EFFECT OF DIETARY ARACHIDONIC ACID LEVEL AND SOURCE ON NEONATAL PIGLET DEVELOPMENT A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Cynthia Tyburczy December 2010 © 2010 Cynthia Tyburczy EFFECT OF DIETARY ARACHIDONIC ACID LEVEL AND SOURCE ON NEONATAL PIGLET DEVELOPMENT Cynthia Tyburczy, Ph. D. Cornell University 2010 Arachidonic acid (ARA) is a long chain polyunsaturated fatty acid that is routinely added to infant formula with docosahexaenoic acid (DHA) to support neonatal growth and development. Optimal levels of ARA in formula remain to be determined and are based largely on mean worldwide values in breast milk. The objective of this dissertation research was to determine the role of dietary ARA in neonatal development and to evaluate the optimal level in formula to support proper growth and development. Two studies with neonatal domestic pigs were carried out to achieve this objective. The purpose of the first study was to determine the effect of the dietary ARA level on growth, clinical chemistry and immune function, and on tissue ARA and DHA accretion. On day 3 of age, formula-reared (FR) piglets were assigned to 1 of 6 milk replacer formulas containing ARA/DHA as follows (% fatty acid (FA)/FA): (a1) 0.1/1.0; (a2) 0.53/1.0; (a3-d3) 0.69/1.0; (a4) 1.1/1.0; (d2) 0.67/0.62; (d1) 0.66/0.33. No significant differences were observed among the FR groups for growth, hemogram, clinical chemistry or measures of immune status. Heart and liver ARA were responsive to dietary ARA while brain and retina were not. Heart ARA was particularly sensitive to dietary ARA. In the second study, we evaluated two ARA oils for potential use in infant formula compared with a third, commercially available ARA oil. Diets were fed on days 3 – 22 of life and supplied ARA at 0.64% FA and DHA at 0.34% FA. We observed no toxicological effects or differences in growth, and concluded that the experimental ARA oils are safe and bioequivalent to the commercially available ARA source. Overall, results from this dissertation research suggest that the dietary ARA level has a negligible effect on growth and development of the central nervous and immune systems when DHA is supplied near the high end of human breast milk levels (1% FA). The unique responsiveness of the heart to dietary ARA level revealed that ARA accretion is limited by dietary supply. Further investigations are warranted to determine the significance of ARA status on immediate and long term cardiac physiology. BIOGRAPHICAL SKETCH Cynthia Tyburczy was born on November 10, 1978 in Red Bank, New Jersey to Kathleen and Joseph Tyburczy. She grew up in Harrington Park, New Jersey and graduated from Northern Valley Regional High School in Old Tappan in 1997. As a child, she enjoyed drawing, playing softball, being a member of the Girl Scouts and cooking with her father. She developed a passion for animals early on that was fostered by frequent trips to zoos and aquariums, and horseback riding lessons. Cynthia attended Rutgers University after high school, but soon decided to suspend her academic training to travel and attend live musical performances. She moved to the mountains of Colorado and on June 2, 2001, was blessed with the friendship of her puppy, Jackson. It was her passion for learning that led her to Cornell University where she finished her B.S. and earned her M.S. in Animal Science under the supervision of Dr. Dale Bauman studying the bioactivity and metabolism of trans 18:1 fatty acids. Cynthia joined the laboratory of Dr. J. Thomas Brenna where she began her doctoral work learning covalent adduct chemical ionization tandem mass spectrometry, and was matched with a project studying arachidonic acid requirements in neonatal pigs, that soon became the primary focus of her dissertation research. Currently, Cynthia has eight first author publications, and has coauthored 18 additional articles, reviews, conference proceedings & abstracts. In her free time, Cynthia enjoys hiking with Jackson, beer, wine and cheese tasting, traveling, attending live musical performances, watching kungfu movies, swimming and going to the beach. Post-graduation she aspires to learn to play the piano and develop a green thumb. iii I dedicate this dissertation to my grandmother, Genevieve Tyburczy, whose battle with heart disease inspired me to pursue a career in lipid nutrition where my contributions could help to reduce the risk of chronic disease in future generations. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. J. Thomas Brenna, for providing the opportunity to learn and study in his laboratory, and for challenging me during my graduate career. I would also like to thank my committee members, Dr. Gerald W. Feigenson, Dr. Ellis R. Loew and Dr. Ling Qi for guidance and helpful discussions. I would like to thank my parents and sister, Kristin, for believing in me, supporting me in my academic pursuits and assuring me that my potential is limitless. I would like to thank my boyfriend, Aaron Guilbeau, for patiently supporting me during my graduate career, for partaking in many adventures with me and sharing a passion for music and travel. I would like to acknowledge Jackson, who has been the greatest friend and companion I have ever had, and who is undoubtedly the world’s most educated dog in the field of lipid nutrition. I would also like to acknowledge my cats, Kitty and Ragamuffin, who have spent numerous hours warming my lap during the preparation of this dissertation. Finally, I would like to acknowledge the members of the Brenna lab, especially Dr. Ying Zhang, Dr. Kumar Kothapalli, Dr. Woo Jung Park, Dr. Rinat Ran-Resseler and Peter Lawerence, for the invaluable friendships and for making the past three years a happy and memorable experience. v TABLE OF CONTENTS Biographical sketch Dedication Acknowledgements Table of contents List of figures List of tables List of abbreviations Chapter One: Neglible effect of dietary arachidonic acid (ARA) levels on growth, clinical chemistry and immune function in domestic piglets Abstract Introduction Materials and methods Animals Diets Sampling Clinical chemistry and hemogram analysis Mycoplasma hyopneumoniae vaccination Enzyme linked immunosorbant assays (ELISA) Statistical analysis Results Clinical observations, feed intake and growth Hemogram and clinical chemistry iii iv v vi ix xi xiv 1 1 2 4 4 5 7 7 7 10 10 11 12 vi Immunization response & measurements of immune status 14 Discussion 17 References 23 Chapter Two: Heart arachidonic acid (ARA) is uniquely sensitive to 27 dietary ARA and docosahexaenoic acid (DHA) in domestic piglets Abstract 27 Introduction 28 Materials and methods 30 Animals and diets 30 Sampling 31 Lipid analysis 31 Statistical analysis 34 Results 34 LCPUFA accretion in liver and heart 34 LCPUFA accretion in neural tissues 35 Tissue ARA and DHA dose-response to diet 37 ARA and DHA competition for tissue incorporation 40 Discussion 42 References 48 Chapter Three: Evaluation of bioequivalency and toxicological 53 effects of three arachidonic acid (ARA) sources in domestic piglets Abstract 53 Introduction 54 Materials and methods 56 vii Animals Diets Sampling Clinical chemistry and hemogram analysis Liver histopathology Fatty acid analysis Statistical analysis Results Formula consumption Clinical observations and growth Hemogram and clinical chemistry Liver histopathology Tissue LCPUFA accretion Discussion References 56 56 59 59 60 60 61 61 61 61 62 63 64 71 76 Appendix I: Supplemental figures and tables for Chapter Two 80 Appendix II: Protocol for the One-step hydrolysis, extraction and methylation procedure for the preparation of fatty acid methyl esters from vertebrate soft tissue 91 viii LIST OF FIGURES 1.1 Body weight measurements by piglet day of age 12 1.2 Relative organ (A) and absolute brain (B) weights from piglets on day 28 of age 13 1.3 Profile of hemogram parameters from piglets on day 28 of age 14 2.1 ARA tissue dose response to dietary ARA content with constant DHA (1.0% total FA) 40 2.2 DHA tissue dose-response to dietary ARA content with constant 41 DHA (1.0% total FA) 2.3 ARA tissue dose-response to dietary DHA content with constant 42 ARA (0.67% total FA) 3.1 Temporal pattern of body weight gain in piglets from day 3 to 22 63 of age 3.2 Relative organ weights of piglets on day 22 of age 64 3.3 Temporal pattern of ARA (A) and DHA (B) content in RBC collected from pigs on days 3, 7, 14 and 21 of life 70 ix S1 DHA tissue dose-response to dietary DHA with constant ARA (0.67% total FA) 80 S2 Neural tissue DHA dose-response to dietary DHA with constant ARA (0.67% total FA) 81 S3 Neural tissue DHA dose-response to dietary ARA with constant DHA (1.0% total FA) 82 x LIST OF TABLES 1.1 Ingredient composition and nutrient analysis of experimental diets 8 1.2 FA composition of milk replacer formulas and sow milk 9 1.3 Summary of clinical chemistry profiles from piglets on day 28 of age 15 1.4 Summary of immune status measurements in piglets fed varying 16 levels of ARA and DHA 2.1 Nutrient composition and analysis of experimental diets 32 2.2 FA composition of milk replacer formulas and sow milk 33 2.3 FA composition of heart and liver from piglets fed varying levels of ARA and DHA from days 3 – 28 of age 36 2.4 FA composition of cerebral cortex, retina and cerebellum from piglets fed varying levels of ARA and DHA from days 3 – 28 of age 38 2.5 FA composition of hippocampus, globus pallidus and inferior colliculus from piglets fed varying levels of ARA and DHA from 39 xi days 3 – 28 of age 3.1 Nutrient composition of experimental diets 57 3.2 FA composition of experimental diets 58 3.3 Summary of hemogram and clinical chemistry parameters from piglets on day 21 of age 65 3.4 FA composition of heart and liver from piglets fed one of three sources of ARA 66 3.5 FA composition of brain (cerebral cortex) and retina from piglets 67 fed one of three sources of ARA 3.6 LCPUFA composition of RBC collected from piglets on days 3, 7, 69 14 and 21 of life S1 FA composition of heart from piglets fed varying levels of ARA and DHA from days 3 - 28 of age 83 S2 FA composition of liver from piglets fed varying levels of ARA and DHA from days 3 - 28 of age 84 S3 FA composition of cerebral cortex from piglets fed varying levels 85 of ARA and DHA from days 3 - 28 of age xii S4 FA composition of retina from piglets fed varying levels of ARA and DHA from days 3 - 28 of age 86 S5 FA composition of cerebellum from piglets fed varying levels of ARA and DHA from days 3 - 28 of age 87 S6 FA composition of hippocampus from piglets fed varying levels of ARA and DHA from days 3 - 28 of age 88 S7 FA composition of globus pallidus from piglets fed varying levels 89 of ARA and DHA from days 3 - 28 of age S8 FA composition of inferior colliculus from piglets fed varying levels 90 of ARA and DHA from days 3 - 28 of age xiii LIST OF ABBREVIATIONS ARA AST DHA FA FAME FR GRAS HP hsCRP HUFA LCPUFA MR MUFA PUFA RAO RBC SAA SFA Arachidonic acid Aspartate aminotransferase Docosahexaenoic acid Fatty acid Fatty acid methyl ester Formula-reared Generally regarded as safe Haptoglobin Highly-sensitive C-reactive protein Highly unsaturated fatty acid Long chain polyunsaturated fatty acid Maternal-reared Monounsaturated fatty acid Polyunsaturated fatty acid Refined arachidonic acid-rich oil Red blood cell Serum amyloid A Saturated fatty acid xiv CHAPTER ONE NEGLIGIBLE EFFECT OF DIETARY ARACHIDONIC ACID (ARA) LEVELS ON GROWTH, CLINICAL CHEMISTRY AND IMMUNE FUNCTION IN DOMESTIC PIGLETS* Abstract In the U.S., infant formulas with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contain these long chain polyunsaturated fatty acids (LCPUFA) in a 2:1 ratio and comprising about 0.67% and 0.32% of total fatty acids (FA). Higher levels of dietary DHA appear to provide some advantages in visual or cognitive performance. This study evaluated the effect of the dietary ARA level on growth, clinical chemistry and immune function when DHA is at the high end of human breast milk levels. On day 3 of age, formula-reared (FR) piglets were matched for weight and assigned to one of six milk replacer formulas. Diets varied in the ratio of ARA/DHA as follows (% FA/FA): (a1) 0.1/1.0; (a2) 0.53/1.0; (a3-d3) 0.69/1.0; (a4) 1.1/1.0; (d2) 0.67/0.62; (d1) 0.66/0.33. A seventh group was maternal-reared (MR) and remained with the dam during the study. Blood collection and body weight measurements were performed weekly and piglets were sacrificed on day 28 of age. No significant differences were found among any of the FR groups for formula intake, growth, hemogram, clinical chemistry or immune status measurements. Mean body weight of MR pigs on day 28 of age was 2.4 kg less than the FR pigs. A few differences in clinical chemistry and immune *Tyburczy, C., Kothapalli, K. S. D., Park, W. J., Blank, B. S., Liu, Y.-C., Nauroth, J. M., Zimmer, J.P., Salem, Jr., N., Brenna, J. T. (2010) Food Chem Toxicol, submitted. 1 status between MR and FR groups likely reflected a difference in growth rate. We conclude that the dietary ARA level has a negligible effect on growth, clinical chemistry and immune function in domestic piglets when DHA is supplied at 1.0% FA, and confirm that DHA has no effect when ARA is at 0.66%. Introduction Arachidonic acid (ARA, 20:4n-6) is a natural component of human breast milk and is routinely added to infant formulas along with docosahexaenoic acid (DHA, 22:6n-3). Both are long chain polyunsaturated fatty acids (LCPUFA) that contribute to normal growth and development during the perinatal period, serving as essential structural components of central nervous tissue and precursors for lipid signaling molecules (1,2). In the U.S., conventional infant formulas include ARA and DHA at target levels of 0.66% and 0.32% of total fatty acids (FA), respectively. World-wide levels in breast milk prove more variable, however, comprising 0.47 ± 0.13% (range 0.24 – 1.0%) and 0.32 ± 0.22% (range 0.06 – 1.4%) of total FA (3). The range of ARA and DHA in breast milk is likely due primarily to maternal diet. As a guide for infant formulas, the world-wide variability in breast milk levels presents a challenge for optimizing the FA composition of formula to support proper development of the infant central nervous and immune systems. Clinical studies in human infants that are designed to determine the optimal level of LCPUFA in formula are limited in the toxicological parameters that they can measure. Here, studies have demonstrated visual and cognitive benefits of LCPUFA intake to formula-fed infants, compared with nonsupplemented formula-fed infants (4). For these studies, DHA was 2 investigated in the range of 0.2 – 1.0% of total FA, and results provide convincing evidence for maintaining DHA in formula at levels ≥ 0.3%. Levels of DHA up to 1.0% of total FA have been reported as generally safe for infants 1-6 months of age, while preclinical studies in weanling rats report no adverse effects on growth or development with DHA intakes up to 5900 mg/kg/d (5). On the other hand, studies addressing the level of ARA or the ARA/DHA ratio in formula are rare and limited to only a few preclinical studies where ARA and DHA were fed together in a 2:1 ratio (6-8). ARA and DHA are thought to compete for tissue incorporation, including liver (9) and may influence each other’s physiological effects (10,11). The effect of this competition on functional outcomes associated with ARA, particularly in the context of high dietary DHA, remains unclear. The absence or presence of DHA and ARA in piglet formula can modulate neonatal immune responses (12), but dose-response effects have not been examined. Three immune status measures were included in this study to examine immunization response, immune maturation and inflammatory state. These measures were selected to be applicable to both piglet and infant immune function. For example, if the dietary treatments modulate immunization response in the piglets, clinical trials could then followup by monitoring vaccine responses in infants. For immune maturation, it is known that early life serum IgG and IgA concentrations increase as exogenous antigen exposures accumulate (13). This applies to piglets as well because gnotobiotic pigs have significantly lower serum IgA and IgG compared to conventionally-raised pigs with a typical gut microflora (14). Thus, serum IgA or IgG may provide a novel but rough comparison of net immunological maturation across dietary treatments. Finally, higher intakes of 3 DHA are anti-inflammatory in humans and animal models (2). It is not known if increasing the dietary ratio of ARA to DHA in piglet formula could produce a relatively pro-inflammatory state. A wide range of clinical and subclinical inflammatory events can trigger the liver to produce serum acute phase proteins. Three different acute phase proteins, highly-sensitive C-reactive protein, serum amyloid A and haptoglobin (hsCRP, SAA and Hp), were monitored because they can differ in sensitivity to detect systemic inflammation (15). To date, there have been no ARA dose-response studies against a background of high dietary DHA in rapidly growing, suckling animals. Our objective was to determine the effect of the dietary ARA level on growth, clinical chemistry and immune function when DHA is constant and near the high end of human breast milk levels (i.e. 1.0% total FA). A dose-response for DHA was also included with normal levels of ARA. Domestic piglets were used because of their metabolic similarities to humans as well as their rates of perinatal brain growth (16). Human breast milk is considered the gold standard of nutrition for the developing infant, and because the FA composition and nutrient content of sow milk closely mimics human breast milk, an additional group of piglets were included as a maternal-reared (MR) reference treatment. Materials and methods Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Cornell University. Domestic piglets were selected for gender and weight from 15 sows at the swine facility at Cornell 4 University. One day before the scheduled, term farrowing date, pregnant sows were injected with 2 cc of Lutalyse (Pfizer Animal Health, Kalamazoo, MI) to obtain a block of piglets with the same birth date. Piglets were processed according to standard facility practices (i.e. intramuscular injection of iron dextran (100 mg) and penicillin G (½ cc), tooth clip, tail dock, ear notch) and remained with the sow until day 3 of age, at which time they were matched for weight and assigned to one of six milk replacer diets (n=8 per diet). The formula-reared (FR) piglets were housed individually in raised metal cages and maintained on a 16/8 hr light/dark cycle. MR piglets remained with their dam at the swine facility for the duration of the study. Diets Milk replacer formula consisting of 60% experimental diet (Research Diets, Inc., New Brunswick, NJ) and 40% Birthright baby pig milk replacer (Ralco Nutrition, Inc., Marshall, MN) was fed to FR piglets on days 3 – 28 of age. Diets were designed to meet or exceed nutrient requirements for growing pigs 3 – 5 kg in body weight (17). The nutrient composition of the Birthright baby pig milk replacer is published elsewhere (18). A detailed ingredient composition and nutrient analysis of the experimental diets is listed in Table 1.1. The four-oil blend consisting of palmolein, soy, coconut and high oleic sunflower oils that is currently used in the commercial human infant formula Enfamil (Mead-Johnson Nutrition, Evansville, IN) made up the base oil for the experimental diets. LCPUFA were supplied by ARASCO and DHASCO single-cell oils (Martek Biosciences, Corp., Columbia, MD), derived from the marine micro-algae C. cohnii and the fungus M. alpina, respectively. As fed, the milk replacer formula had a caloric density of 0.7 kcal/ml and a 5 macronutrient analysis as follows (gram %): protein 26.7%, fat 21.9% and carbohydrate 38.9%. Formulas were constituted daily by adding 1.0 L water to 180 g dry diet and stored at 3-4˚C until feeding. Fresh formula was provided three times per day in stainless steel bowls fixed into the cage doors and water was offered ad libitum. FR piglets were fed at 80% ad libitum intake, based on pilot data, to moderately exceed growth rates of the MR piglets (19). Formula intakes were recorded daily. The FA composition of the milk replacer formula and sow milk (day 14 in lactation) is presented in Table 1.2. Diets varied in the ratio of ARA/DHA as follows (% FA/FA): (a1) 0.1/1.0; (a2) 0.53/1.0; (a3-d3) 0.69/1.0; (a4) 1.1/1.0; (d2) 0.67/0.62; (d1) 0.66/0.33 (conventional infant formula), and corresponded to the following ARA/DHA intakes (mg/mg per 100 kcal of formula): (a1) 3.2/45.3; (a2) 24.0/45.3; (a3-d3) 31.3/45.3; (a4) 49.0/45.3; (d2) 30.4/28.1; (d1) 29.9/14.9. Diets a1-a4 are a dose-response for ARA against DHA constant at 1.0%, while diets d1-d3 are a dose-response for DHA against ARA constant at 0.67%. Diets d2 and a4 contain ARA and DHA at a 1:1 ratio, but at two different dietary levels, 0.67% and 1.0%, respectively. FA composition of the diets and sow milk was determined by gas chromatography. FA methyl esters (FAME) were prepared from dry diet and 50 μl sow milk by the one-step hydrolysis, extraction, and methylation procedure (20) as modified previously (21) and quantified on a 5890 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a BPX70 fused silica column (25 m x 0.22 mm i.d. x 0.25 μm film; SGE Inc., Austin, TX). A detailed protocol for the One-step method is presented in Appendix II. An equal weight FAME mixture was used to verify response factors. FAME were structurally identified by 6 covalent-adduct chemical ionization mass spectrometry on a Saturn 2000 mass spectrometer (Varian, Inc., Walnut Creek, CA) (22). Sampling Body weights measurements were taken weekly. Non-fasted blood samples were collected from the anterior vena cava into vacutainer tubes (BD, Franklin Lakes, NJ) on days 3, 7, 14 and 21 of age. Overnight-fasted blood samples were collected on day 28 of age, and an additional aliquot of blood was collected into EDTA-containing tubes for hemogram analysis. Serum was harvested by centrifuging clotted blood at 2800 rpm and 4˚C for 10 min. Piglets were sacrificed on day 28 of age via an intravenous injection of Fatal Plus (1 ml/4.54 kg body weight; Vortech Pharmaceuticals, Dearborn, MI, USA) followed by exsanguination. Organs were removed, weighed, and samples flash frozen within 10 min of cessation of heart beat. Clinical chemistry and hemogram analysis Serum and EDTA-blood from 28-day-old piglets were delivered to the Animal Health Diagnostic Center (College of Veterinary Medicine, Cornell University) for analysis of clinical chemistry and hemogram parameters. Mycoplasma hyopneumoniae vaccination Piglets were vaccinated against M. hyopneumoniae (RespiSure-One, Pfizer Animal Health, Kalamazoo, MI) on day 7 of age. Serum samples collected on days 21 and 28 of age (14 and 21 days post-immunization) were analyzed for antibodies to M. hyopneumoniae. M. hyopneumoniae immunity protects against a common respiratory disease in swine that is not endemic at 7 Table 1.1. Ingredient composition and nutrient analysis of experimental diets Composition Content Dietary ingredient, % dry matter Dried skim milk 55.0 Oil blend 23.9 Calcium sodium caseinate 9.6 Vitamin mix1 4.8 Mineral mix2 4.8 Xanthan gum 1.4 Methionine, DL 0.5 Nutrient analysis, % kcal Protein 24.3 Carbohydrate 28.7 Fat 47.0 1The mineral mix contained (g/kg): 257.8 sucrose, 615.8 dibasic calcium phosphate, 106.7 sodium chloride, 11.8 ferric citrate, 4.0 zinc carbonate, 3.4 magnesium oxide, 0.4 cupric carbonate, 0.2 manganous carbonate, 0.1 potassium iodate, 0.01 sodium selenite. 