nally, my gratitude is exten ded to all my family members for their support and encouragement throughout t he graduate school and my whole life.


6 TABLE OF CONTENTS Page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 8LIST OF FI GURES .......................................................................................................... 9ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUC TION .................................................................................................... 12Nutrition and Health ................................................................................................ 13Sow Nutrit ion .................................................................................................... 13Neonatal Piglet Nutriti on ................................................................................... 17Weanling piglet nutrition ................................................................................... 21Physiological Chan ges at W eaning .................................................................. 25Immunity ................................................................................................................. 31Innate Imm unity ................................................................................................ 31Acquired Imm unity ............................................................................................ 36Inflammatory Mediator s .................................................................................... 41Nutrition and Immunity ..................................................................................... 45Omega -3 Polyunsaturated Fatty Acids ( n -3 PUFA) in Swi ne Nutrit ion ................... 52Definition and Biosynthesis of n -3 PUFA .......................................................... 52Effects of n3 PUFA on Growth ........................................................................ 55Effects of n3 PUFA on Inflamma tory Res ponses ............................................ 57Mechanisms of Action of n-3 PUFA .................................................................. 612 MATERIALS A ND METHOD S ................................................................................ 66Experimental Anim als and Die ts ............................................................................. 66Measurement of Growth and Feed Intake ............................................................... 68Blood Collection and Analysis ................................................................................. 68Fecal Eval uation ..................................................................................................... 69Statistical A nalysis .................................................................................................. 693 RESULT S ............................................................................................................... 70Body Weig ht ........................................................................................................... 70Average Daily Gain, Feed Inta ke and Feed E fficiency ............................................ 71Plasma IGF-I and TNF........................................................................................ 73Hematological and Fecal Characteri stics ................................................................ 744 DISCUSSI ON ......................................................................................................... 76


7 5 CONCLUS ION ........................................................................................................ 80APPENDIX: LEAST SQUARE S MEANS SIGNIFIC ANT DATA ................................ 81LIST OF RE FERENCES ............................................................................................... 83BIOGRAPHICAL SK ETCH .......................................................................................... 100


8 LIST OF TABLES Table page 1-1 Inflammatory cytokines and chemokines (Murray et al ., 2012) ........................... 422-1 Ingredient and calculated compositions of experimental diets (as-fed basis) ..... 662-2 Fatty acid profile (g/100 g of tota l fat) of experimental diets (as-fed)a ................. 673-1 Hematological traits of weanling pigs fed diets with vegetable oil or n -3 PUFAa................................................................................................................. 743-2 Fecal consistency scoresa of weanling pigs fed diets with vegetable oil or n-3 PUFAb................................................................................................................. 75A-1 Least squares means (+SEM) for body weig hts (kg) of male and female pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) ................... 81A-2 Least squares means (+SEM) for daily body weight gains (g) of weanling pigs fed diets enriched with vegetable oil (n = 20) or n-3 PU FA (n = 20) ............ 81A-3Least squares means (+SEM) for daily f eed intakes (g) of weanling pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) ......................... 81A-4 Least squares means (+SEM) for gain : f eed ratios of weanling pigs fed diets enriched with vegetable oil (n = 20 ) or n-3 PUFA (n = 20) .................................. 82A-5Least squares means (+SEM) for plasma in sulin-like growth factor-I (IGF-I) and tumor necrosis factor alpha (TNFconcentrations in weanling pigs fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) ......................... 82


9 LIST OF FIGURES Figure page 1-1 Omega-3 PUFA biosynthesis (adapt ed from Pereira et al., 2004). ..................... 543-1 Body weights of male (A) and fema le (B) pigs fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) for four weeks after weaning .......... 703-2Average daily gains of w eanling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) ............................................................................. 713-3Average daily feed intakes of wean ling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) ........................................................ 723-4Average gain : feed of wean ling piglets fed diets enr iched with vegetable oil (n = 20) or n -3 PUFA (n = 20) ............................................................................. 723-5 Concentrations of IGF-I (A) and TNF(B) in plasma of weanling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) ......................... 73


10 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science EFFECT OF SUPPLEMENTING AN OMEG A-3 FATTY ACID-CON TAINING PRODUCT TO PIG DIETS ON GROWTH AND IMM UNE RESPONSES OF WEANLING PIGS By Qizhang Li May 2014 Chair: Lokenga Badinga Major: Animal Sciences The objective of this study was to examine the effect of feeding an n -3 PUFAenriched diet on growth and immune response of weanling piglets. Newly weaned pigs (averaging 27 2 days of age and 8.1 0.7 kg of body weight were assigned randomly to receive a control (3% vegetable oil, n = 20) or n -3 PUFA-supplemented (3% Omega n = 20) diet for 28 days post-weaning. A diet gender week interaction was detected ( P 0.04) for body weight. Female pigs consuming the n -3 PUFA-enriched diet were lighter ( P at week 4 post-weaning than their count erparts. Newly weaned pigs gained more weight ( P 0.01), consumed more feed ( P 0.01) and had better feed efficiency ( P 0.01) between day 14 and 28 post-weaning. In piglet s consuming the vegetable oil-enriched diet, plasma TNFconcentration increased ( P 0.04) from 33.7 16.9 pg/mL to 92.7 14.1 pg/mL between days 0 and 14 post-weani ng and remained elevated through out the remaining weeks of the study. T he weaning-induced increase in serum TNFwas completely abolished by adding n -3 PUFA to the piglet’s diet. Fecal consistency scores


11 improved ( P 0.01) with increasing weeks after w eaning, but were similar between the two dietary treatments. Results showed female pigs consuming the n -3 PUFA-supplemented diet were lighter at week 4 post-weaning co mpared with control group. Dietary n -3 PUFA may improve the immune status of weanling pigs, as reflected by considerably lower plasma TNFin pigs consuming n -3 PUFA.


12 CHAPTER 1 INTRODUCTION The original efforts to use early weaning and segregation technologies to obtain pathogen-free piglets were attempted in the early 1980s (Alexander et al ., 1980). The early weaning technique has since been dev eloped and widely utilized in the United States swine industry (Fangman and Tubbs, 1997). This strategy improves the productivity of the sow herd, breaks the cycle of disease trans mission from th e facilities, but exasperates the weanling ch allenges of piglets (Corl et al ., 2008). Nutritional, environmental and immune challenges associat ed with weaning may lead to substantial economic losses to pork producers. This period is generally characterized by decreased voluntary feed intake, altered gut integrity and increased production of inflammatory cytokines (Le Dividich and Seve, 2000; Pie et al ., 2004; Montagne et al ., 2007). These nutritional and physiological ab normalities often result in diarrhea and depression of growth in newly weaned piglets. Restrictions of antibiotic usage in swine have compelled the industry to find alte rnatives that offer both performance enhancement and protection from diseas e (Cromwell, 2002; Vondruskova et al ., 2010). In this regard, Liu et al (2003) reported t hat dietary fish oil reduc ed the release of proinflammatory cytokines in weaned pigs challenged with E. coli lipopolysaccharide, and a more recent study indicated that prenatal exposure to long-chain n -3 PUFA increased postnatal glucose absorpt ion in piglets (Gabler et al ., 2009). Although exact mechanisms by which dietary n -3 PUFA modulate immune and metabolic functions in pigs are yet to be fully elucidated, the above study would indicate that dietary n -3 PUFA may help the piglets adapt quickly to t he rapid change of diet at weaning.


13 Currently, there is very little information regarding the use of n -3 PUFA in the diets of pigs raised under minimal disease and stress conditions. To test the hypothesis that nutritional management strategies that attenuate in testinal inflammation may provide a physiological milieu that is c onducive to optimal growth and lean tissue accretion, this study was designed to examine the effects of dietary n -3 PUFA on growth and immune response of weanling pi gs raised without an added bacterial or environmental challenge. Nutrition and Health Sow Nutrition Maintaining a good physical condition and achieving a high productive efficiency are always key factors of pr ofitability for sow feeding and management. A good health status may prevent disease outbreak and ext end the breeding age of a sow, which may have a shorter period of postweaning anestrus and farrow a litter of heavier piglets. Besides the genotype, management and facilit ies, sow nutrition is one of the prerequisites that determine good health an d productive efficiency. Gestation and lactation are two critical phases during t he reproductive cycle of the sow and thus, optimal nutrition is needed to meet the requirements of macroand micronutrients. During pregnancy, energy is partitioned bet ween maintenance, fetal growth and body tissue accretion. The priority is gi ven to maintenance, fetal development and mammary gland development. If nut rients are below these requirements, body reserves will be mobilized. Conversely, if nutrient intake exceeds t hese requirements, the excess will contribute to the sow’s body reserves (van Milgen et al ., 2008). Energy requirements for pregnancy vary according to BW, housing conditions, and the amount of maternal gain and lactation BW loss desired by the producer (Noblet et al ., 1990).


14 The National Research Council (NRC, 2012 ) suggested that the daily maintenance energy requirement was 105 kcal ME/kg metabolic body size (BW0.75), but the factorial approach is used to precisely determine feedi ng requirements of individual sows. As fetal growth and mammary development occur mainly in late gestation, the 2-phase feeding strategy may be more appropriate t han the constant diet single-phase feeding (Ji et al ., 2005; Mcpherson et al ., 2004). In addition, insuffici ent energy intake may allow successful lactation but will increase the weig ht loss of the sow at weaning and delay post-weaning estrus (Trottier and Johnston, 2001). Excess feed intake during gestation increases the growth of the fetus and the deposition of body fat and protein. But it reduces energy intake and increases weight loss during lactation. So, when increasing feed intake during the last 30 days to support t he rapid fetal growth, it is still necessary to limit energy intake and avoid excess weight gain by the sow for the entire gestation (NRC, 2012). During lactation, energy is partitioned betw een maintenance, milk production and lipid and protein mobilization from ma ternal body reserves (van Milgen et al ., 2008). The lactating sow requires 5-10% greater energy than during pregnancy (NRC 2012) and increased amounts of nutrients for milk production which is esti mated to be at least 78% of the net energy (NE) r equired for the modern prolific sow (Boyd and Kensingen, 1995). The sow’s feed intake directly affe cts the growth and dev elopment of the neonatal piglets via the milk produced, and also influences the reproductive efficiency of the sow. The energy needed daily is the sum of energy for maintenance and energy for lactation, and the energy amount associated with milk produc tion can be calculated as following:


15 Milk Energy (GE, kcal/day) = (4.92 ADG) (90 LS) (1-1) where GE means gross energy, ADG = average da ily gain of litter (g), and LS = number of pigs per littler (NRC, 2012). Improvements in energy intake may be achieved by supplementing diets with fat, which may im prove litter weight gain and subsequent reproductive performance of the sows (Rosero et al ., 2011). In addition, increasing the level of soluble fiber in the diet improves ener gy digestibility of the sow (Renteria-Flores et al ., 2007). Dietary amino acid requirements of t he pregnant sow vary depending on the rate of protein accretion and are influenced by their requirements for maintenance and protein deposition in maternal and fetal tiss ues. The factorial approach is useful to define amino acid requirements of sows (T rottier and Guan, 2000). Sows fed extremely low protein levels during gestation could fa rrow a litter with normal size and weight, but it had a dramatic negative effect on milk production during the subsequent lactation and on the return to estrus. During gestation, so ws fed a high dietary protein level gained more lean tissue and less fat than those fed lower-protein diets (Pettigrew and Yang, 1997). Also, prolific sows nursing large litte rs need high levels of dietary protein to maximize milk production, prevent excessive BW loss during even a relatively short lactation period, and prevent delayed return to estrus following weaning of litters (Cromwell, 2009). Consequently, high protei n diets are recommended for lactating sows. Lysine is considered to be the first limiting amino acid for establishing an ideal amino acid balance for both gestation and lactat ion. The daily requirement for lysine is the sum of the requirements fo r maintenance and protein accr etion during gestation, or


16 the sum of the requirement s for maintenance, protein accretion and milk production during lactation. The daily maintenance requir ement for true ileal digestible lysine is considered to be 34.79 mg/kg BW0.75 of gestation sow and 46.26 mg/kg BW0.75 for lactating sow (NRC, 2012). The requirement for protein accretion is thought to be 0.129 g of true ileal digestible lysine above ma intenance per g of accreted protein. The requirement for milk production is suggested to be 22 g of apparent ileal digestible lysine/kg of litter weight gain (NRC, 2012). T he lysine requirement seems to be greater during late than early gestation and the lysine requirement of heavy sows is less than that of light sows at a similar ME intake (Pettigrew and Yang, 1997). Requirements for the essential amino acids other t han lysine are also considered to consist of separate components for maintenance and prot ein deposition. Calculations are based on the ideal protein system in which requir ements for each of the other amino acids throughout gestation resu lt in overfeeding amino acids in early gestation and underfeeding amino acids in late gestation. So, phase feeding sows in gestation will more closely meet the demands for nutrients (Levesque et al ., 2011). The relative ideal ratios for Lys : Thr : Val : Leu are 100:79:65:88 for day 0 to 60 of gestation, and changed to 100:71:66:95 from day 60 to day 114 of gestation (Kim et al ., 2009). Dietary supplementation with 1% Glutam ine between d 90 and 114 of gestation improves fetal growth in gilts, reduces preweaning mortality of piglets and enhances milk production by lactating sows (Wu et al ., 2010). Sows vary greatly in their mineral and vi tamin requirements, which largely reflect the nutritional demands during different phases of their reproductive cycle. Minerals and vitamins are needed for conceptus format ion, mammary secretion and growth and


17 maintenance, and therefore, the sow’s mineral and vitami n requirements are higher during late gestation (Mahan, 1990). F eeding sows organic trace minerals may improve their reproductive performance (Pet ers and Mahan, 2008). Dietary addition of excess levels of vitamins A and D has been dem onstrated to have toxic effects in swine. In contrast, very few toxicity signs have been reported for the vitamin E, K and vitamin B group. Massive riboflavin s upplementation of the sow’s diet during early pregnancy may increase the number of farrowings, but does not increase the litter size (Pettigrew et al ., 1996). Neonatal Piglet Nutrition Body condition and health status influenc e the survival rate of piglets, and determine the growth potential and carcass c haracteristics of growing-finishing pigs (Gondret et al ., 2005). Piglets heavier at birth have higher post-natal survival rates than lighter piglets, and keep their weig ht superiority after weaning (Smith et al ., 2007). The growth of low-birth weight piglets can be enhanced by feeding a high protein diet but the provision of a high-protein diet that exc eeds the protein requirement does not further increase protein synthesis or the expression of translation in itiation factor activation (Frank et al ., 2005). So, the appropriate feeding and management of neonatal piglets are critical to maintaining efficient weig ht gain and good health condition. Also, heavier and healthier piglets tend to a have stronger i mmune system that pr otects them from pathogens and prevents disease out breaks. Less disease and fast growth rate will allow the growing-finishing pig to reac h the market weight rapidly. Piglets are usually born indoors a nd the temperatur e is around 15-30oC. They are poorly insulated because of sparse hai r covering and a lack of subcutaneous fat, thus, they need to increase their heat pr oduction to avoid hypothermia. Shivering


18 thermogenesis predominates as there is littl e or no brown adipose tissue in new-born piglets. Before suckling co lostrum, the body glycogen and pr otein reserve is the only energy source for heat production (Noblet and Le Dividich, 1981). A new born piglet must get energy to meet its maintenance requirements, including thermoregulation, physical activity and growth, and the mainte nance requirements could be as high as 275 KJ/kg BW, it has been estimated t hat the extra energy required for thermoregulation averages 2 KJ/kg BW/h per degree centigrade below the lowest critical temperature. Thus, the energy and nutrient requirem ents of piglets are maximal at birth, which is estimated at 2.6 time s higher than those at weaning (Le Dividich et al ., 1994). Standing costs 9.5 KJ/kg BW/h and the minimal energy needed for physical activity is about 105 KJ/kg BW during t he first 24 h following birth (Le Dividich et al ., 1994). The average energy associated with BW gai n at the first day of birth is 70 67 g/kg BW (Le Dividich et al ., 2005). Under thermoneutral c onditions, the lowest net energy required for survival of a 1.0 kg pigl et is approximately 700 KJ during the first 24 h after birth, and could be as high as 900950 KJ in a moderately cold surrounding. Based on recent studies (NRC, 2012), the to tal lysine requirements for neonatal piglets are estimated to be 1.10%, 1.10%, 1.35% a nd 1.30% for a 5, 10, 15 and 20 kg piglet. The other amino acids are calculated from lysine using the ratios established for maintenance and protein accretion on a tr ue ileal digestible basis (NRC, 2012). The primary source of energy for new bor n piglets is their body reserve. This energy is derived from protein, glycogen and fat oxidation. As body protein catabolism occurs at a very low rate at birth, glycoge n reserves, estimated at 30 to 38 g/kg BW at birth, are depleted rapidly. Under norma l environmental conditions, 75% of liver


19 glycogen and 41% of muscle glycogen are ut ilized by 12 hours postpartum (Elliot and Lodge, 1977). However, available energy derived from glycogen is still as low as 100.38 Kcal/kg BW. Because a large proportion of fat is structural fat and not available for mobilization, energy produced from thes e substrates does no t meet the energy requirement for maintenance and physical activi ty during the first day of life (Mellor and Cockburn, 1986). As first suckling occurs 10 to 30 min afte r birth, colostrum provides energy and maternal antibodies for piglet survival. Colo strum is the first 24-30 h secretion of the mammary gland. It is very digestible and both colostral energy and nitrogen (N) are retained with a very high efficiency. Compared with milk, colostrum has higher concentrations of dry matter and crude prot ein and lower concentrations of fat and lactose. The protein concentrations vary widely because of the variation in immunoglobulin concentration. Fats are the main source of energy, accounting for 40 to 60% of the total energy supplied by colost rum, and the content and the composition of fat are both largely dependent on the fat composition of the diet fed to the sow during late gestation. In contrast, lactose c oncentration of colostrum and milk is only marginally related to the diet of the sow. Amino acid composition of colostrum is relatively constant, but more threonine is contained in colo strum than milk. Colostrum contains less minerals than milk. Colostrum and milk contain a variety of growth factors and hormones, including IGF-I, EGF, TGFand leptin (Estienne et al ., 2000). Both colostrum and milk are used remarkably well by piglets with ratios of ME to GE from 0.93 to 0.98, and a ratio of N retained to N intake between 0.88 and 0.91 (Le Dividich et al ., 1999). The true


20 digestibility of amino acids is about 92% and is lower than their apparent digestibilities (Mavromichalis et al ., 2001). The efficiency of ME utilization for total energy and for energy retained as protein are higher for co lostrum than for milk (0.91:0.72; retention 0.91:0.56), and colostrum stimulates more muscle protein synthes is compared with mature milk. Because piglets have low immunity at birth, the most important function of colostrum is to provide immunoglobulins that transfer the passive immunity to the neonatal piglets. Colostrum contains more IgG than mature milk. Because gastric and pancreatic proteolytic enzymes do not di gest immunoglbulins, these molecules are absorbed directly into the blood stream to build the piglet’s immunity (Le Dividich et al ., 2005). Colostrum also plays an important role in the development of the gastrointestinal tract of piglets (Xu and Wang, 1996). The mini mal enteral nutrient intake necessary to sustain normal mucosal proliferation and grow th is 60% of the total nutrient intake (Burrin et al ., 2007). As a high-quality colostrum results in gr eater growth rate of piglets, early and increased intake of good quality of colostrum is very important. The production and composition of colostrum and milk are influenced by the parity and genotype. The endocrine status of the sow al so affects the process of colostrogenesis and changes in the sow’s endocrine status can have an impac t on the quantity and quality of colostrum produced. Nutrition is undoubtedly a major fact or that could be used as a tool to alter colostrum and milk composition with fat cont ent being the most affected by the diet (Farmer and Quesnel, 2008). In order to in crease the production of colostrum and milk, the prenatal stress of the sow must be r educed before, during and after farrowing. The


21 appropriate diet should be fed and one must ensure that the sow has unrestricted access to fresh drinking water at all times. Colostrum and milk consumption by individu al piglets varies considerably. It is independent of birth order, but related positively to birth we ight (increased by 26-37 g per 100 g increased in BW). Colostrum and m ilk consumption is negatively related to litter size. During the first postnatal day, ingestion of 160-170 g colostrum/kg BW is required to ensure survival of piglets (Le Dividich et al ., 2004), whereas 280 g/kg BW is recommended (Le Dividich et al ., 1994). Milk arginine is usually insufficient for supporting the maximal growth of young pigs, and oral administration of N-carboam oylglutamate will enhance plasma arginine level and weight gain (Wu et al ., 2004). Piglets develop an iron deficiency anemia during the first weeks of life, which will lead to slow-growth and weakness. Because 21 mg Fe/kg BW are needed to maintain Fe status (Braude and Newport, 1977), neonatal pigs are usually injected with a chelated form of Fe within 3 d of age to prevent anemia (Kegley et al ., 2002). Weanling piglet nutrition Natural weaning occurs at 70 days or so of age without human intervention. The improvement of feeding and management technologies for the young pig has not only provided the opportunity for im provement of the productivity of the sow herd, but has also provided a means of breaking the cycle of disease transmission from the facilities. Although it is possible to successfully wean pigs at 14 to 21 days of age, it is more challenging to keep these young, newly we aned pigs healthy and growing than pigs weaned at older ages. Weani ng can often be a time associated with a lag in performance that includes depressed gain and feed intake and increased disease


22 occurrence and mortality. Unless managed pr operly, this post-weaning lag will be more magnified in pigs weaned at younger ages, bec ause their gastrointestinal tracts have not been fully developed to digest and absorb solid feeds. The competence of the digestive system of the suckled pig to handl e a non-milk diet begins to develop between 14 and 28 days of age. Under conditions of gradual diet change from liquid to solid feed, growth is likely to be fully supportable, so feeding of a mixture of milk and externally sourced solid feed to the gut of the young pig is relevant to natural development. Besides the diet change, piglets are exposed to a series of stressors that can lead to poor performance and increased mortality. Reduction of feed intake results in proportionate decline in metabolic body heat generation, making the pig more susceptible to chilling at the time of weaning. Gastrointest inal maladies are the most severe diseases because the requirements of the intestine are dramatically increased at weaning. Also the intestine serves as the first line of defense against both viral and bacterial pathogens that are ubiquitous in the postnatal environment (Corl et al ., 2008). In addition to the stresses of nutrition and disease, the impact of a sub-optimal environment and disturbances to the previous social order must not be underestimated. The Prairie Swine Centre have demonstrated that about 30% of the variation in age to market can be attributed to the time it takes a pig to reach a body weight of 23 kg. So, it is very critical to understand and meet t he nutritional requirements of weaned pigs. The suckling pig partitions nutrients to ward lipid deposition to reach 150-160 g per kg of BW at the time of weaning, and t he ratio of lipid to protein in the body is around 1:1 at weaning. Post-weaning feed inta ke inadequacies, together with stress and disease challenges, cause a rapid loss of body lipid in support of maintenance and


23 protein synthesis. So, adequate water must be provided ad libitum. The young pig has a high potential for growth (Hodge, 1974). Among numerous factors which influence the extent to which this potential is express ed, energy intake is the most important. The energy requirements of the weaned pig include maintenance (MEm), protein and fat deposition (NRC, 2012). Metaboliz able energy required to maintain a weaned pig is the same as that required to maintain the suckling pig (112.33 kcal ME/kg BW0.75 per day), with the energy for activity accounting for 15-30% of the total MEm (Halter et al. 1980). Heat production (HP) increases to meet t he energy requirement fo r thermoregulation when piglets are in an environment where te mperature is below the lower critical temperature (LCT). The extr a ME requirement for thermo regulation is about 3.8-4.3 kcal/kg BW0.75 per C at the time of weaning (Le Dividich et al. 1998). Although energy intake is the first limiting factor, the choice of dietary ingredients for a weaned pig must fit its digestive capacity, maintain gut heal th and promote feed intake. Once adaptation to digestion of solid feed is complete, t he piglet can be consi dered a growing pig with regard to the net energy system (NRC, 2012). The ability of the young pig to use diets of variable energy concentration is more closely related to its tolerance of the fiber used for the dilution of energy, and of the fat used to provide energy. Fat and fiber are known to decrease the rate of passage of the digesta in the upper part of the digestive tract, particularly at the stomach le vel, and, thus, cause a decrease in total energy intake (McConnell et al. 1982). Therefore, it is important to add an appropriate ratio of fat while reducing the fiber quantity in the diet. Shields (2009) suggested that glycerol is a potential valuable and economical energy source for swine diets.


24 The protein requirement of the weaner pig has to be considered differently because raw materials used in diets desi gned for this age supply not only N and essential amino acids but also Immunoglobu lins. In order to m eet the amino acid requirements of the weaner pig, a two-st age approach is used to achieve the ideal protein level. Early additional pr otein supply is necessary to achieve the growth potential of the pig (Sve and Ballvre, 1991), and the fi rst-limiting amino acid requirements vary with genotype, sex and livew eight. van Lunen and Cole ( 1998) suggested that the requirement for hybrid pigs could be 1.2 g lysine/MJ, which is higher than the value of Agricultural Research Council (ARC, 1981). Lysine, methionine and threonine are commonly used as supplements in weaner diets to achieve the ideal balance of amino acid. Protein sources commonly used in weaner feeds include milk proteins, crystalline amino acids, vegetable proteins and animal protein sources. Porcine plasma would appear to be the beneficial protein supplement in simple diets containing raw cereals and soybean products (Cole and Sprent, 2001). Vitamin B supplementation is necessary to maximize growth performance of early weaned pigs (James et al ., 2002), and it appears that adding a dietary supplement of B6 by 50 mg/kg saturates the B6 in red blood cell pool of weaned pigs. Additionally, vitamin B6, and along with vitamin B2, influences the glucose homeostasis through the entero-insular axis (Matte et al ., 2005) Because weaned pigs are susceptible to vitamin E deficiency, feeding a high level of vitamin E has been suggested to improve the immune response of weanling pigs (Bonnette et al ., 1990). Carlson (1995) suggested that both earlyand traditionally weaned pigs need to be fed pharmacological c oncentrations of Zn provided as ZnO for


25 a minimum of 2 wk immediately after weani ng to enhance growth. Addition of 250 ppm Cu to the diet stimulates intestinal lipas e and phospholipase A activities, leading to an improvement of dietary fat digestibility in weanling pigs (Luo and Dove, 1996). Physiological Changes at Weaning Rapid increases in intestinal dimensi ons of piglets occur during the early postnatal period, particularly the first 6 h of suckling (Zhang et al. 1997). The onset of suckling stimulates a rapid gr owth of the neonatal intesti ne that is supported by a high rate of protein synthesis (Burrin et al. 1992). The ingestion of co lostrum promotes the maturation of the developing in testinal epithelium (Burrin et al. 1992) and stimulates crypt cell proliferation (Zhang et al. 1997). The non-nutritive colo stral factors that elicit faster intestinal growth include both immunogl obulins and biologically active substances such as insulin-like growth factor-I (IGF-I) (Xu and Wang, 1996) that can enhance macromolecular absorption and increase circ ulating IGF-I levels in newborn pigs (Wester et al. 1998). The enterocytes of the small intestine of newborn piglets possess complex apical endosomal systems comp rising tubular endosomes and associated vesicles that are involved in the extensiv e uptake of maternal immunoglobulins and other colostral constituents (Murata and Na mioka, 1977). An age-related decrease in ion and nutrient fluxes takes place in t he immediate postnatal period (Sangild et al. 1993), but the total nutrient transport c apacity increases with age due to a large increase in intestinal mass (Pacha, 2000). Much of the decline in carrier-mediated nutrient absorption during the first 24 h of su ckling is caused not by loss of transporters but instead by the rapid increase in the ti ssue mass that effectively ‘dilutes’ the transporters (Buddington et al. 2001). The decline in carrier-mediated amino acid and glucose transport also coincides with the post natal replacement of the fetal enterocytes,