2The vitamin mix contained (g/kg): 910.1 sucrose, 75.0 choline bitartrate, 6.2 vitamin E acetate (50%), 3.4 vitamin A acetate (500,000 IU/g), 1.5 vitamin D3 (100,000 IU/g), 1.3 vitamin B12 (0.1% mannitol), 0.9 pantothenic acid (d, calcium), 0.9 niacin, 0.4 biotin (1%), 0.1 riboflavin, 0.1 thiamine HCl 0.03 menadione sodium bisulfite, 0.01 folic acid, 0.01 pyridoxine HCl. 8 Diet ARA/DHA Σ SFA + MUFA ARA DHA Σ n-6 Σ n-3 18:2n-6 18:3n-3 18:2n-6/18:3n-3 Table 1.2. FA composition of milk replacer formulas and sow milk1 a1 a2 a3-d3 a4 d2 d1 0.1/1.0 0.53/1.0 0.69/1.0 1.1/1.0 0.67/0.62 0.66/0.33 79.86 0.09 79.94 0.53 79.43 0.69 FA (% total FA) 79.18 80.02 1.06 0.67 80.02 0.66 1.00 17.50 1.02 17.52 1.01 17.95 1.04 18.15 0.62 17.76 0.33 18.00 2.53 2.43 2.49 2.51 2.10 1.86 17.25 1.53 16.83 1.41 17.11 1.48 16.93 1.47 16.90 1.48 17.19 1.53 11.3 12.0 11.6 11.5 11.4 11.3 MR 0.74/0.01 83.94 ± 3.16 0.74 ± 0.02 0.01 ± 0.01 15.21 ± 3.08 0.71 ± 0.04 12.05 ± 2.98 0.57 ± 0.06 21.1 1Milk from two sows taken on day 14 of lactation; MR piglets remained with the sow for the duration of the study. 2SFA, saturated FA; MUFA, monounsaturated FA. 9 the Cornell University swine facility or the Large Animal Research and Teaching Unit. Respisure-One is intended to induce a protective antibody titer 14-21 days after a single injection at day 7 (23). Enzyme linked immunosorbant assays (ELISA) Serum samples were thawed immediately prior to use. Commercial ELISA kits were used to quantify serum total IgA, IgG, and IgM, (Bethyl Laboratories, Montgomery, TX) and the acute phase proteins: hsCRP and Hp (Kamiya Biomedical Company, Seattle, WA), and SAA (Tri-Delta Development LTD, Ireland) following the manufacturer’s instructions. Antibodies to M. hyopneumoniae were detected by semi-quantitive ELISA following the manufacturer’s instructions (HerdChekTM, IDEXX Laboratories, Westbrook, ME). Serum samples were tested in duplicate, with the exception of the Hp ELISA in which two different serum dilutions (1:10,000 and 1:20,000) were averaged. Statistical analysis Statistical analysis was carried out using the Fit Model platform of JMP (2008 SAS Institute, 8.0) to fit mixed models. Fixed effects were diet, gender, day 3 of age body weight and the full factorial of interactions. Interaction effects were considered significant at P < 0.10 and fixed effects at P < 0.05. For each parameter analyzed, effects not considered significant were removed from the final model. Random effects were litter and animal nested within litter for repeated measures of body weight. Significance of pairwise comparisons was determined using the Student’s t-test. Linear regression analysis was performed to determine the relationship between clinical chemistry and 10 hemogram parameters and the dietary content of ARA and DHA. Values are reported as means ± SD. Results Clinical observations, feed intake and growth All 56 piglets remained on the study until day 28 of age, and only one piglet (diet a3-d3) required antibiotic treatment during the study, which was due to developing an abscess on its toe. Total formula intakes averaged 51.6 ± 0.1 L and were unaffected by diet. There were small differences in body weight among the FR groups on day 7 of age with a1 piglets (0.1/1.0% ARA/DHA) weighing significantly less than all other FR groups, as shown in Figure 1.1. By day 14 of age, this difference in body weight was abolished and the a1 piglets showed body weights similar to all other FR groups for the remainder of the study. Further, there were no differences in body weight among any of the FR groups on days 14, 21 or 28 of age. The MR group was heavier than the FR groups on day 7 of age, but the MR group weighed the least at days 21 and 28 of age. On day 28 of age, mean weight for all FR piglets was 10.3 ± 0.6 kg, while MR piglets weighed 7.9 ± 0.7 kg. Relative organ weights (weight as a percentage of final body weight) from piglets on day 28 of age are presented in Figure 1.2 (Panel A). There were no differences in relative organ weights among any of the FR groups; however, compared with the MR group, FR piglets had greater relative weights for liver. Absolute brain weights (Figure 1.2, Panel B) were similar across all diets, while relative brain weight was greatest for MR piglets compared with the FR groups. 11 12 a1 a2 a3-d3 10 a4 d2 d1 MR 8 * * Body Weight (kg) 6 4 2 0 Day 3 Day 7 Day 14 Day 21 Day 28 Figure 1.1. Body weight measurements by piglet day of age. Values (means ± SD) not sharing a common letter are significantly different (P < 0.05); * values significantly different between maternal-reared (MR) piglets and all formula-reared groups (P < 0.05). Hemogram and clinical chemistry Data for the hemogram analysis are presented in Figure 1.3. Mean values for hematocrit and red blood cell count were 35 ± 4% and 6.7 ± 0.6 mill/μl, respectively, and were unaffected by diet. Small differences were apparent among the FR groups for mean cell hemoglobin and red cell distribution width. There were no differences among the FR groups for hemoglobin, mean cell hemoglobin concentration and mean cell volume; however, FR groups significantly differed from the MR piglets for these values with the MR group presenting a more favorable hemogram status. For all piglets, platelet count averaged 1022 ± 259 thou/μl and was unaffected by treatment. Total white blood cell count averaged 9.8 ± 3.3 thou/μl and was unaffected by diet. There were no differences among diets for segmented neutrophils, monocytes, eosinophils or basophils (data not presented). 12 Lymphocyte count was highest in a4 piglets; however, the range of means (4.9 – 6.8 thou/μl) across all diets was within the normal reference range for nursery piglets (24). Weight (% of body weight) Weight (g) A 3.5 3.0 2.5 * a1 a2 a4 d2 MR a3-d3 d1 B 100 75 2.0 1.5 50 1.0 * 0.5 25 0.0 0 Brain Liver Heart Lung Kidney Spleen Brain Figure 1.2. Relative organ (A) and absolute brain (B) weights from piglets on day 28 of age. *Values (means ± SD) significantly different between maternal-reared (MR) piglets and all formula-reared groups (P < 0.05). Consistent with the hemogram analysis, only small differences in the clinical chemistry profile were apparent among the FR groups, including values for serum chloride, albumin and globulin, as listed in Table 1.3. While statistical differences were observed for these parameters, values fall within the normal reference ranges for nursery piglets (25,26) and thus were likely to be biologically insignificant. Also consistent with the hemogram analysis, clinical chemistry values related to serum iron status (i.e. serum iron, total iron binding capacity and percent saturation) were different between MR and FR groups and indicated a more positive serum iron status for the MR piglets. Additional serum clinical chemistry parameters that were not significantly different among any of the dietary treatments were creatinine, aspartate 13 aminotransferase, glutamate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, sorbitol dehydrogenase and creatinine kinase. Mean value (specified units) 70 a1 a2 a3-d3 60 a4 d2 d1 50 MR 40 * 30 bc b c c b bc a 20 * 10 0 * ab b ab ab b a c ab b ab b b a c MPV (fL) RDW (%) MCV (fL) MCH (pg) MCHC (g/dL) Hemoglobin (g/dL) Figure 1.3. Profile of hemogram parameters from piglets on day 28 of age. Values (means ± SD) not sharing a common letter are significantly different (P < 0.05); * values significantly different between maternal-reared (MR) piglets and all formula-reared groups (P < 0.05). MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; MPV, mean platelet volume; MR, maternal-reared; RDW, red cell distribution width. Immunization response & measurements of immune status Table 1.4 presents a summary of immune status measurements. All piglets were negative for antibodies to M. hyopneumoniae on day 28 of age and there were no significant differences in titers to M. hyopneumoniae by dietary treatment. Serum IgA levels in the MR piglets were significantly higher on day 28 of age than all FR groups (P < 0.001), but IgA levels did not differ among any of the FR groups. Serum IgM and IgG levels on day 28 of age were unaffected by diet. There were minor differences in SAA levels on day 7 14 Table 1.3. Summary of clinical chemistry profiles from piglets on day 28 of age1,2 Diet a1 a2 a3-d3 a4 d1 d2 MR ARA/DHA (%, FA/FA) 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Glucose (mg/dL) 158 ± 30 152 ± 32 159 ± 27 156 ± 33 162 ± 27 169 ± 32 131 ± 8 NS BUN (mg/dL) 6±1 6±2 6±2 6±2 6±1 6±2 8±1 NS Creatinine (mg/dL) 0.7 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.9 ± 0.1 NS Total protein (g/dL) 5.2 ± 0.3 5.0 ± 0.3 5.0 ± 0.3 5.1 ± 0.4 5.0 ± 0.3 5.3 ± 0.3 4.7 ± 0.3 NS Total bilirubin (mg/dL) 0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.2 NS Na (mEq/L) 143 ± 4 143 ± 3 144 ± 3 145 ± 3 143 ± 2 144 ± 2 141 ± 2 NS K (mEq/L) Cl (mEq/L) 5.9 ± 0.7 100 ± 2c 6.2 ± 0.7 101 ± 1bc 6.2 ± 0.8 102 ± 2ab 6.2 ± 0.8 101 ± 2ab 5.9 ± 0.8 100 ± 2bc 5.9 ± 0.5 100 ± 2c 5.2 ± 0.9 105 ± 2a NS 0.02 Bicarbonate (mEq/L) 19 ± 5 21 ± 5 20 ± 4 20 ± 6 21 ± 4 19 ± 5 23 ± 2 NS Ca (mg/dL) 11.3 ± 0.7 11.3 ± 0.6 11.1 ± 0.5 11.2 ± 0.7 11.1 ± 0.4 11.3 ± 0.3 10.6 ± 0.2 NS P (mg/dL) 10.8 ± 1.0a 11.0 ± 0.9a 11.1 ± 0.8a 10.9 ± 0.8a 11.3 ± 1.1a 10.9 ± 0.7a 8.1 ± 0.5b <.0001 Mg (mEq/L) 1.9 ± 0.3 1.9 ± 0.3 1.9 ± 0.2 1.9 ± 0.3 2.0 ± 0.4 2.0 ± 0.2 1.9 ± 0.2 NS Fe (μg/dL) TIBC (μg/dL) 22 ± 8b 814 ± 57a 25 ± 9b 764 ± 67a 18 ± 2b 818 ± 86a 24 ± 6b 803 ± 77a 23 ± 4b 771 ± 59a 41 ± 50b 800 ± 100a 90 ± 40a 397 ± 50b <.0001 <.0001 Saturation (%) 3 ± 1b 4 ± 1b 2 ± 0b 3 ± 1b 3 ± 1b 6 ± 7b 23 ± 10a <.0001 Albumin (g/dL) Globulin (g/dL) 4.1 ± 0.2ab 1.1 ± 0.3a 4.1 ± 0.2ab 1.0 ± 0.2c 4.0 ± 0.3b 1.0 ± 0.2abc 4.1 ± 0.3ab 1.0 ± 0.2abc 4.0 ± 0.2ab 1.0 ± 0.2bc 4.3 ± 0.2a 1.0 ± 0.2a 3.4 ± 0.3c 0.0002 1.3 ± 0.4abc 0.02 Albumin:Globulin 4.1 ± 1.2 4.4 ± 1.0 4.2 ± 1.1 4.2 ± 0.6 4.4 ± 0.9 4.3 ± 0.8 2.9 ± 1.0 NS 1Values (means ± SD) not sharing common superscript are significantly different (P < 0.05); NS, not significant (P > 0.05). 2Reference ranges for nursery piglets have been previously reported (25,26). 3BUN, blood urea nitrogen; TIBC, total iron binding capacity. 15 Table 1.4. Summary of immune status measurements in piglets fed varying levels of ARA and DHA1 Diet a1 a2 a3-d3 a4 d2 d1 MR ARA/DHA (% FA/FA) 0.1/1.0 0.53/1.0 0.69/1.0 1.06/1.0 0.67/0.62 0.66/0.33 0.74/0.01 M. hyopenmoniae (sample/positive) 0.1 ± 0.1 IgG (μg/ml) 4866 ± 935 0 ± 0.1 4439 ± 967 0.1 ± 0.1 4293 ± 1018 0.1 ± 0.2 4952 ± 933 0.1 ± 0.1 4158 ± 781 0.1 ± 0.1 4865 ± 942 0.2 ± 0.3 5903 ± 1031 IgM (μg/ml) 540 ± 254 470 ± 186 551 ± 257 525 ± 161 534 ± 216 481 ± 176 768 ± 186 IgA (μg/ml) 160 ± 71b 167 ± 95b 168 ± 63b 157 ± 70b 164 ± 69b 132 ± 56b 408 ± 99a hsCRP (μg/ml) Day 3 16.4 ± 10 17 ± 10.3 20.1 ± 10.6 12.5 ± 6.2 10.6 ± 7 11.1 ± 5.9 17.7 ± 9.4 Day 7 10.5 ± 2.5 10 ± 3.4 11 ± 6 11.3 ± 3.4 8.8 ± 3.4 8.9 ± 1.6 10.6 ± 2.7 Day 14 9 ± 3.3 11.3 ± 4.8 10.4 ± 3.5 9 ± 3.7 6.6 ± 2.8 10.6 ± 1.8 12.5 ± 2.5 Day 21 14.4 ± 6.8 16.2 ± 11.9 12.7 ± 4.5 13.1 ± 3 10.2 ± 5.1 14.8 ± 4.3 13 ± 2.6 Day 28 13.5 ± 6 13 ± 4.6 14.6 ± 6.2 12.4 ± 3.4 10.6 ± 5.3 15.8 ± 6.8 15.2 ± 4.6 SAA (μg/ml) Day 3 23.4 ± 64.4 19.5 ± 29.2 38.6 ± 56.4 26.1 ± 47.9 5.3 ± 14.4 1.6 ± 2.5 17.7 ± 49.3 Day 7 17 ± 29.0a 9.5 ± 7.3b 21.1 ± 20.4ab 15.5 ± 23.0b 6.8 ± 4.6b 10.2 ± 9.9b 6.4 ± 7.1b Day 28 2.5 ± 3.8 2.8 ± 3.8 5.7 ± 5.1 1.9 ± 1 2 ± 1.9 3±4 0.9 ± 0.4 Hp(µg/ml) 807 ± 818a 235 ± 176b 1137 ± 874ab 355 ± 516b 593 ± 1033ab 812 ± 706a n/a2 P NS NS NS 0.0008 NS NS NS NS NS NS 0.03 NS 0.02 1Hp, haptoglobin; hsCRP, highly-sensitive C-reactive protein; SAA, serum amyloid A. 2Values below limit of detection. 16 of age, with the a1 (low ARA) group having significantly higher levels than most other groups. SAA levels, however, were unaffected by diet on day 3 and 28 of age. Likewise, there were minor, statistically significant differences in serum Hp levels among FR groups on day 28, although no discernable pattern among diets was apparent. Hp in the MR piglets was below detectable limits. Serum hsCRP levels were similar among all groups on days 3, 7, 14, 21 and 28 of age. Discussion Human breast milk is considered the gold standard of nutrition for the developing infant. There is broad consensus that human milk composition serves as a model for infant formula. All breast milks naturally contain ARA and DHA but the range of reported concentrations is wide and thus studies of optimal levels within the natural range are relevant to setting composition guidelines. With interest in optimizing the formula content of ARA and DHA for proper development of the infant central nervous and immune systems, it is critical to determine the interaction between these two LCPUFA and their resulting effect on growth and development. The present study was carried out to determine if there were any safety concerns associated with the dietary ARA level on growth, clinical chemistry and immune function in domestic piglets when DHA is constant and near the high end of human breast milk levels. Human term and preterm infants are capable of synthesizing ARA and DHA from the dietary precursors linoleic acid (18:2n-6) and linolenic acid (18:3n-3), respectively, although rates of endogenous biosynthesis and tissue accretion are insufficient to support optimal neural and visual development in 17 western countries in which studies have been conducted (27). Formulas supplemented with single-cell triglyceride oils serve as highly bioavailable sources of LCPUFA and enhance plasma and red blood cell content of ARA and DHA compared with infants consuming LCPUFA-free formula, and within the range of breast-fed infants (28). However, plasma and red blood cell ARA levels serve as surrogate biomarkers for tissue status, especially with regard to neural tissue (29). Functional outcomes, including those associated with safety, are thus necessary to develop a complete picture of their effects. Growth is considered one of the most useful clinical markers of infant development, and over the last two decades, growth outcomes have been intimately linked as a basis for addition of ARA to infant formula. Early studies showed a correlation between blood ARA and growth in preterm infants fed formula (30), and prompted investigation of the hypothesis that plasma ARA levels are crucial to growth outcomes (31). Recent studies, however, indicate that LCPUFA supplementation has a negligible effect on growth (32,33). In the present study, piglets fed 0.1% ARA had marginally lower, but statistically significant different body weights on day 7 of age compared with all other FR and MR piglets. These differences in body weight were abolished by day 14 of age and the 0.1% ARA piglets grew as well as all other FR pigs for the remainder of the study. For all piglets, the rapid rates of growth and concomitant demand for nutrients may have exacerbated the requirement for ARA during the first two weeks of life. The approximately eight-fold greater growth rate of piglets compared to human infants would be expected to amplify effects of ARA on growth, if any (34). Thus, results from the present study are consistent with recent reports in human infants and suggest a negligible effect of ARA level on growth. 18 M. hyopneumoniae titers did not differ among any of the dietary treatment groups. Titers were also relatively low compared to the manufacturer’s data on RespiSure-One (23). The HerdChekTM ELISA kit is intended to monitor pig herds for outbreaks of M. hyopneumoniae and focuses on dividing sample titers into positive versus negative cases relative to a single positive control sample, and may have limited ability to detect subtle changes. Future studies using this system may benefit from different immunizations or immunization schedules to induce higher serum titers and a detection test with a broader dose-response curve. MR piglets had significantly higher levels of serum IgA than the other dietary treatment groups. One possible explanation is that some or all of the IgA is maternally derived. However, by 24-36 hours after birth, piglets no longer transport milk immunoglobulins from the gut into circulation (35). And, based on 125I labeled immunoglobulin clearance rates (36), the average halflife of maternally-derived IgA is 2.7 days. Thus, the elevated IgA in the serum of MR controls is unlikely to be of maternal origin and may reflect differences in the rate of maturation. Both serum IgG and IgM showed groupwise comparison trends similar to IgA but failed to reach significance. The lack of significant differences among the FR groups indicates altering the dietary amounts or ratios of ARA to