26 leading to a redistribution of transport func tions along the crypt villus axis (Buddington et al. 2001). The detection of intestinal glucosidas e (sucrase, maltase) in the suckling pig, albeit at low activities, suggests that pigs have a limited capacity to process carbohydrates other than lactose (Zhang et al ., 1997). The microbial colonization of the pig gut by non-pathogenic micr oorganisms greatly modifies the gut structure and function and their stimulation of the intestin al immune system results in a constitutive, low-level inflammation and epi thelial changes that can have both negative and positive effects on nutrient and energy absorption in the young (Gaskins, 1997). Microbial colonization is a complex process of natural selection and ecological succession (Rolfe, 1996) and is influenced by numerous regulatory factors of both bacterial and host origin, including bacterial antagonisms, animal genotype and physiology and, importantly, nutrition (Conway, 1999). The col onization pattern of the suck ling pig is similar for most animals, with lactic acid bacteria, enter obacteria and streptococci appearing first, followed by obligate anaerobes (Conway, 1999) Piglets are immunodeficient at birth and are only able to generate limited T and B cell responses when challenged with pathogens, so they are highly dependent up on a supply of both specific and nonspecific immune factors present in maternal colostrum and milk for passive immunity for protection, development and survival (Stokes et al ., 2004). Weaning is a critical period in the pig’ s life. The pig must cope with separation from the sow, the trans ition from highly dige stible milk to a less digestible and more complex solid feed, separation from litterma tes, and exposure to unfamiliar pigs. These stressors cause the reduction of growth dur ing the first week and are associated with changes in the histology and biochemistry of t he small intestine such as villous atrophy


27 and crypt hyperplasia. These changes resu lt in decreased digestive and absorptive capacities and contribute to post -weaning diarrhea (Pluske et al ., 1997). Hampson (1986) reported that following weaning at 21 days of ag e, the villous height was reduced to around 75% of pre-weaning va lues within 24 hours (940 to 694 m). Subsequent reductions in villous height were smaller but continued to occur until the fifth day after weaning, at which point the villous height at most sites along the gut was approximately 50% of initial values found at weaning. Five to eight days after weaning villous height began to increase. Crypt elongation occurs after two days post-weaning because the number of cells in the crypt increases steadily from the third to eleventh day after weaning (Hampson, 1986). As a result of these changes in villous height and crypt depth, the villous height : cr ypt depth ratio in weaned pigs is markedly reduced and is manifested in a change from the longer, finerlike villi seen in newborn and sucking pigs to wider leaf-like or tongue-like villi (Pluske et al ., 1997). The preand post-weaning periods are characterized by major devel opmental and diet-i nduced changes in the expression of intestinal brus h-border enzymes. The specific activity (activity per unit mass of tissue) of lactase which hydrolyses m ilk lactose, reaches a peak in 3-week-old pigs but declines rapidly over the wean ing period. Also, sucrase and isomaltase activities fall by at least 50% by five days and recover by 11 days after weaning (Miller et al ., 1986). Thus, the intestines of weaned pigs have high sucrase and maltase activities and are characterized by corres pondingly high rates of glucose and fructose transport across the apical membranes of enterocytes (Puchal and Buddington, 1992). Carrier-mediated amino acid absorption declines significantly in the post-weaned pig intestine. This decline is consistent with a sh ift to an adult diet that is lower in protein


28 (Buddington et al ., 2001). The exception is the post-weaning increase in the rate of carrier-mediated absorption of l ysine, the principal amino ac id limiting the performance of early weaned pigs (Thacker, 1999). After weaning, the net absorption of fluid and electrolytes in the small intestine of pigs is temporarily decreased (Nabuurs et al. 1994). The primary factor for the compromised villus to cr ypt ratio and the intestinal function at weaning is inade quate feed intake during the post-weaning period (Hampson et al ., 1986). A compromised mucosal barrier caused by acute fasting may allow the passage of nutritional or bacterial antigens in to the lamina propria where activation of immune responses can occur. When feed intake patterns retu rn to normal, intestinal inflammation subsides and epithelial morphol ogy improves in the young pig (McCracken et al ., 1999). During the postnatal development, alteration s in the diet are believed to induce a succession of related changes in the gut mi crobial ecosystem (Conway, 1999). In the immediate post-weaning period, the balanc e between the development of healthy commensal microbiota and the establishment of a bacterial intestinal disease can be easily tipped towards disease expressi on (Hopwood and Hampson, 2003). The microbial colonization changes more in the il eum than in cecum or colon. The obligate anaerobic bacteria become numerically dominant, particularly in the hindgut of the pig, and high levels of such bacteria are also found in the ileum (Conway, 1997). Enterococci and Enterobacteriaceae dramatically decrease from the fi fth to the eleventh day post-weaning. Yeast counts decrease fi ve days post-weaning but recover by the eleventh day. The number of lactobacilli de creases on the first day post-weaning and recovers within five days to initial levels (Pieper et al ., 2006). These changes may


29 predispose the weaner pig to malabsorpt ion, dehydration, diarrhea and enteric infections (McCracken et al. 1999) because of the signific ant increase of potentially pathogenic coliforms such as Enterotoxigenic E. Coli (ETEC) and rotaviruses. ETEC produce heat-labile enterotoxins or heat-stable enterotoxins that cause the inflammation and diarrhea in weanling pigs (Nakazawa et al ., 1987). Intestinal ischaemia is a key predisposing factor to postweaning diarrhea in weaned pigs and it has been suggested that it may lead to intestinal acidosis and increase permeability to ETEC toxins (Nabuurs et al ., 1994). Weaning is associated with changes in the regulation of the ly mphoid cells in the mucosal immune system, and proand anti-in flammatory cytokines are increased due to intestinal inflammation (Stokes et al ., 2004). The newborn piglet has very few lymphocytes in its intestinal epithelium or lamina propria. Cluster s of lymphocytes are present in the mucosa, in the areas that will subsequently develop into Peyer’s patches (Pabst, 1988). In the first two weeks of life, the intestine rapidly becomes colonized with lymphoid cells. These cells express the CD2+ surface marker but do not co-express CD4+ or CD8+. The Peyer’s patches begin to get organized during this period, reaching a relatively “adult architecture” by 10 to 15 days after birth (Stokes et al ., 2004). In piglets 2-4 weeks old the intestinal mucosa becomes colonized by CD4+ T cells, primarily in the lamina propria. CD8+ cells are still largely absent. Small numbers of B cells appear, preferentially expressing IgM. Early weaning at 3 weeks of age is associated with a transient r eduction in the ability of intraepithelial lymphocytes to respond to mitogens and splenic T cells to secrete IL-2 (Bailey et al ., 2005), and 4 days after weaning at 3 weeks of age, there are increases in CD2+ and granulocyte cells in


30 proximal small intest inal villi (Vega-Lopez et al ., 1994). Thus, the young piglet is capable of active immune responses to liv e viruses and to dietary components by 3 weeks of age, but the mucosal immune system remains relatively immature throughout the weaning period (Stokes et al ., 2004). Also, weaning is associated with a transient increase of pro-inflammatory cytokines in th e small intestine and the colon, including TNF, IL-1 and IL-6. Increased mRNA expression of these cytokines are detected in the spleen, thymus, tonsil and me senteric lymph nodes (Stokes et al ., 2004). These changes could reflect an inflammatory reaction occurring in the lymphatic organs and immune system in response to a pathogenic challenge and reduced feed intake. Nutritional, psychological and environmental stressors associated with weaning contribute to the subsequent reduction in grow th rate, and the nutritional status has a strong influence on the expression and secr etion of a variety of growth-related hormones, including growth hormone (G H), insulin-like growth factorI (IGF-I) and thyroid hormones. Serum concentration of GH sharply declines during the first 4 days of postnatal development, serum IG F-I concentrations are inversely correlated with levels of GH but positively correlated with average daily gain (Carroll et al ., 1998), while weaning does not affect IGF-I, IGF-2 or GH receptor mRNA levels in the liver (Matteri et al ., 2000). In summary, the weaning process and the development of the gastrointestinal tract of the pig have a ma rked effect on feed digestion, nutrient absorption and protection from pathogenic challenges. Diets formu lated for young pigs should take into account the changes that occur at weani ng and utilize ingredients that the young pig can better absorb, thus, support intestinal health.


31 Immunity Innate Immunity Pigs are constantly exposed to infectious agents, but, in most cases, they are able to overcome these infections. It is their immune system t hat enables them to overcome infections. The immune system is composed of two major components: the innate or non-specific immune system and the adaptive or specific immune system. The innate immune system is the first line of def ense against invading organisms, whereas the adaptive immune system acts as a second li ne of defense and also offers protection against re-exposure to the same pathogen. Ea ch of the major subdivisions of the immune system has cellular and humoral com ponents by which they carry out their protective function. In additi on, the innate immune system al so has anatomical features that function as barriers to infection. Al though these two arms of the immune system have distinct functions, there are in teractions between these systems (Male et al. 2006). The elements of the innate (non-spec ific) immune system include anatomical barriers, secretory molecu les and cellular components. The infectious agent must overcome innat e host defenses to establish a focus of infection (Janeway, 2005). The anatomical ba rriers consist of the skin, the internal epithelial layers, movement of the intestines and the oscill ation of broncho-pulmonary cilia. Associated with these protective surf aces are chemical and biological agents. The desquamation of skin epithelium helps re move bacteria and other infectious agents that have adhered to the epithelia l surfaces. Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria and destabilize bacterial membranes. The mucosal epithelium covering the orifices of the body is protected by mucous secretions and cilia


32 (Murray et al ., 2012). Movement of c ilia and peristalsis help keep air passages and the gastrointestinal tract free of microorganisms. The trapping effect of the mucus that covers the respiratory and gastr ointestinal tracts helps protect the lungs and digestive systems from infection. The low pH of sweat and gastric secretions prevents growth of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal tract have antimicrobial activi ty. Surfactants in the lung act as opsonins (substances that promote phagocytosis of parti cles by phagocytic cells). The microflora of the skin and that of the gastrointestinal tract can prevent the colonization of pathogenic bacteria by secreting toxic subs tances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces (Male et al. 2006). If the microorganisms are not suppress ed by nonpathogenic bacteria or chemical factors, they may cross the epithelial barrier and begin to replicate in the tissues of the host (Janeway, 2005), ultimately causing acute inflammation (Male et al. 2006). Inflammation plays three essential roles in com bating infection. The first is to deliver additional effector molecules and cells to the sites of infection to augment the killing of invading microorganisms by the front-line macrophages. The second role is to provide a physical barrier in the form of microvascular coagulation to prevent the spread of the infection in the bloodstream. The third role is to promote the repair of injured tissue (Janeway, 2005). Humoral immunity is the as pect of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins and certain antimicrobial peptides. Humoral factors play an important role in inflammation, which is characterized by edem a and the recruitment of phagocytic cells. These humoral factors are found in serum or are formed at the site of infection. The


33 complement system is an alarm and a weapon against infection, especially bacterial infection. The complement system is acti vated directly by bacteria and bacterial products (alternate or properdin pathway), by lectin bi nding to sugars on the bacterial cell surface (mannose-binding protein), or by complexes of antibody and antigen (classical pathway). Activation by either pathway initiates a cascade of proteolytic events that cleave the proteins into “a” and “b” subunits. The “a” subunits (C3a, C5a) attract (chemotactic factors) phagocytic and inflammatory cells to the site of infection, allow access to soluble molecules and cells by increasing vascular permeability (a naphylactic C3a, C4a, C5a) and activate re sponses. The “b” subunits are bigger and bind to the invading agents to promote thei r phagocytosis by opsoniz ation together with antibody, and build a molecular drill that c an directly kill the infecting agent (Murray et al ., 2012). Pathogens also can be recognized by the mononuclear phagocytes without the help of complement. Sites of infection ar e reinforced by the recruitment of large numbers of neutrophils which are attracted by the molecules secreted by mononuclear phagocytes. Both of these phagocytic cells hav e a key role in the innate immunity, because they can recognize, ingest and des troy many pathogens without the aid of an adaptive immune response. Interaction of m any cell-surface receptors on phagocytic cells by pathogens leads to phagocytosis of the pathogen (Janeway, 2005). After attachment of a bacterium, the phagocyt e begins to extend pseudopods around the bacterium. The pseudopods eventually surr ound the bacterium and engulf it, and the bacterium is enclosed in a phagosome. Duri ng phagocytosis, the granules or lysosomes of the phagocyte fuse with t he phagosome and empty their c ontents. The result is a


34 bacterium engulfed in a phagolysosome which c ontains the contents of the granules or lysosomes. After activation of phagocytes bacteria will be killed inside the phagocyte by hydrolytic enzymes and oxygen metabo lites (Male, 2006). The most important phagocytes (macrophages) are formed from blood monocytes and contain opsonin receptors which promote phagocytosis of microbes, PAMP receptors which initiate activation and response, and cytokine recept ors which promote their activation. Macrophages also express MHC II proteins for antigen presentation to CD4 T cells. Macrophages can be activated by IFN(classical activation) produced by NK cells and CD4 and CD8 T cells as part of the TH 1 response and are then able to kill phagocytosed bacteria. These are called M1 macrophages (Murray et al ., 2012). There is an increase in glucose and oxygen consumption within M1 macrophages, and this is referred to as the respiratory bur st inside M1 macrophages. The function of the respiratory burst is to generat e a number of oxygencontaining compounds which kill the bacteria being phagocytosed. This is re ferred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse wit h the phagosome. This is referred to as oxygen-independent intracellular killing (Male et al ., 2006). On the other hand, activated M1 macrophages produce cytoki nes, enzymes, and other molecules to promote antimicrobial action. They also reinforce local inflammatory reactions by producing various chemokines to attract neutrophils, immature dendritic cells (iDC), Natural Killer cells (NK), and activated T ce lls. Activation of the macrophages makes them more efficient killer s of phagocytosed microbes, virally infected cells, and tumor cells.