DHA has no adverse effect on immunological maturation. Serum IgG levels, in the present study, were marginally lower than previously reported values for sow-fed piglets (37,38) but consistent with other formula-fed piglets (37). Serum IgM levels were comparable to previous studies (37,38). Serum acute phase protein levels showed few significant differences among dietary treatment groups. Values were highly variable, especially for 19 Hp and SAA, had inconsistent patterns of between-group differences and, in some cases, tended to be either higher or lower than previously published data (39,40). The lack of detectable Hp and low concentrations of SAA in the MR pigs are inconsistent with previous studies of early life acute phase protein concentrations in piglets. For example, in (39), , SAA levels were slightly higher than values for our MR pigs, but showed a similar trend of decreasing levels with age. Where significant differences did exist among dietary treatment groups, e.g. day 28 Hp and day 7 SAA, they were not associated with higher concentrations of ARA relative to DHA. Thus, with a battery of three acute phase protein tests, we could not find evidence that increasing ARA relative to DHA produces a detectable pro-inflammatory state in the piglets. We have previously examined the safety of ARA and DHA for use in formula at 0, 1, 3 and 5 times the levels currently used in conventional infant formulas (7). For the 5x diet, levels of ARA and DHA corresponded to 3.15 and 1.66% of total FA, respectively, and across the full range of ARA and DHA intakes tested, no toxicological effects were observed for growth, clinical chemistry or liver histopathology. Others have observed a similar negligible effect on growth outcomes when ARA and DHA were fed 3x the level in conventional formulas (8). Safety concerns associated with providing ARA and DHA at different ratios, particularly with regard to growth and immune status, led us to conduct the present study. Here, we observed negligible differences among all the FR groups, indicating that the dietary ARA level, when DHA is constant at 1.0% of total FA, does not influence functional outcomes associated with ARA during the neonatal period. One possible explanation for a lack of difference is that fact that the dietary content of 20 linoleic acid (18:2n-6), roughly 17% of total FA and at a ratio of 11.5:1 with linolenic acid (18:3n-3), supported the endogenous biosynthesis of ARA at levels sufficient for normal growth and immune function. A superficially striking observation from the present study was the large difference in growth, hemogram and clinical chemistry parameters between the MR and FR piglets. These results were not unexpected, however, as the diet and rearing environment differed tremendously between the two groups. In terms of growth, we opted to set intakes for the FR piglets at 80% ad libitum intake based on reports of growth patterns for MR piglets (19) and data from a pilot study. This would enable a moderately faster rate of growth for the FR piglets, which was observed with a 2 kg difference in body weight between FR and MR piglets on day 28 of age. Remarkably, absolute brain weights were unaffected by diet. However, relative brain weights expressed as brain weight as a percentage of body weight were greatest for the MR piglets, and driven entirely by the larger carcasses of the FR groups. Here, excess nutrients beyond those required for brain growth were partitioned to the FR carcass, highlighting the brain’s protected growth during the perinatal period. Differences between MR piglets and the FR groups were also observed for biomarkers of blood iron status, including hemoglobin, serum iron, total iron binding capacity and percent saturation. Furugouri (41) demonstrated that rapidly growing piglets exhibit high levels of erythropoesis and have a heightened daily requirement for available iron. In the present study, the faster rates of growth observed for the FR piglets, compared with the MR group, corresponded to an elevated demand for iron that was apparently unmet and manifested as poorer blood iron status. 21 Current FDA GRAS Notices indicate that ARA be included in infant formula at levels at least equal to DHA (42,43), confirmed recently by recommendations of an expert panel (44). Panel recommendations also suggest that the formula content of DHA not exceed 0.5% of total FA due to a lack of evidence showing further benefit beyond the 0.3% level. Data from the present study show no toxicological effects of DHA when supplemented up to 1% of total FA, and extend the current body of literature to show that comparable levels of ARA are not required to match high DHA to support normal growth and immune function. In Chapter Two, we examine the influence of dietary ARA level on tissue ARA and DHA accretion. It would be of further interest to determine the competition between these LCPUFA at the tissue level and to determine if dietary ARA level influences functional outcomes associated with DHA, such as visual acuity or cognition. In conclusion, milk replacer formulas supplemented with varying levels of ARA and DHA equally supported normal growth, development and immune function of rapidly growing domestic piglets up to 28 days of age. Dietary ARA level, comprising 0.1 – 1.0% of total FA, and fed concomitantly with 1.0% DHA produced no toxicological effects on any hemogram, clinical chemistry or immune function parameters. The lack of adverse or toxicological effects in piglets consuming the low ARA diet (0.10% total FA) may be due to adequate endogenous biosynthesis of ARA from 18:2n-6. 22 REFERENCES 1. Carlson, S. E. (2001) Docosahexaenoic acid and arachidonic acid in infant development. Semin Neonatol 6: 437-449. 2. Calder, P. C., Krauss-Etschmann, S., de Jong, E. 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(2007) Agerelated changes in pro-inflammatory cytokines, acute phase proteins and cortisol concentrations in neonatal piglets. Neonatology 91: 44-48. 40. Romagosa, A., Ruiz, J. L., Manteca, X., Menjón, R., Bollo, J. M., Ibañez, A. M., Calvo, E. & Fariñas, F. (2009) Effect of Mycoplasma hyopneumoniae vaccination on acute phase serum proteins (PigMAP® and haptoglobin) in weaned piglets. XXX Simposium ANAPORC, Parador de Segovia. 41. Furugouri, K. (1974) Kinetics in iron metabolism in piglets. J Anim Sci 38: 1249-1256. 42. Rulis, A. M. & Lewis, C. J. (2001) Agency Response Letter GRAS Notice No. GRN 000041. CFSAN/Office of Premarket Approval. 43. Rulis, A. M. (2001) Agency Response Letter GRAS Notice No. GRN 000080. CFSAN/Office of Premarket Approval. 44. Koletzko, B., Lien, E., Agostoni, C., Bohles, H., Campoy, C., Cetin, I., Decsi, T., Dudenhausen, J. W., Dupont, C. et al. (2008) The roles of longchain polyunsaturated fatty acids in pregnancy, lactation and infancy: review of current knowledge and consensus recommendations. J Perinat Med 36: 5-14. 26 CHAPTER TWO HEART ARACHIDONIC ACID (ARA) IS UNIQUELY SENSITIVE TO DIETARY ARA AND DOCOSAHEXAENOIC ACID (DHA) CONTENT IN DOMESTIC PIGLETS* Abstract Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are commonly added to infant formula to support neonatal growth and development. Higher levels of these long chain polyunsaturated fatty acids (FA) in formula may provide some advantage in visual or cognitive performance. This study determined the sensitivity of heart and brain ARA to the dietary ARA level in a dose-response design with constant, high DHA. On d3 of age, formula-reared (FR) pigs were assigned to 1 of 6 milk replacer formulas that varied in ARA/DHA as follows (%, FA/FA): (a1) 0.1/1.0; (a2) 0.53/1.0; (a3-d3) 0.69/1.0; (a4) 1.1/1.0; (d2) 0.67/0.62; (d1) 0.66/0.33. A seventh group was maternal-reared. On d28 of age, piglets were sacrificed and organs harvested. Higher levels of dietary ARA were associated with increased heart and liver ARA, while brain ARA remained unaffected. Dietary ARA had no effect on tissue DHA accretion. Higher levels of dietary DHA resulted in higher DHA in the heart, liver and neural tissue, as well as reduced liver ARA. The heart was particularly sensitive, with pigs in the intermediate groups having different ARA values (a2, 18.6 ± 0.7%; a3, 19.4 ± 1.0%) and a 0.17% increase in dietary ARA resulted in a 0.84% increase in heart ARA. We *Tyburczy, C., Kothapalli, K. S. D., Park, W. J., Blank, B. S., Bradford, K. L., Zimmer, J.P., Butt, C. M., Salem, Jr., N., Brenna, J. T. (2010) J Nutr, submitted. 