35 Dendritic cells (DC) provide the bri dge between the innate and the immune responses. The cytokines they produce dete rmine the nature of the T-cell response. Monocytes and precursor myeloid DC circulate in the blood and then differentiate into immature dendritic cells (iDC) in tiss ue and lymphoid organs. Immature DC are phagocytic and, upon activation by danger signals, release an early cytokine-mediated warning system and then are converted to Ma ture DC. Mature DC are the ultimate antigen-presenting cells, one of the antigen-presenting cells th at can initiate an antigenspecific T-cell response. These cells expres s different combinations of danger sensors that can detect tissue trauma (adenosine triphosphate, adenosine, reactive oxygen species, heat shock proteins (Murray et al ., 2012). Natural killer (NK) cells are innate lym phoid cells that provide an early cellular response to a viral infection. They have antitumor activity, and amplify inflammatory reactions after bacterial infection. Natural k iller cells are also responsible for antibodydependent cellular cytotoxicity, in which they bind and kill ant ibody-coated cells. The NK cell sees every cell as a potential victim, espe cially those that appear in distress, unless it receives an inhibitory signal from the target cell by binding to carbohydrates and surface proteins on the cell surface. The inte raction of a class I ma jor histocompatibility complex ( MHC I) molecule on the target cell with a killer-cell immunoglobulin-like (KIR) inhibitory receptor is like communicating a se cret password indicating that all is normal, and this provides an inhibitory signal to prevent the killing of the target cell by NK cells. Virus-infected and tumor cells express “stress -related receptors” an d are often deficient in MHC I molecules and become NK-cell tar gets. The NK cells neither recognize a specific antigen nor require presentation of antigen by MHC molecules. Additionally,


36 the NK system does not involve memory or requi re sensitization, and, therefore, cannot be enhanced by specific immunization (Murray et al ., 2012). Natural killer T (NKT) cells and / T cells reside in tissue and in the blood and differ from other T cells because they have a limited repertoire of T-cell receptors. Unlike other T cells, NKT and / T cells sense non-peptide antigens, including bacterial glycolipids (mycobacteria) and phos phorylated amine metabolites from some bacteria (Escherichia coli, mycobacteria) but not others (streptococci, staphylococci). These T and NK cells produce IFN, which activate macrophages and DC to enforce a protective TH1 cycle of cytokines and local cellular inflammatory reactions. The NKT cells also express NK-cell receptors and large amount of TH2 cytokines (Murray et al ., 2012). In general, as the first line of defens e against pathogenic infection, the innate immune system uses several components and me chanisms to recognize and respond to pathogens. Some of these components di rectly stimulate phagocytosis, other molecules are secreted and promote the phagocytosis of pathogens by opsonization and intensify inflammation, while other co mponents can initiate the adaptive immune system later on (Janeway, 2005). Acquired Immunity Acquired immunity is induced after pat hogens multiply within the host, and is mediated by B and T lymphocytes which have specific surface receptors for the antigens. Thus, acquired immunity is also called antigen-specific immunity and extends the hosts protection provided by innate i mmunity. The B and T cells are specific immune cells that distinguish and fight s pecific antigens. The memory capacity of


37 lymphocytes allows them to respond more r apidly and effectively to re-exposure of the same antigen (Male et al ., 2006). The acquired immunity includes humoral an d cell-mediated immunity. During the first exposure to the pathogens, the antigen is recognized by and binds to the receptors on the antigen presenting cells (APCs). T hese APCs then move into the secondary lymphoid organs where they encounter with T cells. After processing of the antigen within the APC, the specific class I and II ma jor histocompatibility complex (MHC I and MHC II) appears on the surface of APCs and engages the T cells through T cell receptor. Development of an antigen-spec ific immune response progresses from the innate responses through dendritic cells (DC), and results in activation of the T which trigger the necessary responses. Dendritic cells provide the br idge between the innate and the acquired immune responses, and the cytokines they produce determine the nature of the T-cell response. The DC have octopus -like arms with large surface area (dendrites), produce cytokines, and have an MHC -rich cell surface to present antigen to the T cells. Macrophages and B cells also c an present an antigen to the T cells, and activate a naive T cell to initiate a new i mmune response. The activated helper T cells (CD4) extend and control immune and inflammato ry responses by specific cell-to-cell interactions and by releasing cytokines (s oluble messengers). The repertoire of cytokines secreted by a specific CD4 T cell in response to an antigenic challenge defines its lineage. Initially, T helper 0 (TH0) cells produce cytokines to promote expansion of the cellular response and develop to wards either a T helper 1 and T helper 2 cell lineage that express different cytokines The TH0 cells are converted to helper 1 (TH1) cells by interleukin-12 (IL12). The TH1 produce interferon(IFN) to activate


38 macrophages and DC and promote responses that are especially important for controlling intracellular (mycobacterial and vira l) and fungal infections and for promoting certain types of IgG production. The T hel per 2 (TH2) cells are formed in the absence of IL-12 and promote antibody responses. The T helper 17 (TH17) cells are a separate lineage that secrete interleukin (IL)-17 to activate neutrophils and promote antibacterial and antifungal responses and inflammation. A separate class of CD4 T cells are immune regulatory T (Treg) cells that express CD4, CD25 and Foxp3 to prevent excessive activation of T cells, and contro l the immune response. The cytokines produced by each of these T cell types re inforce their own production but may antagonize other responses (Murray et al ., 2012). Upon conversion from TH0 cell, TH2 cells produce IL-4, IL-5, IL-6, IL-10, and IL13, which activate naive B cells to secrete antibody, enhance IgG production and promote antibody switch to IgE or IgA. The TH2 response also promotes terminal differentiation of B cells to plasma-cell antib ody factories. Antibodies are the primary protection against extracellular bacteria and reinfection and prevent the spread of bacteria in the blood. The antibody prom otes complement activation, opsonizes bacteria for phagocytosis, blocks bacterial adhesion, and neutralizes exotoxins and other cytotoxic proteins produced by bacteri a. Immunoglobulin M (IgM) is produced early in the antibacterial response, bind to bacteria and trigger the classical complement cascade, promoting both the direct k illing of gram-negative bacteria and the inflammatory responses. I mmunoglobulin M is usually the only antibody produced against capsular carbohydrates. The large size of IgM limits its ability to spread into the tissue. Later in the immune response, the T-cell promotes the di fferentiation of the B


39 cells and immunoglobulin class switching to produce Immunoglobulin G (IgG), which is the predominant antibody, especially on re challenge. Immunoglobulin G fixes complement and promotes phagocytic uptake of the bacteria through Fc receptors on macrophages. The production of Immunoglobulin A (IgA) requires TH2 cytokines and other factors. Immunoglobulin A is the prim ary secretory antibody and is important for protecting mucosal membranes. Secretory Ig A acquires the secretory component that promotes interaction and passage of IgA through mucosal epithelial cells. Immunoglobulin A prevents the spreading of bacteria and neutralizes their toxins at epithelial cell surfaces (Murray et al ., 2012). The CD8 T cells include cytotoxic T lym phocytes (CTLs) and suppressor cells. The CTL are important for eliminating vira lly infected cells and tumor cells. The CTL response is initiated when naive CD8 T ce lls in the lymph node are activated by antigen-presenting DC and cytokines produc ed by TH1 CD4 T cells. Class I MHC molecules are found on all nucleated cells and are the major marker for CD8 to distinguish normal from abnormal cells. They display antigen to CD8 T cells which can divide and differentiate into mature cytotoxic T lymphocytes (CTL). They bind through interactions of the T cell re ceptor (TCR) with ant igen-bearing class I MHC proteins and adhesion molecules on both cell s. Granule-containing to xic molecules, granzymes (esterases), and a pore-forming pr otein (perforin), move to t he site of interaction and release their contents into the immune sy napse formed between the T cell and target cell. Perforin generates holes in the tar get cell membrane to allow the granule contents to enter and induce apoptosis in the target cell. Apopt osis is characterized by degradation of the tar get cell DNA into discrete fragm ents of approximately 200 base


40 pairs and disruption of internal membranes. The cells shrink into apoptotic bodies, which are readily phagocytosed by macrophages and DC. Suppressor T cells provide the antigen-specific r egulation of helper T-ce ll function through inhibitory cytokines and other means. Like CTLs, suppressor T cells interact with class I MHC molecules (Janeway, 2005). The activated T helper cells also acti vate macrophages. Macrophages require two signals for activation: one of these is provided by interferon (IFN), the other can be provided in different ways. It is also needed for the sensitizat ion of the macrophage to respond to IFN. The TH1 cells can deliver both signals. Interferon is the most important cytokine produced by TH1 cells upon interacting with their specific target cells, whereas the CD40 ligand expressed by t he TH1 cell delivers the sensitizing signal by interacting with CD40 on the macrophage. The CD8 T cells are also an important source of IFNand can activate macrophages to present antigens derived from cytosolic proteins. Macrophages c an be made more sensitive to IFNby very small amounts of bacterial lipopolysaccharide, and the latter pathway may be particularly important when CD8 T cells are the primary s ource of IFN. It is also possible that membrane-associated TNFor TNFcan substitute for CD40 ligand in macrophage activation. The TH2 cells are inefficient macrophage activators, because they produce IL-10, a cytokine that can deactivate ma crophages. However, they do express CD40 ligand, and can deliver the contact-dependent signal required to sensitize macrophages to respond to IFN(Janeway, 2005). After activation by the TH1 cells, macrophages have the capacity to kill the engulfed microbes and secrete cytokines to activate the acute-phase response (Janeway, 2005).


41 In addition to immediately eliminating the pathogens in the host, all activated T and B cells are converted to memory T and B cells which have longer half-lives and circulate in the whole body. Upon encounter ing the antigens, they react more rapidly and more efficiently to the second infection. Inflammatory Mediators Inflammation represents a protective def ense reaction induced by external or internal trauma such as tissue injury, infection, or tumor growth. It is caused by several mediators released by fibroblasts, neurons, and immune cells. These mediators include neurotrophins, histamine, brad ykinin, prostanoids, serotoni n, protons, free radicals, chemokines and cytokines (Table 1; Dray, 1995). The mediators can have a direct action, stimulate the release of other chem icals, activate the immune system, facilitate vasodilatation and plasma exudation or s ensitize the nocicept ive system (Marnet et al ., 2002). It is generally accept ed that the main inflammatory mediators induced by bacteria and their cell wall components are chemokines and cytokines (Wang et al ., 2000). Chemokines represent a sophisticat ed communication system used by all the cell types. Chemokine messages are decoded by specific receptors that initiate the signal transduction events leading to a mult itude of cellular responses, leukocyte chemotaxis and adhesion at infection sites. Infectious microorganisms can directly stimulate inflammatory chemokine producti on by tissue dendritic cells and macrophages as well as by many parenchymal and stroma l cells (Rot and von Andrian, 2004). Inflammatory chemokines function mainly as chemoattractants for leukocytes, monocytes, neutrophils and other effector cells.


42 Table 1-1. Inflammatory cyt okines and chemokines (Murray et al ., 2012) Factor Source Major Target Function Innate and acute-phase responses Interleukin 1 Interleukin 1 Macrophage, DC1, fibroblasts, epithelial cells T cells, B cells, PMN2, tissue, central nervous system, liver Promotion of inflammatory and acute-phase responses, fever, activation of T cells and macrophages Tumor necrosis factorSimilar to interleukin 1 Macrophages, T cells, NK3 cells, epithelial Similar to IL-1, and also antitumor, wasting functions, sepsis, endothelial activation Interleukin 6 DC, Macrophages, T and B cells, fibroblasts, epithelial cells, endothelial cells T and B cells, hepatocytes Stimulation of acute-phase and inflammatory responses, T and B ell growth and development Interleukin 12, Interleukin 23 DC, Macrophages NK cells, TH1, TH17 cells Activation of Tcell-mediated and inflammatory responses, IFNproduction Chemokines -chemokines: CXC chemokines (IL-8, IP-10, GRO, GRO, GRO) Many cells Neutrophils, T cells, macrophages Chemotaxis, activation -Chemokines: CC chemokines (MCP1, MIP, MIP, RABTES) Many cells T cells, macrophages, basophils Chemotaxis, activation 1DCs, dendritic cells; 2PMN, pol ymorphonuclear leukocyte; 3NK, natural killer cell; TH, T helper cell; IP, interferonprotein; RANTES, regulated on activation. Some inflammatory chemoki nes activate cells to initiate an immune response or promote wound healing. Inflammatory chem okines include chemokine ligand 2 (CCL2), CCL3 and CCL5, interleukin 1 (IL-1 or CXCL1) IL-2 and IL-8. Inte rleukin 8 (IL-8) is produced by macrophages and other cell types such as epithelial cells, airway smooth


43 muscle cells and endothelial cells and is one of the most effective c hemo-attractants for neutrophils in the innate immunity (Graham and Locati, 2013). Interleukin-8 has two primary functions: the first one is to i nduce chemotaxis of neutrophils and other granulocytes, causing them to migrate toward the site of infection, and the second function is to induce the phagocytosis of phagocyt es. Interleukin-8 induces a series of physiological responses such as increased intracellular Ca2+, exocytosis, and respiratory burst, which are required for migration and phagocytosis (Modi et al ., 2004). Cytokines are proteins made by cells t hat affect the behavior of other cells. When secreted by lymphocytes, cytokines are ca lled lymphokines or interleukins. They act via specific cell surface receptors (J aneway, 2005). The cytokines secreted by macrophages play important roles in innate re sponses. These include tumor necrosis factor(TNF), interleukin-1 (IL-1 ) and interleukin-6 (IL-6). The T helper 1 cells (TH1) not only activate macrophages to secrete interferon(IFN), but also sensitize them to respond to IFN. After activation, macrophages begin to release cytokines in the liver, spleen and other systemic sites that have long-range effects that contribute to the host defense. Tumor necrosis factor, IL-1 and IL-6 are referred to as endogenous pyrogens, because they cause fe ver and are derived from an endogenous source rather than from bacterial components Fever is generally beneficial to the host defense. Most pathogens grow better at normal body temperatures and adaptive immune responses are more intense at elevat ed temperatures. The host cells are also protected from the delet erious effects of TNFat elevated temperatures. Tumor necrosis factorinduces the migration of DC to the lymph nodes which initiate the acquired immune responses. Tumor necrosis factoralso causes vasodilation, which


44 leads to a loss of blood pressure, increased vascular permeability and increased migration of IgG, complement and phagocytes to the tissue. However, increased vascular permeability can cause a loss of plasma volume and, thus, the septic shock of the host. In septic shock, a random intrav ascular coagulation can also be triggered by TNF, leading to the generation of clots in many small vessels. This condition frequently causes the failure of vital organs su ch as the kidneys, the liver, the heart, and the lungs, and often results in a very high mortality rate (Janeway 2005). Interleukin-1 triggers the responses of vascular endot helium and lymphocytes, increases tissue permeability, and allows more effector cells to access the infectio n site. Interleukin-1 also induces the secretion of IL-6 which can activate the lymphocytes and increase antibody production. These cytokines also have other biological activities that help coordinate the body’s responses to infecti on. They activate bone marrow endothelium to release neutrophils that intensify the phagocyt osis at the infection site. They increase protein and energy mobilization in the muscl e and fat tissues and allow a rise in body temperature and a decrease in viral and bacterial replication. They also increase the antigen processing and acquired immune res ponse. One of the most important functions of the cytokines is the initiation of acute-phase response. This involves a shift in the proteins secreted by the liver. Du ring the acute-phase response, concentrations of some proteins in plasma decrease, wher eas levels of others increase markedly. The proteins whose synthesis is induced by the cytokines are called acute-phase proteins. Several of these proteins are of particular interest, because they mimic the action of antibodies, but, unlike antibod ies, these proteins have a broader specificity for pathogen-associated molecular patterns (Janeway, 2005).


45 P rostaglandins (PG) are sma ll-molecules derived from ar achidonic acid (AA) by the action of cyclooxygenases (COX) and PG synthases. P rostaglandin E2 (PGE2) has been shown to regulate multiple functions of immune cells (Phipps et al. 1991). It mediates inflammation, promot es local vasodilatation, an d causes local attraction and activation of neutrophils, macrophages, and mast ce lls at early stages of inflammation. The ability of PGE2 to trigger the synthesis of IL -10 and to suppress the production of multiple pro-inflammatory cytokines allows it to limit nonspecific inflammation and to promote the immune suppression associated with chronic inflammation and cancer. Although PGE2 can promote the activation, matu ration, and migration of DC to the lymph nodes, it has been shown to suppre ss both innate and antigen-specific immunity at multiple molecular and cellular levels, earning PGE2 the paradoxical st atus of a proinflammatory factor with immunosuppr essive activity (Kalinski, 2012). Although inflammatory mediators i nduce the innate and immune responses against infection at the early stage of infla mmation, the excessive production of these molecules often will cause a malaise and damage the host tissue. Nutrition and Immunity It is generally accepted that nutrition is an important determinant of immune response. Deficiency of one or several nutrients can negatively affect the host’s immune defense system. Incorporation of nutri ents into the cell membrane affects the membrane structure and fluidity, which may have a major effect on transmembrane signaling. The production of antibodies or cytok ines is altered by the availability of proteins or limiting amino acids. Additionally, defic iency of nutrients suppresses cell proliferation through its e ffect on DNA replication and cell-cycle regulation. The antimicrobial and antitumor functions of macrophages are known to be modified by


46 nutrients that promote the syn thesis of reactive oxygen or nitrogen intermediates. Prostaglandin synthesis by phagocytes is rela ted to the types and quantities of dietary fatty acids. These reports collectively indi cate that nutrients can affect immune system at several levels (Kubena et al ., 1996). Lymphoid atrophy is known to result fr om protein-energy malnutrition. Under these conditions, the size and we ight of the thymus are r educed, and there is a loss of corticomedullary differentiation; there are fe wer lymphoid cells and the Hassall bodies are enlarged, degenerated and occasi onally calcified. These changes lead to a primary immune deficiency, such as DiGeorge syndr ome (Roitt and Brostoff, 1991). Under condition of protein-energy defic iency, there is a significant reduction of lymphocytes in the lymph nodes. Poor nutrition leads to a marked decrease in the proportion of CD4+ on the surface of T helper cells. There also is a moderate reduction in the number of suppressor cytotoxic CD8+ cells. Thus, the ratio of CD4+ and CD8+ cells are significantly lower in malnourished subjec ts compared with well-n ourished subjects. Moreover, co-culture experiments showed a reduction in the number of antibodyproducing cells and in the am ount of immunoglobulin secreted (Chandra, 1997). This may be due to decreased help provided by T lymphocytes. In undernourished subjects, lymphocyte proliferation and DNA synt hesis are reduced, especially when an autologous plasma from a patient is used in cell cultures. This may be caused by inhibitory factors as well as deficiency of e ssential nutrients in the patient’s plasma. Serum antibody responses are generally intact in protein-energy malnutrition. However, antibody affinity is decreased in patients who are malnourished. This may provide an explanation for a higher frequency of anti gen-antibody complexes found in such


47 patients. Concentrations of secretory immu noglobulin A antibody also are lower after immunization with viral vaccines. Phagocytos is is also affected in protein-energy malnutrition. Because of the reduction in C3 C5, factor B and total hemolytic activity, there is a slight reduction in opsonic activi ty of plasma, and metabolic activation and intracellular destruction of bacteria are r educed. Malnutrition not only decreases the production of several cytokines, includi ng interleukins 1 and 2 and interferon but also alters the ability of T lymphocytes to res pond appropriately to cytokines. Poor nutrition has negative effects on the integrity of ph ysical barriers, the quality of mucus and several other innate immune responses, whic h ultimately leads to immune failure (Chandra, 1997). Deficiencies of essential amino acids can also suppress the synthesis of proteins, including those which contribute uni quely to host defense. These proteins encompass all of the cytokines produced by lymphocytes, macrophages, and other cells. In addition, essential amino acid deficiencies decrease the production of complement proteins, numerous tissue enzymes and metalloenzymes that are activated during acute phase responses. All of the acute phase glycoproteins produced by the liver are also reduced by amino acid deficiencies (Beisel, 1996). Fatty acids may influence the immune system in a number of ways. Alteration in membrane composition is known to result in changes in flui dity and transmembrane signal transduction in immune ce lls. Arachidonic acid metabolism may be altered by the quantity and quality of dietary fat, ultima tely resulting in changes in highly immunomodulatory prostagland in production (Calder and Newsholme, 1993). Immunosuppression associated with fatty acids, particularly polyunsaturated fatty acids,


48 may be beneficial in conditions involving an overactive immune response. When vitamin E is added to the diet, the inhi bition of lymphocyte proliferation by polyunsaturated fatty acids is reduced (Martinez-Alvarez et al ., 2005). On the contrary, excessive selenium combined with fat consumpt ion can increase the inhibitory effect of polyunsaturated fatty acids on an tibody circulation by polyunsaturated fatty acids (HarikKhan et al ., 1993). Adequate fiber ingestion is needed to suppor t the growth of microbiota in the host. Short chain fatty acids (SCFA) are end products of microbial fermentation, and exert marked effects on host immune responses Low levels of butyrate modify the cytokine produced by TH cells and promote intest inal epithelial barrier integrity, which in turn can help limit the exposure of the mu cosal immune system to luminal microbes and prevent aberrant inflammatory responses. Production of another SCFA acetate, by the microbiota promotes the resolution of intest inal inflammation by the G-protein-coupled receptor GPR43. Acetate production plays an important role in pr eventing infection with the enteropathogen Escherichia coli (0157:H7). This effect is linked to the ability of acetate to maintain gut epithelial barrier function. Also, intestinal microbiota can synthesize several vitamins involved in various aspects of microbial and host metabolism. These include cobalamin (vitam in B12), pyridoxal phosphate (the active form of vitamin B6), pantothenic acid (vitam in B5), niacin (vitamin B3), biotin, tetrahydrofolate and vitamin K. Most of these vitamins are involved in the immune system (Kau et al ., 2011). Vitamins and minerals also play a cruc ial role in the maintenance of immunecompetence. These include vitamin A, betacarotene, folic acid, vitamin B6, vitamin


49 B12, vitamin C, vitamin E, riboflavin, iron, zinc and selenium (Grimble, 1997). Dietary deficiencies of vitamin A have been shown to induce impairment of mitogen-induced proliferation, reduce ant ibody responses to pneumococcal polysaccharide, and decrease NK cell and IFNacivity (Kubena et al ., 1996). Vitamin C is an antioxidant that plays a pivotal role in maintaining t he antioxidant/oxidant balance in immune cells and in protecting them from oxidative stress (Victor et al 2004). Deficien cies of vitamin C induces a number of subtle changes in immune system and lymphocyte functions, but the major effect of vitamin C defici ency occurs in phagocytic cells (Anderson et al ., 1990). In both clinical and experimental scu rvy, the locomotion of phagocytic cells is severely impaired, possibly because these ce lls cannot produce tubulin (Beisel, 1996). This vital intracellular prot ein allows cells to change shape, and to move about. Without an adequate tubulin infrastructure, phagocytic ce lls cannot migrate to sites where an inflammatory process is needed in order to prevent the disseminat ion of a localized infection. Although the microbicidal activity of phagocytes generally remains effective in the presence of ascorbic acid deficien cy, the cells cannot move toward the microorganisms to engulf and destroy them (Beise l, 1996). Vitamin E is the major lipidsoluble antioxidant in the body and is requir ed for protection of membrane lipids from peroxidation. Since free r adicals and lipid peroxidation are immunosuppressive, it is believed that vitamin E may act to optim ise and even enhance the immune response. In fact, vitamin E deficiency decreased spleen lymphocyte proliferati on, natural killer cell activity, antibody production following vacc ination, and phagocytosis by neutrophils (Meydani and Beharka, 1998). On the other hand, vitamin E supplementation of the diet of laboratory animals enhances antibody production, lymphocyte proliferation,


50 natural killer cell activity, and macrophage ph agocytosis. In another report, vitamin E prevented the retrovirus-i nduced decrease in production of IL-2 and interferon(IFN) by spleen lymphocytes and in natural killer cell activity (Wang et al 1994). The high level of vitamin E caused increased IL-2 and IFNproduction of spleen lymphocytes (Han and Meydani.1999). Vitamin D is an im portant immune system regulator. The active form of vitamin D, 1, 25-dihydroxyvitamin D3 [1, 25(OH)2D3], has been shown to inhibit the development of aut oimmune diseases, including inflammatory bowel disease (IBD). An additional factor that determines the effect of vitamin D status on immune function is dietary calcium. This mineral has independent effects on the severity of IBD severity, and addition of 1, 25 (OH)2D3 to low-calcium diets was shown to improve IBD symptoms (Cantorna et al ., 2004). Selenium is essential for the efficient operation of many as pects of the immune system in both animals and humans. Low dietary intake of selenium and consequent deficiency in farm animals can result in a wide range of diseases that are often associated with a concurrent vitamin E defic iency (Turner and Finc h, 1991). Selenium can influence the cell function through antioxidant activities, thyroid hormone metabolism and regulation of the activity of redox-active proteins (McKenzie et al ., 2002). All of these general effects on metabo lism can be associated with more specific processes that will affect the immune system Thus, selenium influences both the innate and the acquired immune systems. Selenium-defici ent lymphocytes are less able to proliferate in response to mi togen, and in macrophages, leukotriene B4 synthesis, which is essential for neutrophil chem otaxis, is impaired by this deficiency. The humoral system is also affected by se lenium deficiency where by a decrease in


51 production of IgM, IgG and IgA is observed. One of the most widely investigated associations between selenium and the immune system is the effect of the micronutrient on neutrophil function. Neutrophils produce s uperoxide-derived radicals that contribute to killing of microbes. Although selenium deficiency does not affect neutrophil numbers in many species, certain aspects of t heir function because subnormal (Turner and Finch, 1991). For example, neutrophils from selenium-defic ient mice, rats and cattle are able to ingest pathogens in vitro, but are less able to kill them than are neutrophils from selenium-sufficient animals (Boyne and Arthur 1986). Selenium defi ciency also impairs thyroid hormone metabolism and attenuates t he ability of neutrophils to respond to foreign organisms. Selenium deficiency ca n favor the formation of pro-inflammatory mediators that would predispose to di seases. Magnesium functions in the development, distribution, and function of immune cells and soluble factors that are critical for humoral and cell-mediated immuni ty. Magnesium not only affects more than 300 enzymes, but it also influences the met abolism of many other nutrients. Single nutrient deficiencies of copper and iron are know n to adversely affect several aspects of the immune response, because they serve as important cofactors for enzymes that are vital for lymphocyte and macrophage func tion (Boyne and Arthur, 1986). Zinc deficiency in animals is associated with a wide range of immune impairments (Fraker et al ., 1993). In effect, a deficiency of zinc in the diet leads to a marked decrease in the number of nucleated cells and a reduction in the number and proportion of lymphoid cell precursors (Osati-Ashtinani, 1998). Reduced intestinal zinc absorption can cause thymic atrophy, impair lymphocyte develop ment, decrease the numbers of CD4 cells and reduce lymphocyte responsiveness (Fraker et al ., 1986). Moderate or mild zinc


52 deficiency or experimental zinc deficiency results in decreased thymulin activity, decreased natural killer cell activity, lo wered ratio of CD4:CD8, and decreased lymphocyte proliferation (S hankar and Prasad, 1998). Exper imental Zinc deficiency decreased IL-2, IFNand TNFproduction by mitogen-stim ulated lymphocytes (Beck et al ., 1997). Low plasma zinc level causes t he development of lower respiratory tract infections and diarrhea among infants (Bahl et al ., 1998). There are now a number of studies showing that zinc supplementati on decreases the incidence of childhood diarrhea and respiratory illness (Jackson, 2000) Zinc administration to preterm lowbirth-weight infants increas ed the number of circulating T lymphocytes, stimulated lymphocyte proliferation, increased cell-mediated immune function and decreased the incidence of gastrointestinal and upper respiratory tract infections (Lira et al ., 1998). Omega -3 Polyunsaturated Fatty Acids ( n -3 PUFA) in Swine Nutrition Definition and Bi osynthesis of n -3 PUFA Omega -3 fatty acids ( n -3 fatty acids) refe r to a group of polyuns aturated fatty acids (PUFA) that includes -linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The term n 3 is a structural descriptor, which refers to the position of the double bond that is closest to the methyl terminus of the acyl chain of the fatty acid. All n -3 fatty acids have this double bond on carbon 3, counting the methyl carbon as carbon one. Like other fatty acids, n -3 fatty acids have systematic and common names, but they are frequently refe rred to by a shorthand nomenclature that denotes the number of carbon atoms in the chain, the number of double bonds, and the position of the first double bond relative to the methyl carbon. The simplest n -3 fatty acid is -Linolenic acid (ALA; C18:3 n -3). Alpha-Linolenic acid is synthesized from linoleic acid (C18:2 n -6) by delta-15 desaturase. Animals do not possess delta 12 and


53 delta 15 desaturase enzymes and, therefore, cannot synthesize ALA. This fatty acid must be supplied in the diet. Although ani mals cannot synthesize ALA, they can metabolize it by further desat uration and elongation (Figure 1) Desaturations occur at carbon atoms below carbon number 9 (counting from the carboxyl carbon) and occurs mainly in the liver. Alpha-Linolenic acid can be converted to stearidonic acid (C18:4 n -3) by delta-6 desaturase and then stearidonic ac id can be elongated to eicosatetraenoic acid (C20:4 n -3), which is subsequently desaturat ed by delta-5 desaturase to yield EPA(C20:5 n -3). It is important to note that the conversi on of ALA to EPA is in competition with the conversi on of linoleic acid to arachidonic acid (AA; C20:4 n6), because the same enzymes are used. The pref erred substrate for delta-6 desaturase is ALA. Linoleic acid is much more prevalent in most human diets than ALA, and thus metabolism of n -6 fatty acids is quantitatively more im portant. The activities of delta-6 and delta-5 desaturases are regulated by nutritional status, hormones and by feedback inhibition by the end products. The conversi on of EPA to docosahexaenoic acid (DHA; C22:6 n -3) involves addition of 2 carbons to EPA to form docosapentaenoic acid (DPA; C22:5 n -3). This molecule is then desaturat ed at the delta-6 position to form tetracosahexaenoic acid (C24:6 n -3), which is subsequently translocated from the endoplasmic reticulum to peroxisomes, w here 2 carbons are removed by limited oxidation to yield DHA (Calder, 2012). Shor t term studies with isot opically-labeled ALA and long term studies using greater amount s of ALA have demonstrated that the conversion of ALA to EPA, DPA and DHA is generally poor in humans (Arterburn et al ., 2006). Eicosapentaenoic acid, DPA and DHA ar e found at high concentration in fish that consume algae.