27 conclude that the heart has a unique responsiveness to the dietary ARA level. Further investigations are warranted to determine the clinical significance of heart ARA level in the developing neonate. Introduction Arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) are two long chain polyunsaturated fatty acids (LCPUFA) that contribute to normal growth and development during the perinatal period. Both are natural components of breast milk and since 2002, these LCPUFA have been added to infant formulas in the U.S. at target levels of 0.64% and 0.32% of total fatty acids (FA) (1). Mean worldwide levels of ARA and DHA in breast milk average 0.47 ± 0.13% total FA (range: 0.24 – 1.0% FA) and 0.32 ± 0.22% FA (range: 0.06 – 1.4% FA), respectively, with individual variability in DHA attributed largely to maternal diet (2). Inevitably, the wide variation in breast milk LCPUFA as well as the relative inaccessibility to human infant tissue presents a major challenge for optimizing levels of ARA and DHA in formula to support neonatal development. It is well-established that the addition of ARA and DHA to formula enhances blood LCPUFA levels equivalent to those of breastfed infants and improves visual acuity and cognitive performance compared with infants fed LCPUFA-free formula (3-6). Functional outcome studies with infants provide clear evidence for the addition of DHA at 0.32% FA (7,8), while higher DHA levels may offer further benefit for neural development, especially in target populations (9-11). Optimal levels of ARA remain to be determined at any DHA intake and are based largely on mean global levels in breast milk (12). ARA comprises approximately 10 – 12% total FA in human infant central 28 nervous tissue (cerebral cortex and retina) appears to be influenced to a greater extent by postnatal age than dietary ARA supply (13). Animal models suggest a relative resistance of brain and retina ARA to dietary ARA intake, possibly as a mechanism for regulating its potent bioactivity (14,15). ARA levels in the heart, liver and blood-borne pools vary with ARA intake and may compete with n-3 LCPUFA for tissue incorporation (3,16-23), especially in the liver (20,23). The sensitivity of heart ARA to dietary ARA content has been reported a few times but has not been systematically studied (17-19,21). In pigs (21), dietary ARA and DHA were maintained at a constant 2:1 ratio and total levels varied up to 5 times the target levels in formula. While liver ARA increased with only the highest ARA intakes, heart ARA responded in a direct doseresponse manner to each incremental increase in dietary ARA. Further, heart ARA more than doubled with the highest ARA level compared with pigs fed LCPUFA-free formula. We tested the hypothesis that heart and brain ARA is sensitive to dietary ARA in a dose-response design with DHA constant at a high physiological level. We examined four levels of ARA ranging from 0.09 to 1.0% total FA against a background of 1.0% DHA. Intermediary levels were 0.53 and 0.67% total FA, where 0.53% is slightly above the worldwide ARA mean in human breastmilk and 0.67% is among the highest values reported. A DHA dose-response was also included to determine the influence of dietary DHA level on tissue ARA incorporation when ARA levels are comparable to those added in conventional formula (0.65% ARA). A randomized maternal-reared (MR) reference group was included for comparison. 29 Materials and methods Animals and diets All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Cornell University. On day 3 of life, domestic piglets were matched for weight and gender and assigned to one of six milk replacer diets (n = 8 per diet). Piglets in the formula-reared (FR) groups were then transferred to the Large Animal Research and Teaching Unit, where they were housed individually in raised metal cages and maintained on a 16/8 hr light/dark cycle. The seventh group was MR and remained with the dam at the swine facility for the duration of the study. Intake and growth performance is reported in Chapter One. Milk replacer formula consisting of 60% experimental diet (Research Diets, Inc., New Brunswick, NJ) and 40% Birthright baby pig milk replacer (Ralco Nutrition, Inc., Marshall, MN) was fed to FR piglets on days 3 – 28 of age. The ingredient composition and nutrient analysis of the base experimental diet is presented in Table 2.1. A detailed nutrient composition and analysis of the Birthright baby pig milk replacer has been reported previously (24). As fed, the milk replacer formula had a caloric density of 0.7 kcal/mL and a nutrient composition as follows (% wt/wt): protein 26.7, fat 21.9 and carbohydrate 38.9. The FA composition of the milk replacer formulas and sow milk (day 14 in lactation) is presented in Table 2.2. Diets varied in the ratio of ARA/DHA as follows (%, FA/FA): (a1) 0.1/1.0; (a2) 0.53/1.0; (a3-d3) 0.69/1.0; (a4) 1.1/1.0; (d2) 0.67/0.62; (d1) 0.66/0.33 (conventional infant formula). Diets a1 – a4 are a dose-response for ARA against DHA constant at 1.0%, while Diets d1 – d3 are a dose-response for DHA against ARA constant at 0.67%. 30 Sampling On day 28 of age, piglets were sacrificed via an intravenous injection of Fatal Plus (1 mL/4.54 kg body weight; Vortech Pharmaceuticals, Dearborn, MI, USA) followed by exsanguination. Organs were immediately removed, weighed and samples flash frozen in liquid nitrogen within 10 min of cessation of heart beat. Cerebral cortex and cerebellum samples were harvested from the left hemisphere of each brain prior to submerging individual hemispheres in liquid nitrogen. Internal brain regions were sampled from partially thawed left hemispheres at a later period. Tissues were stored at -80°C until FA analysis. Lipid analysis Fatty acid methyl esters (FAME) were prepared from ~50 mg of tissue or dry diet and 50 μL sow milk according to the One-step hydrolysis, extraction and methylation procedure (25) with previous modifications (26). A detailed protocol for the One-step procedure is presented in Appendix II. Retinas, which were suspended in physiological saline at necropsy, were first dried using a Savant SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA). FAME were quantified on a 5890 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a BPX70 fused silica column (25 m x 0.22 mm i.d. x 0.25 μm film; SGE Incorportated, Austin, TX). FAME were structurally identified by covalent-adduct chemical ionization mass spectrometry on a Saturn 2000 mass spectrometer (Varian, Inc., Walnut Creek, CA) (27). An equal weight FAME mixture was used daily to verify response factors. 31 Table 2.1 Nutrient composition and analysis of experimental diets Composition Content Dietary ingredient, % dry matter Dried skim milk 55.0 Oil blend 23.9 Calcium sodium caseinate 9.6 Vitamin mix1 4.8 Mineral mix2 4.8 Xanthan gum 1.4 Methionine, DL 0.5 Nutrient analysis, % kcal Protein 24.3 Carbohydrate 28.7 Fat 47.0 Caloric density (kcal/g) 4.67 1The mineral mix contained (g/kg): 257.8 sucrose, 615.8 dibasic calcium phosphate, 106.7 sodium chloride, 11.8 ferric citrate, 4.0 zinc carbonate, 3.4 magnesium oxide, 0.4 cupric carbonate, 0.2 manganous carbonate, 0.1 potassium iodate, 0.01 sodium selenite. 