54 Figure 1-1. Omega-3 PUFA biosynthes is (adapted from Pereira et al., 2004). Because ALA is produced in plants, green leaves and some plant oils, nuts and seeds contain moderate to hi gh amounts of ALA. Alpha-linolenic acid is the major n -3 fatty acid consumed in most human diets. However, the main PUFA in most Western


55 diets is linoleic acid which is typically c onsumed in 5 to 20-fold greater amounts than ALA. Sea foods are a good source of long chain n -3 PUFA. These fatty acids are found in the flesh of both lean and oily fish with much greater amounts in the latter, and in the livers of some lean fish. Humans who consume small amount of fish in their diet deposit less than 0.2 to 0.3 g of long chain n -3 PUFA per day (British Nutrition Foundation, 1999). A single lean fish meal could provide about 0.2 to 0.3 g of long chain n -3 PUFA, while a single oily fish meal can provide 1.5 to 3. 0 g of these fatty acids. Fish oil is prepared from the flesh of o ily fish or from the livers of lean fish. In a typical fish oil supplement, EPA and DHA make about 30% of the total fatty acids. Consequently, a one gram fish oil capsul e will provide about 0.3 g of EPA and DHA. However, the amount of n -3 PUFA can vary between fish oils, as can the relative proportions of the i ndividual long chain n -3 PUFA. In fish oil capsules, fatty acids are usually present in the form of ethyl esters (Calder, 2012). Effects of n3 PUFA on Growth Polyunsaturated fatty acids (PUFA) comp rise a group of fatty acids with more than one double bond in their carbon chains. These fatty acids have the ability to affect a diverse number of physiological processes, including the regulation of plasma lipid concentration, immune function and retinal dev elopment. There ar e two major classes of PUFA found in the diet, n3 and n6. Both n3 and n6 PUFA are essential, but their metabolic products elicit diffe rent cellular functions (Meers et al ., 2004). The long-chain n3 PUFA, eicosapentaenoic acid (EPA, 20:5 n3) and docosahexaenoic acid (DHA, 22:6 n -3), are found in fish oil and have a wid e range of biological effects that are believed to confer benefits to human health (B ritish Nutrition Foundation, 1999). As a


56 result, it is generally recommended that long-chain n3 PUFA consumption be increased in the human diet. Feeding pigs with a diet containing a high -linolenic acid level leads to significant enrichment of animal tissues with n3 fatty acids. Because unsaturated fatty acids inhibit lipogenesis than saturated fatty acids do, dietary fish oil may reduce the adipocyte size and fat pad weights in domestic animals (Fickova et al ., 2002). Supplemental fish oil does not appear to a ffect growth responses (feed intake, daily gains, feed conversion efficiency) or ca rcass quality (slaughter weight, dressing percentage, lean percentage, nutrient composition of the loin) in pigs when the ratio of n6 : n3 PUFA is higher than 2 (Jaturasitha et al ., 2002). In one study (Duan et al ., 2014), growth performance of pigs fed with diets with an n6: n3 PUFA ratio of 5:1 was the best, but the grou p fed diets with an n6: n3 PUFA ratio of 1:1 had the highest muscle mass and the lowest adipose tissue mass. Excessive dietary n3 fatty acids can cause soft pork, but also increase the susceptibil ity of the meat to ox idation. Therefore, concomitant supplementation of vitamin E is necessary to prevent fishy flavor due to lipid oxidation (Wood and Enser, 1997). It is well documented that lipids play an important role in skeletal biology and bone health. Dietary fat may influence bone met abolism by altering the biosynthesis of prostaglandins. Prostaglandins are loca lly produced in osteogenic cells and regulate both bone formation and bone resorption. Eico sapentaenoic acid treated-cells tended to have increased levels of alkaline phosph atase activity and osteocalcin, suggesting that PGE2 may decrease bone mass by inhibiting ost eoblast activity. Additionally, PGE2 may modulate IGF-1 synthesis and affect it s action to support anabolic responses in the


57 bone (McCarthy et al ., 1994). Thus, dietary supplementation of n3 PUFA can affect bone remodeling by directly inhibi ting the biosynthesis of PGE2 and indirectly influencing IGF-1 secretion. On the other hand, cells exhibit greater collagen synthesis when enriched with n3 PUFA (Watkins et al ., 2001). The fibroblast also displays a high collagen formation when exposed to moderate levels of EPA (Hankenson et al ., 2000). Because DHA is involved in the fluidity, ph ysical flexibility and compressibility of cell membranes, n3 fatty acids are needed to maintain the development of brain and the normal function of retina (Neuringer et al ., 1988). Furthermore, n3 PUFA are known to increase the litter size, and reduce the preweaning mortality of piglets. They also affect the conception rate and the number of pigs born alive (Reese, 2003). Effects of n3 PUFA on Inflammatory Responses Long chain n3 PUFA decrease the expression of adhesion molecules on the surface of monocytes (Hughes et al ., 1996), macrophages (Miles et al ., 2000), lymphocytes (Sanderson et al ., 1998) and endothelial cells (Yamada et al ., 2008). Eicosapentaenoic acid reduces the expressi on of VCAM-1, ELAM-1 and ICAM-1 on the surface of LPS-stimulated human umbilical vein endothelial cells (Calder, 1997). Murine peritoneal macrophages cultured in t he presence of EPA or DHA were less adherent to artificial surfaces than those cu ltured with other fatty acids (Calder, 1997). Consumption of 1.8 g EPA and DHA per day by patients with peripheral vascular disease decreased the adhesive interacti on of their monocytes to endothelial monolayers in culture (Luu et al ., 2007) Chemotaxis is the process by which leukocytes move towards the infection sites in response to the release of specific chem icals. These chemicals, also known as chemokines, include IL-8 and the arachidoni c acid-derived eicosanoid Leukotriene B4


58 (LTB4). Studies with fish oil supplementat ion of healthy humans have demonstrated a decrease in chemotaxis of neutrophi ls and monocytes towards various chemoattractants including LTB4, bacterial peptides and human serum (Luostarinen et al ., 1992). The near-maximum inhibition of chemot axis occurs at an intake of 1.3 g EPA and DHA per day (Schmidt et al ., 1991). Long chain n3 PUFA also suppress the produc tion of arachidonic acid-derived eicosanoids including PGE2 and LT (Peterson et al ., 1998). Arachidonic acid is released from the phospholipids th rough the action of phospholipase A2 enzyme, which is activated by inflammatory stimuli. The free arachidonic acid t hen acts as a substrate for cyclooxygenase (COX), lipoxygenase (L OX) or cytochrome P450 enzymes. Cyclooxygenases lead to PG and throm boxane formation, whereas lipoxygenases lead to LT formation. Cytochrome P450 enzymes catalyze the formation of hydroxyeicosatetraenoic and epox yeicosatrienoic acids. A ll these eicosanoids have long been recognized as key mediators and r egulators of inflammation, acting via G protein-coupled receptors (Tilley et al ., 2001). An EPA intake of 2.7 g per day significantly decreases PGE2 production (Rees et al ., 2006). Eicosapentaenoic acid is also a substrate for the COX, LOX and cytochrome P450 enzymes that produce eicosanoids, but the mediat ors produced have different structures and functions from those made from arachidonic acid. In stead of serving as substrate for PGE2 and LTB4 biosynthesis, EPA is converted to PGE3 and LTB5 which are much less biologically active than those produced from arachidonic acid (Bagga et al ., 2003). Omega -3 PUFA compete with n6 fatty acids not only for enzymes, but also for the receptors on the leukocytes. They decrease the producti on of inflammatory eicosanoids from


59 arachidonic acid and increase the production of anti-infla mmatory eicosanoids (Wada et al ., 2007). Moreover, long chain n3 PUFA lead to the formation of endocannabinoids, which have marked anti-inflammatory properti es in cell culture systems (Meijerink et al ., 2011). Resolvins and protectins were recently discovered as lipid mediators produced from long chain n3 PUFA. The synthesis of resolvins and protectins also involves the COX and LOX pathways and leads to the resolution of inflammation. Resolvin E1, D1 and protectin D1 all inhibi t transendothelial migration of neutrophils, ultimately preventing the infiltration of neutrophils into the sites of inflammation. Resolvin D1 inhibits IL-1 production, whereas protectin D1 inhibits TNFand IL-1 production (Serhan et al ., 2002). Through their effects on eicosanoid production, n3 PUFA affects the concentrations of cytokines in biologica l fluids. Eicosapentaenoic acid and DHA (2 g/day) suppress the secretion of TNF, IL-1 and IL-6 by endotoxin-stimulated macrophages, and decrease circulating TNF, IL-1 and IL-6 concentrations in blood (Sadeghi et al ., 1999). Lymphocyte-derived cytokine pr oduction (IL-2, IL-4 and IL-10) is also reduced by long chain n3 PUFA (Calder, 1997). Besides the humoral immunity, n3 PUFA influence the cell mediated immunity as well. A number of studies have s hown that ALA, EPA and DHA inhibit the proliferation of lymphocytes in ly mphoid tissues and peripheral blood. Eicosapentaenoic acid appears to be the most i nhibitory. Eicosapentaenoic acid and DHA have been reported to suppr ess phorbol ester-stmulating superoxide generation by neutrophils. Docosahexaenoic acid allegedly inhibits IFN-stimulated tumoricidal


60 action of macrophages by suppressing IFNdependent signals. Furthermore, n3 PUFA affect the killing of microbial or tu mor cells in macrophages by modulating the production of reactive oxygen species and ni tric oxide (Calder, 1997). Not only do n3 PUFA affect the response of lymphocytes to ant igen, but they may also affect the ability of antigen presenting cells to present antigen (Calder, 1997). T he activities of cytotoxic cells (CLT) and natural killer cells are diminished by n3 PUFA as well, because n3 PUFA inhibit NK cell activity directly and suppress the degranulat ion of CLT. In addition, there is evidence that circ ulating levels of immunoglobulin G and immunoglobulin M may be decreased by dietary supplementation of n3 PUFA (Calder, 1997). The health benefits of n3 PUFA may come at a pr ice. The ability of n3 PUFA to improve the host’s survival from gram-posit ive and gram-negative bacterial infections is due to their inhibitory effects on excessive immune responses caused by exotoxin and endotoxin produced by these extr acellular pathogens. However, n3 PUFA can diminish the host’s resistance to intrace llular bacteria, because they inhibit the production of certain key cytokines (IL-12 and IFN) that play essential roles in the host’s defense against intracellular pathogens. Omega -3 PUFA can have a direct cytotoxic action on certain parasites, but they may affect the host’s antiviral defense in both positive and negative ways, and the magnit ude of their effects appears to be much smaller for antiviral than ant ibacterial responses. Ther efore, more studies are needed to investigate the mechanism of n3 PUFAs benefit the host immunity against a large variety of pathogens (Ander son and Fritsche, 2002).


61 Mechanisms of Action of n-3 PUFA Omega -3 PUFA affect inflamma tion by regulating prostaglandin biosynthesis and their effects on nuclear factor kappa(NF-kb) and peroxisome pr oliferator-activated receptor(PPAR) activation which may be mediated via the G-protein coupled receptor GPR 120 (Calder, 2012). Because the primary PUFA in cell membranes is arachidonic acid (AA), most eicosanoids pro duced in cells are prostanoids of the 2 and 4 series (PGE2, PGF2 and leukotriene B4). Eicosapentaenoic acid is a substrate for prostanoids of the 3 and 5-series. In general, AA-derived eicosanoids have proinflammatory effects (Calder and Kew, 2002 ), whereas EPA-derived eicosanoids have anti-inflammatory effects (Calder and Kew, 2002). High intakes of n -3 PUFA result in their incorporation into membrane phosphol ipids, where they partially replace AA (Crawford, 2000). By dec reasing the availability of AA, EPA suppresses the biosynthesis of AA-derived eicosanoids and favors the formation of EPA-derived 3series prostanoids and 5-series leukotrienes. Additionally, n -3 PUFA compete with n -6 PUFA for desaturases and elongases, and have greater affinities for these enzymes than n -6 PUFA. Therefore, dietary intake of n -3 PUFA reduces the desaturation and elongation of linoleic acid (LA) to AA (Rose and Connolly, 1999) and decreases the production of AA-derived eicosanoids. Furthermore, n -3 PUFA suppress COX enzymes or compete with n -6 PUFA for eicosanoid biosynthesis (Ringbom et al ., 2001). Compared with AA, EPA is t he preferential substrate fo r lipoxygenase; hence an increased EPA intake leads to greater form ation of EPA-derived lipoxygenase products at the expense of AA-derived lipoxygenase products (Grimm et al ., 2002). Finally, n -3 PUFA enhance eicosanoid catabolism, wh ich is postulated to be mediated through


62 induction of peroxisomal enzymes (von Schacky et al ., 1993). Changes in fatty acid composition of inflammatory cell membrane phospholipids alters the availability of substrates for synthesis of eicosanoids endocannabinoids, resolvins and protectins (Calder, 2012). A second aspect of the alteration of ce ll membrane phospholipid fatty acids with marine n -3 PUFA involves the so called ‘lipid rafts ’ which are now fairly well studied in T cells (Yaqoob, 2009). Rafts are structures that are formed by the movement of receptors, accessory proteins and enzymes within the plane of the cell membrane. This movement occurs in response to cell activa tion and is essential for the intracellular signals to be properly transduced into the cytos ol and nucleus. Lipid rafts are intimately involved in T-lymphocyte responses to ac tivation (Harder, 2004). Cell culture and animal feeding studies have shown that exposure to marine n -3 PUFA modifies the raft formation in T-cells in a way that impairs the intracellular signaling mechanisms (Zeyda and Stulnig, 2006). Dietary PUFA and their metabolites may exer t some of their ant i-tumor effects by affecting gene expression or activity of si gnal transduction molecules involved in the control of cell growth, differ entiation and apoptosis (Larsson et al ., 2004). Nuclear factor kappa B (NFkBs) is the main transcr iption factor involved in up-regulation of the genes encoding inflammatory cytokines, adhesio n molecules and COX-2 (Siga, 2006). Nuclear factor kappa B are normally confined in the cytoplasm through their association with IkBs. When cells are activated by infl ammatory stimuli, the IkBs are rapidly phosphorylated and degraded to free the NF-kBs. The free NF-kBs then migrate to the nucleus where they bind to cognate DNA bi nding sites and activate inflammatory gene


63 transcription (Calder, 2012). Eicosapentaenoic acid or fish oil decreased endotoxininduced activation of NF-kBs in human monocytes and this was associated with decreased IkB phosphorylation (Zhao et al ., 2004). Likewise, DHA reduced NF-kBs activation in response to endotoxin in cultured macrophages (Lee et al ., 2001) and dendritic cells (Kong et al ., 2010), an effect that involv ed decreased IkB phosphorylation (Lee et al ., 2001). These observations suggest that marine n -3 PUFA may inhibit inflammatory gene expression via inhibition of activation of the transcription factor NFkBs in response to exogenous infl ammatory stimuli (Calder, 2012). Another transcription factor that has been identified as being regulated by fatty acids is PPAR(Three isoforms: PPAR1, PPAR2, and PPAR3. Jump, 2002). This is a member of the PPAR superfa mily, which also includes PPARand PPAR. These ligand-activated transcription factors were first found to be implicated in the regulation of lipid metabolism and homeosta sis but have recently been shown to be involved in cell proliferation, cell differentiation, and infla mmatory responses (Grimaldi, 2001). The preferred nat ural ligands of NFare PUFA, including LA, -LNA, AA, and EPA (Houseknecht et al ., 2002). Proliferato r-activated receptorknockout mice show enhanced susceptibility to chemica lly-induced colitis (Desreumaux et al ., 2001) and PPARagonists reduce colitis in other mice models (Su et al ., 1999). Thus, upregulation of PPARis a likely target for cont rolling inflammation (Desreumaux et al ., 2001). While PPARdirectly regulates inflammatory ge ne expression, it also interferes with the translocation of NFto the nucleus (Vanden Berghe et al ., 2003). Eicosapentaenoic acid has been shown to significantly increase PPAR1 messenger RNA concentrations in isolated adipocytes (Chambrier et al ., 2002). These effects were


64 linked to decreased production of the inflammatory cytokines TNFand IL-6 upon endotoxin stimulation (Kong et al ., 2010). Thus, activation of PPARmay be one of the anti-inflammatory mechanisms of action of marine n -3 PUFA and this may be linked to the inhibition of NFactivation described above (Calder, 2012). Several G-protein coupled cell membrane receptors can bind fatty acids. These include GPR40 and GPR120. Inflammatory macrophages express GPR120 abundantly but do not express GPR40 (Oh et al ., 2010). A synthetic agonist of GPR120 inhibited the macrophage response to endotoxin, an effect that involved main tenance of cytosolic Ik and a decrease in production of TNFand IL-6 (Oh et al ., 2010). These effects are similar to those of EPA and DHA and indicate that GPR120 may be involved in antiinflammatory signali ng. Oh et al. (2010) studied the effect of EPA and DHA on GPR120-mediated gene activation and found that both EPA and DHA enhanced the anti-inflammatory signaling. The ability of DHA to inhibit the responsiveness of macrophages to endotoxin was abolished in GP R120 knockout cells. These findings indicate that the inhibitory effect of DHA (and probably also of EPA) on NFoccurs in part via GPR120. Thus, there appear to be at least two mechanisms by which marine n -3 PUFA inhibit NFactivation-one involving GPR120 and the other involving PPAR, although the two may be linked (Calder, 2012). Besides the mechanisms discuss ed above, incorporation of n -3 PUFA in cell membrane phospholipids increases its flui dity, flexibility, compressibility and permeability (Neuringer et al ., 1988). Additionally, the acti vity of a number of enzymes are affected by changes in membrane fatty acid composition, including ornithine decarboxylase, 3-hydroxy-3-methylglut aryl coenzyme-A reductase, COX-2 and


65 lipoxygenases (Larsson et al ., 2004). Further studies are needed to identify new mechanisms and verify these mechanisms in animal models to better understand the effects of n -3 PUFA intake on animal health in vivo.


66 CHAPTER 2 MATERIALS AND METHODS Experimental Animals and Diets Forty crossbred pigs (averaging 27 2 d of age and 7.8 0.6 kg of BW) were balanced for initial BW and gender across tw o treatment groups in a complete randomized block design. The animal protocol for this research was approved by the institutional Animal Research Committee (ARC) of the Universi ty of Florida. To avoid potential differences due to farrowing seas on, the study was conducted using piglets born within one week at the Swine Research Unit of the University of Florida (Gainesville, FL) during the month of March 2013. Piglets were housed in 8 pens (5 animals per pen; size = 2.4 m x 1.8 m). Table 2-1. Ingredient and calculated composit ions of experimental diets (as-fed basis) Composition Experimental dietsa Ccontrol Omega Corn 61.90 61.90 Soybean meal 25.00 25.00 Vegetable oil 3.00 Gromega 3.00 Min.Vit. mix 10.00 10.00 L-Lysine.HCL 0.10 0.10 Calculated composition ME, kcal/kg 3282.38 3282.38 CP,% 19.53 19.53 CF,% 3.39 3.39 Lysine,% 1.40 1.40 Calcium,% 0.78 0.97 Phosphorus,% 0.63 0.63 aDiets were: Control (3% vegetable oil) and omega (3% Gromega Ultra 345, provided by JBS United, Inc., Sheridan, IN).


67 Four pens were assigned to receive a contro l diet (3% vegetable oil, n = 20), while the other 4 pens were assigned to receive t he n-3 PUFA -supplemented (3% Gromega, JBS United, Sheridan IN, n = 20) diet for f our weeks. Piglets we re provided ad libitum access to feed and water and the nursery te mperature was maintained within the thermoneutral zone for the piglets. Table 2-2. Fatty acid profile (g/100 g of total fat) of experimental diets (as-fed)a Experimental Dietsb Fatty acid Control Omega C14:0 0.21 2.61 C15:0 0.00 2.61 C16:0 14.68 19.76 C16:1,9c 0.32 2.98 C17:0 0.13 0.42 C17:1 0.00 0.39 C18:0 4.26 4.71 C18:1,9c 24.72 23.89 C18:2n-6 49.79 37.04 C18:3n-3 4.53 2.15 C18:4n-3 0.00 0.47 C20:0 0.38 0.40 C20:1n-9 0.00 0.71 C20:5n-3 0.00 1.30 C22:0 0.41 0.26 C22:5n-3 0.00 0.26 C22:6n-3 0.00 0.96 C24:0 0.26 0.34 49.79 37.04 4.53 5.54 10.99 6.69 20.33 28.79 79.36 70.15 aFatty acid analysis was performed by the Univer sity of Missouri Anal ytical Laboratory. bDiets were: Control (3% vegetable oil) and omega (3% Gromega Ultra 345, provided by JBS United, Inc., Sheridan, IN).


68 The n-3 PUFAand vegetable oil-enriched di ets were formulate d to meet the NRC (2012) nutrient requirem ents, and were based primarily on corn, soybean meal, vitamin and mineral premix and L-lysine supp lements which were calculated to contain 3,282.38 Kcal/g ME, 19.53% CP, 3.39% CF and 1.40% Lys. Complete ingredient compositions and FA profiles of experimental diets are su mmarized in Tables 2-1 and 2-2, respectively. Measurement of Growth and Feed Intake Individual piglet body we ights and pen feed consumptio n were recorded weekly throughout the 4-week experiment. These obser vations were used to calculate average daily gain and average daily feed intake. T he G:F ratios were calc ulated to estimate feed efficiency. Blood Collection and Analysis On d 0, 14 and 28 of the experiment, jugul ar venous blood samples (~8 mL from each experimental pig) were collected into evacuated heparinized tubes (BD Franklin Lakes, NJ) and centrifuged (3,000 x g for 15 min) to separate plasma. The plasma samples were stored at -80oC until analysis. Concentrations of IGF-I and TNFin plasma were analyzed using commercially ava ilable ELISA kits (R&D Systems, Inc., Minneapolis, MN). Hormone and cytokine analyses were performed in single assays and intra-assay CV were 4. 0 and 4.7% for IGF-I and TNF, respectively. The least detectable concentrations were 0.06 ng/ mL for IGF-1 and 5.50 pg/Ml for TNF, respectively. On d 27 of the experiment, additional blo od samples were collected for complete blood cell counts (CBC), and hemat ological traits were determined as described by Quiroz-Rocha et al (2009).


69 Fecal Evaluation Fecal scores were measured at each pen on weeks 1, 2, 3, and 4 post-weaning. The scale used to assess fecal consistency was based on a numerical scale of 1 to 3, where 1 represented a normal (hard) feces, 2 represented a soft (moist) feces, and 3 represented diarrhea (watery liquid). The final score for each pen was calculated by averaging the two fecal consistency scores. Statistical Analysis Effects of diets on growth, IGF-I, TNFand fecal characteristics were analyzed using the MIXED procedure of SAS with repeated measures (Littell et al ., 1998). For individual measurements (body weight), fixed effects incl uded diet, sex, diet sex interaction, week after weaning, diet week interaction, sex week interaction and diet sex week interaction. The pig, nes ted within sex and die t, was considered a random variable, and therefore the pig variance was used to test the effects of diet, sex, and diet x sex interaction. A similar model was used to test the effect of diet on plasma IGF-I and TNFconcentrations, except that week a fter weaning was replaced by day of blood sample collection. For collective measurements (feed intake average daily gain, feed efficiency, and fecal consistency score s), the statistical model included the independent effects diets, pen (diet), week relati ve to weaning, diet x week interaction. In these models, the pen was used as experimental unit to test the main effect of diet. Single blood samples were collected for CB C counts, and, therefor e, the statistical models for blood counts contained only the main e ffect of diet. Significant differences were declared at P < 0.05 and tendencies were discussed at P greater than 0.05 but less than 0.10.


70 CHAPTER 3 RESULTS Body Weight Body weight increased ( P < 0.01) from 8.0 0.6 to 16.5 0.6 kg between weeks 0 (week 0 = week of weaning) and 4 in male pigs fed the vegetable oil-enriched diet and from 8.0 0.6 to17.6 0.6 kg in the males consuming the n -3 PUFA-supplemented diet (Figure 3-1A). B A B Males Body weight (kg) 8 10 12 14 16 18 Vegetable oil Omega Females Weeks post-weaning 012345Body weight (kg) 8 10 12 14 16 18 Vegetable oil Omega A B* Figure 3-1. Body weights of male (A) and female (B) pigs fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) for f our weeks after weaning. Asterisk indicates significant differenc e between the least squares means at the specified collection week.


71 Corresponding values for fe male piglets were 7.9 0.6 and 17.7 0.6 kg for the vegetable oil-enriched diet, and 7.4 0.6 to 15.4 0.6 kg for the n -3 PUFAsupplemented diet (Figure 3-1B ). Female piglets fed the n -3 PUFA-enriched diet were lighter ( P < 0.04) than those consum ing the vegetable oil-enriched diet at week 4 postweaning (Figure 3-1B). Average Daily Gain, Feed In take and Feed Efficiency Newly weaned pigs gained ( P < 0.01) 529.6 9.8 g between weeks 2 and 4 post-weaning (Figure 3-2). Corresponding val ues for average daily feed intake and gain feed ratio were 800.6 12.6 and 0.66 0.02, respectively (Figures 4 and 5). Under conditions of this study, inclusion of 3% n -3 PUFA in the diet did not affect daily gain, feed intake or feed efficiency. Days post-weaning 0-1414-280-28Average Daily Gain (g) 0 100 200 300 400 500 600 Vegetable oil Omega a b c Figure 3-2. Average daily gains of weanling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20). Pairs of hi stograms with differ ent superscripts differ at P < 0.01. There were no differences in daily weight gain due to the diet.


72 Days post-weaning 0-1414-280-28Average Daily Feed Intake (g) 0 200 400 600 800 1000 Vegetable oil Omega a b c Figure 3-3. Average daily feed intakes of weanli ng piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20). Pairs of histograms with different superscripts differ at P < 0.01. There were no differences in daily feed intake due to the diet. Days post-weaning 0-1414-280-28Gain : Feed 0.0 0.2 0.4 0.6 0.8 Vegetable oil Omega a b c Figure 3-4. Average gain : feed of weanling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20). Pairs of hi stograms with differ ent superscripts differ at P < 0.01. There were no differences in gain : feed ratio due to the diet.


73 Plasma IGF-I and TNFConcentrations of IGF-I and TNFin plasma are presented in Figures 3-5A and 3-5B. Peripheral IGF-I concentration decreased ( P < 0.01) from 87.2 17.0 ng/mL to 68.3 21.1 ng/mL between days 0 and 14 pos t-weaning, and then increased to 155.2 20.9 ng/mL at d 28. There were no differences in per ipheral IGF-I concentration due to diet or gender of the piglet (Figure 3-5A). 01428IGF-I (ng/mL0 0 50 100 150 200 250 Vegetable oil Omega Days post-weaning 01428TNF( g/mL) 0 20 40 60 80 100 120 Vegetable oil Omega A B * Figure 3-5. Concentrations of IGF-I (A) and TNF(B) in plasma of weanling piglets fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20). Plasma IGF-I concentration was affected by day of blood collection ( P < 0.01), but not dietary treatment ( P > 0.44). Plasma TNFconcentration was affected by dietary treatment ( P < 0.01), but not day of blood collection ( P > 0.28). Asterisks indicate significant difference ( P < 0.01) between the least squares means at the specified collection week.


74 There was a positive correlation between plasma IGF-I concentration and body weight (Pearson’s correlation coefficient =+0.56, P < 0.01). This positive relationship persisted after adjustment for th e dietary effect (partial co rrelation coefficient = +0.56; P < 0.01). In piglets fed the vegetable oil-enriched diet, became elevated ( P < 0.01) plasma TNFconcentration between days 0 and 14 post-weaning (37.6 14.5 pg/mL < 102.9 16.6 pg/mL; Figure 3-5B). Contrastly, pigs fed the n -3 PUFA diet showed no increase in TNFconcentration between day 0 and 14 and t hus, a diet effect (P < 0.05) was noted for TNF(Figure 3-5B). Hematological and Fecal Characteristics Blood platelet and eosinophils concentration was higher ( P < 0.05) in weanling piglets fed the n -3 PUFA-enriched diet as compared to those consuming the vegetable oil-enriched diet (674.0 80.4 103 / mm3 > 378.5 80.4 103 / mm3, P < 0.05; 0.438 0.05 % > 0.208 0.05 %, P < 0.05). Dietary n -3 PUFA had no detectable effects on the other hematol ogical traits (Table 3-1). Table 3-1. Hematological traits of wean ling pigs fed diets with vegetable oil or n -3 PUFAa Experiment Dietsb Trait Control Omega SEM PC WBCd 03 / mm3 9.7 15.7 2.3 0.13 Lymphocytes, % 5.9 6.5 0.5 0.44 Neutrophils, % 7.6 8.0 1.0 0.81 Eosinophils, % 0.2 0.4 0.0 0.02 Monocytes, % 0.4 0.7 0.1 0.20 RBCe x 103 / mm3 9.7 6.6 2.0 0.32 Hemoglobin 10.4 9.2 0.7 0.28 Hematocrit, % 34.1 31.4 2.1 0.37 Platelets x 103 / mm3 378.5 674.0 80.4 0.04 aMeans represent 4 pigs per dietary treatment. bDiets were: Control (3% vegetable oil) and omega (3% Gromega Ultra 345, provided by JBS United, Inc., Sheridan, IN). cP-values for control compared to Omega diet. dWhite blood cells. eRed blood cells.


75 Table 3-2. Fecal consistency scoresa of w eanling pigs fed diets with vegetable oil or n3 PUFAb Experimental dietsc Week Post-weaning Control Omega SEM Pd 1 2.6 2.5 0.2 0.67 2 1.9 1.9 0.2 1.00 3 2.0 1.9 0.2 0.68 4 1.3 1.1 0.2 0.68 aThe scale used for assessing fecal consisten cy was based on a numerical scale of 1 to 3, where 1 represented a norma l (hard) feces, 2 represent ed a soft moist feces, and 3 represented diarrhea (watery liquid). bMeans represent average fecal scores for 4 pens per dietary treatment. cDiets were: Control (3% vegetable oil) and omega (3% Gromega Ultra 345, provided by JBS United, Inc., Sheridan, IN). dP-values for control compared to Omega diet. Fecal consistency score decreased ( P < 0.01) from 2.6 0.1 at week 1 to 1.2 0.1 at week 4, but did not differ between the two dietary treatments (Table 3-2). There were no differences in fecal consistency scores due to the diets.


76 CHAPTER 4 DISCUSSION Weaning imposes tremendous stress on pi glets and is accompanied by marked changes in gastrointestinal physiology, microbiology and immunology (Pluske et al ., 1997; Brooks et al ., 2001). The biochemical and histolog ical changes that occur in the small intestine cause excessive secretion of pro-inflammatory cytokines and induce severe intestinal inflammation. Omega -3 PUFA are known to possess antiinflammatory properties in humans (Calder, 2010, 2012), swine (Carroll et al ., 2003; Liu et al ., 2003; Mateo et al ., 2009) and chickens (Korver and Kl ansing, 1997). To test the hypothesis that nutritional m anagement strategies that atte nuate intestinal inflammation may repartition nutrients to tissue accreti on, we examined the effects of dietary n -3 PUFA on growth and immune responses of weanling pigs raised without an added bacterial or environmental challenge. Inclusion of 3 % n -3 PUFA in the weanling piglet ’s diet did not significantly improve average daily gain, aver age daily feed intake or gain:f eed ratio. Our results are consistent with an earlier study (Eastwood et al ., 2009) which detected no linear or quadratic effects of dietary flax seed m eal (rich in alpha linolenic acid, an n -3 PUFA on basal body weight gain, feed in take or feed efficiency in w eanling pigs. These findings indicate that, in the absence of signifi cant immunological challenges, dietary n -3 PUFA do not affect feed intake and growth response in nursery pigs (Liu et al ., 2003). Additionally, the fact that the control diet contained a relatively high amount of n -3 PUFA (Table 3) may have prevented us from detecting true n -3 PUFA effects on growth parameters. The improvement of body weight gain detected between days 14 and 28 post-weaning (Figure 3) likely resulted from an increase in feed intake and a decrease


77 in basal inflammatory challenges during the second phase of growth. The observation that female piglets consuming the n -3 PUFA-supplemented diet were lighter at week 4 than those consuming the v egetable oil-enriched diet, the mechanism warrants further investigation. Peripheral concentration of IGF-I decr eased immediately fo llowing weaning and increased again by day 28 post-weaning. Thes e findings indicate that weaning causes a significant metabolic stress in weanling pigs and that this stress decreases with increasing weeks after weaning. There is little information on the effect of dietary n -3 PUFA on peripheral concentration of growth factors in the pig. In the present study, inclusion of 3 % n -3 PUFA into the piglet’s diet had no detectable effects on plasma IGF-I concentration during the first four wee ks after weaning. These observations are consistent with previous studies (Carroll et al ., 2003; Liu et al ., 2003) which showed no beneficial effects of dietary fish oil on basal IGF-I concentration in weaned pigs. Thissen and Verniers (1997) reported that interleukin-6 (IL-6) and TNFdecreased both growth hormone (GH) and IGF-I mRNA in rat hepatocyte primary cultures. We did not examine GH or IGF-I transcript modulation by inflammatory cytokines, and therefore, whether or not the lack of n -3 PUFA effects on plasma IGF-I concentration detected in the present study was indicative of cytokine-mediated uncoupling of GH and IGF-I gene expression in weanling pigs warrants further investigation. Tumor necrosis factor, a cytokine produced primarily by monocytes and macrophages, is thought to be one of the principal mediators of inflammation (Bemelmans et al ., 1994). In the present study, plasma TNFconcentrations were lower in weanling piglets supplemented with n -3 PUFA than those fed the vegetable oil


78 supplement. These findings are consis tent with previous in vitro (Lo et al ., 1999; Novak et al ., 2003; Zhao et al ., 2004) and in vivo (Carroll et al ., 2003; Gaines et al ., 2003; Malekshahi Moghadam et al ., 2012) studies and suggest that n -3 PUFA inclusion in the diet could significantly improve the piglet s’ immune status. Whereas exact mechanisms of n -3 PUFA suppression of TNFare yet to be fully eluci dated, we speculate that suppression of TNFproduction by n -3 PUFA may be attribut ed, in part, to their inhibitory effects on the nuclear factor kappa B (NFB) activation and or translocation to the nucleus (Chong-Jeh et al ., 1999; Novak et al ., 2003; Zhao et al ., 2004). Nuclear factor-kBs are normally confined in the cytopl asm through their asso ciation with IkBs. When cells are activated by inflammatory st imuli, the IkBs are rapidly phosphorylated and degraded to free the NF-kBs. The free NF-kBs then migr ate to the nucleus where they bind to cognate DNA binding sites and activate inflammatory gene transcription (Calder, 2012). On the other hand, eicosapentaenoic acid is a substrate for prostanoids of the 3 and 5-series, which are less infla mmatory than arachidonic acid (AA)-derived prostaglandins. Additionally, eicosapentaenoic acid was shown to significantly increase PPAR1 messenger RNA levels in isolated adipocytes (Chambrier et al ., 2002). These effects were linked to decreased producti on of the inflammatory cytokines TNFand IL-6 upon endotoxin stimulation (Kong et al ., 2010). Thus, activation of PPARmay be another intracellular mechanism by which marine n -3 PUFA regulate NFactivation and TNF-a production in cell models (Calder, 2012). Hematological traits of swine are infl uenced by a variety of environmental and physiological factors including diet, age, sex and housing (Wilson et al ., 1972; Friendship et al ., 1984). In the present study, mo st of the blood characteristics


79 examined did not differ between pigs fed the two dietary tr eatments (Table 4). Blood samples for complete blood cell count (CBC) were collected at 4 weeks after weaning, and it is possible that by this sampling time the weanling piglets had already recovered from most physiological and dietary challe nges normally associated with weaning in pigs. Alternatively, the piglets used in this study were raised in a thoroughly cleaned environment (pressured washed followed by steam cleaning and then sprayed with a disinfectant) and may not have acquired adequate intestinal t hat would cause clinical disease. This hypothesis was further supported by our inability to detect salmonella and enterotoxigenic E. coli in fecal samples collected at week 4 post-weaning (data not shown).


80 CHAPTER 5 CONCLUSION In the pig, the period following weaning is generally characterized by sub-optimal growth, deteriorated feed efficiency, and a high incidence of diarrhea (Heo et al ., 2012; Pluske et al ., 2013). Results of this st udy provided no evidence for n -3 PUFA modulation of growth or hem atological parameters of nurs ery piglets raised in the absence of significant immunological and env ironmental challenges, but might decrease the female piglets’ growth rate. However, dietary n -3 PUFA may improve the immune status of weanling pigs, as reflect ed by considerably lower plasma TNFin pigs consuming n -3 PUFA than those fed vegetable o il. Furthermore, the gradual increase of body weight, feed intake and feed efficiency following weaning may reflect a slow adaptation to post-weaning diets and a gradual improvement of t he gastrointestinal microbiota.


81 APPENDIX LEAST SQUARES MEANS SIGNIFICANT DATA Table A-1. Least squares means (+SEM) for body weights (kg) of male and female pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) Week post-weaning Male pigs Female pigs Vegetable oil Omega Vegetable oil Omega 1 8.0 + 0.6 8.0 + 0.6 7.9 + 0.6 7.3 + 0.6 2 9.2 + 0.6 9.6 + 0.6 9.9 + 0.6 8.8 + 0.6 3 12.2 + 0.6 13.3 + 0.6 13.5 + 0.6 12.2 + 0.6 4 16.5 + 0.6 17.6 + 0.6 17.7 + 0.6 15.4 + 0.6 Table A-2. Least squares means (+SEM) for daily body weight gains (g) of weanling pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) Days post-weaning Vegetable oil Omega 0-14 97.0 + 13.9 90.5 + 13.9 14-28 538.4 + 13.9 520.9 + 13.9 0-28 317.7 + 13.9 298.7 + 13.9 Table A-3. Least squares means (+SEM) for daily feed intakes (g) of weanling pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) Days post-weaning Vegetable oil Omega 0-14 258.8 + 17.8 241.1 + 17.8 14-28 796.6 + 17.8 804.7 + 17.8 0-28 527.7 + 17.8 522.9 + 17.8


82 Table A-4. Least squares means (+SEM) for gain : feed ratios of weanling pigs fed diets enriched with vegetable oil (n = 20) or n-3 PUFA (n = 20) Days post-weaning Vegetable oil Omega 0-14 0.37 + 0.02 0.38 + 0.02 14-28 0.68 + 0.02 0.65 + 0.02 0-28 0.61 + 0.02 0.58 + 0.02 Table A-5. Least squares means (+SEM) for pl asma insulin-like grow th factor-I (IGF-I) and tumor necrosis factor alpha (TNFconcentrations in weanling pigs fed diets enriched with vegetable oil (n = 20) or n -3 PUFA (n = 20) Days post-weaning IGF-I TNFVegetable oil Omega Vegetable oil Omega 0 93.8 + 22.7 80.6 + 25.4 37.6 + 14.5 33.0 + 17.2 14 84.9 + 26.0 51.7 + 33.2 102.9 + 16.6 29.1 + 21.7 28 142.3 + 25.4 168.1 + 33.2 99.0 + 17.2 36.7 + 21.4


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100 BIOGRAPHICAL SKETCH Qizhang Li was born in Nanji ng, China, in 1987. She is daughter of Xiaosu Li and Minglang Xia. She graduated from Nanji ng NO.13 high school in 2006, and started her Bachelor of Agriculture that same year at Yangzhou University in Animal Sciences Department. She graduated in 2010 and after that, she spent one year working as a laboratory technician in the same department. S he joined the University of Florida In fall 2012, and began working on her Master of Sc ience degree under the guidance of Dr. Lokenga Badinga. She graduated with a M.S. in animal sciences in 2014. Her research at the University of Florida has focused on the effects of omega -3 polyunsaturated fatty acid on growth and immunity of weanling piglets.