2The vitamin mix contained (g/kg): 910.1 sucrose, 75.0 choline bitartrate, 6.2 vitamin E acetate (50 g/100 g), 3.4 vitamin A acetate (500,000 IU/g), 1.5 vitamin D3 (100,000 IU/g), 1.3 vitamin B12 (0.1g/100 g mannitol), 0.9 pantothenic acid (d, calcium), 0.9 niacin, 0.4 biotin (1 g/100 g), 0.1 riboflavin, 0.1 thiamine HCl 0.03 menadione sodium bisulfite, 0.01 folic acid, 0.01 pyridoxine HCl. 32 Diet ARA/DHA Σ SFA + MUFA ARA DHA Σ n-6 Σ n-3 18:2n-6 18:3n-3 18:2n-6/18:3n-3 Table 2.2. FA composition of milk replacer formulas and sow milk1 a1 a2 a3-d3 a4 d2 d1 0.1/1.0 0.53/1.0 0.69/1.0 1.1/1.0 0.67/0.62 0.66/0.33 79.86 0.09 79.94 0.53 79.43 0.69 FA (% total FA) 79.18 80.02 1.06 0.67 80.02 0.66 1.00 17.50 1.02 17.52 1.01 17.95 1.04 18.15 0.62 17.76 0.33 18.00 2.53 2.43 2.49 2.51 2.10 1.86 17.25 1.53 16.83 1.41 17.11 1.48 16.93 1.47 16.90 1.48 17.19 1.53 11.3 12.0 11.6 11.5 11.4 11.3 MR 0.74/0.01 83.94 ± 3.16 0.74 ± 0.02 0.01 ± 0.01 15.21 ± 3.08 0.71 ± 0.04 12.05 ± 2.98 0.57 ± 0.06 21.1 1Milk from two sows taken on day 14 of lactation; MR piglets remained with the sow for the duration of the study. 2SFA, saturated FA; MUFA, monounsaturated FA. 33 Statistical analysis Values are reported as means ± SD. Statistical analysis was carried out using the Fit Model platform of JMP (2008 SAS Institute, 8.0) to fit mixed models and tested the hypothesis that all mean treatment values are not equal. Fixed effects were diet, gender, day 3 of age body weight and the full factorial of interactions. The random effect of litter was included the model. Interaction effects were considered significant at P < 0.10 and fixed effects at P < 0.05. For each parameter analyzed, effects not considered significant were removed from the final model. Significance of pairwise comparisons was determined using the Student’s t-test. Linear regression analysis was performed to determine the dose-response of tissue LCPUFA to the dietary ARA and DHA level. Results LCPUFA accretion in liver and heart Table 2.3 presents the FA composition of heart and liver. A detailed composition of these tissues is presented in Appendix I (Tables S1 and S2). Heart ARA increased with increasing dietary ARA. Heart ARA levels were higher in the a3 group compared with Diet a2 (P = 0.04) such that a 0.17% increase in dietary ARA corresponded to an increase of 0.84% in heart ARA between the two treatments. Mean heart ARA was 40% higher in the a4 group compared with a2 pigs. Liver ARA increased with increasing dietary ARA, although the greatest increase in liver ARA was observed between Diets a1 and a2. DHA accretion in the liver reflected dietary intake with a1 – a4 pigs having the highest liver levels, d1 and d2 pigs having intermediary levels and 34 MR pigs having the lowest liver DHA (Table 2.3). Similarly, DHA levels in the heart reflected dietary DHA content, with a1 – a4 pigs having the highest, d1 and d2 pigs having intermediary and MR pigs having the lowest heart DHA levels (P < 0.0001), although for Diets a1 – a4, a2 pigs had higher heart DHA levels than the a1 and a3 groups (P < 0.05). MR pigs had a strikingly low DHA content (0.75 ± 0.08%) that was 6-fold lower than DHA levels in the a2 group. In both the heart and liver, the sum of 22-carbon HUFA reflected tissue DHA content and was lowest in d1 and MR pigs (P < 0.0001; Table 2.3). Compared with the d3 group, d1 pigs had lower liver DHA and higher liver levels of 22:4n-6, 22:5n-6 and 22:5n-3 (P < 0.001). In the heart, however, d1 pigs showed increases in 22:4n-6 and 22:5n-3 compared with d3 pigs (P < 0.0001), while trace levels of 22:5n-6 were detected in all samples. LCPUFA accretion in neural tissues Tables 2.4 and 2.5 present the FA composition of neural tissue from pigs fed varying levels of ARA and DHA from day 3 – 28 of age. The detailed FA composition of each neural tissue is presented in Appendix I (Tables S3 – S8). ARA levels in the cerebral cortex, hippocampus, globus pallidus and inferior colliculus were similar across all dietary treatments. ARA levels in the cerebellum were significantly influenced by diet (P = 0.007) but showed no distinct pattern associated with ARA or DHA intake. A1 pigs had the lowest retina ARA levels of the FR groups, although differences among the means only reached significance for the a4, d1 and d2 groups (P < 0.05). 35 Table 2.3. FA composition of heart and liver from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet ARA/DHA3 a1 0.09/1.00 a2 0.53/1.02 a3-d3 0.69/1.01 a4 1.06/1.04 d2 0.67/0.62 d1 0.66/0.33 MR2 0.74/0.01 P Liver ARA 22:4n-6 22:5n-6 20:5n-3 22:5n-3 DHA ∑ 22C HUFA4 13.92 ± 1.49d 1.08 ± 0.29d 0.03 ± 0.06d 0.61 ± 0.14a 0.94 ± 0.11c 10.77 ± 1.77a 12.82 ± 1.86a FA (% total FA) 16.14 ± 0.87c 16.12 ± 1.10bc 16.88 ± 1.50b 17.36 ± 0.63b 17.87 ± 1.46a 1.15 ± 0.22cd 1.27 ± 0.22c 1.36 ± 0.22bc 1.28 ± 0.20bc 1.50 ± 0.10b 0.06 ± 0.05d 0.04 ± 0.04d 0.06 ± 0.07d 0.20 ± 0.09c 0.37 ± 0.11b 0.42 ± 0.15bc 0.45 ± 0.10b 0.36 ± 0.11cd 0.34 ± 0.05cd 0.31 ± 0.04d 0.91 ± 0.09c 0.94 ± 0.12c 0.94 ± 0.14c 1.01 ± 0.07c 1.11 ± 0.16b 11.43 ± 1.22a 10.49 ± 1.41a 10.45 ± 1.79a 9.65 ± 1.01b 7.52 ± 0.69c 13.55 ± 1.26a 12.74 ± 1.44ab 12.81 ± 1.90ab 12.14 ± 1.21b 10.51 ± 0.81c 17.00 ± 1.36abc <.0001 2.27 ± 0.35a <.0001 1.89 ± 0.53a <.0001 0.18 ± 0.06e <.0001 1.35 ± 0.20a <.0001 2.37 ± 0.30d <.0001 7.88 ± 1.22d <.0001 Heart ARA 22:4n-6 22:5n-6 20:5n-3 22:5n-3 DHA ∑ 22C HUFA 14.76 ± 1.07d 18.57 ± 0.67c 0.63 ± 0.09e 0.71 ± 0.09e 0.00 ± 0.01b 0.00 ± 0.00b 0.75 ± 0.09a 0.54 ± 0.09b 0.59 ± 0.07d 0.59 ± 0.08d 4.81 ± 0.24b 5.20 ± 0.49a 6.04 ± 0.25bc 6.50 ± 0.51a 19.41 ± 1.00b 20.90 ± 0.93a 19.98 ± 1.38ab 19.09 ± 1.88bc 20.26 ± 1.20ab 0.71 ± 0.06de 0.82 ± 0.09cd 0.88 ± 0.13bc 0.96 ± 0.12b 1.33 ± 0.13a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.02 ± 0.03b 0.08 ± 0.01a 0.49 ± 0.04b 0.41 ± 0.04c 0.42 ± 0.04c 0.32 ± 0.03d 0.28 ± 0.06d 0.57 ± 0.04d 0.57 ± 0.04d 0.71 ± 0.07c 0.83 ± 0.13b 1.24 ± 0.07a 4.78 ± 0.43b 5.01 ± 0.25ab 4.20 ± 0.64c 3.00 ± 0.50d 0.75 ± 0.08e 6.06 ± 0.4abc 6.40 ± 0.23ab 5.78 ± 0.72c 4.81 ± 0.59d 3.40 ± 0.15e <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (%, FA/FA). 3∑ 22C HUFA = ∑ 22:4n-6, 22:5n-6, 22:5n-3, DHA. 36 Pigs fed 1.0% DHA had significantly higher DHA levels in the cerebral cortex, cerebellum and retina than the d1 and MR groups, while d2 pigs showed intermediary levels (P < 0.0001; Tables 2.4 and 2.5). DHA accretion in the hippocampus, globus pallidus and inferior colliculus was greatest for pigs fed 1.0% DHA and lowest for the MR group (P < 0.05). For these tissues, DHA levels were 17%, 15% and 3% higher, respectively, in the d3 compared with d1 pigs, although differences were not statistically significant. The sum of the 22-carbon HUFA, corresponding to the sum of 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3 and DHA, was similar across all dietary treatments in all neural tissues reported (Tables 2.4 and 2.5). For these tissues, the magnitude of the increase in non-DHA 22-carbon HUFA was greatest for 22:5n-6. Differences in the composition of 22-carbon HUFA between Diets d1 and d3 were significant and apparent, with d1 pigs having higher 22:5n-6 in cerebral cortex, cerebellum and retina (P < 0.01), and higher 22:4n-6 in cerebellum and retina than d3 pigs (P < 0.001). Tissue ARA and DHA dose-response to diet Figure 2.1 summarizes the linear regression analysis for the tissue ARA dose-response to the dietary ARA level (Diets a1 – a4). ARA levels in the heart and liver were highly responsive to the dietary ARA content (P < 0.0001), whereas ARA levels in the cerebral cortex and retina were not esponsive to ARA intake. Regression analysis for the tissue DHA dose-response to dietary DHA content (Diets d1 – d3) is presented in Appendix I (Figure S1). Cerebral cortex, retina, liver and heart DHA levels increased with dietary DHA content (P < 0.05). For heart and liver, however, differences were most pronounced between Diets d1 and d3, as increasing the 37 Table 2.4. FA composition of cerebral cortex, retina and cerebellum from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Cerebral cortex FA (% total FA) ARA 11.95 ± 0.29 11.82 ± 0.60 11.80 ± 0.35 12.04 ± 0.63 11.93 ± 0.40 11.77 ± 0.68 12.33 ± 0.00 NS 22:4n-6 4.03 ± 0.52 4.06 ± 0.16 4.09 ± 0.35 4.21 ± 0.60 4.21 ± 0.32 4.19 ± 0.26 4.81 ± 0.30 NS 22:5n-6 4.49 ± 0.40c 4.44 ± 0.47c 4.68 ± 0.29c 4.61 ± 0.56c 5.52 ± 0.64b 5.49 ± 0.25b 8.26 ± 0.18a <.0001 22:5n-3 0.20 ± 0.02c 0.19 ± 0.02c 0.19 ± 0.04c 0.19 ± 0.03c 0.20 ± 0.02c 0.25 ± 0.05b 0.32 ± 0.03a <.0001 DHA 12.10 ± 0.83a 11.91 ± 0.74ab 11.98 ± 0.69ab 11.90 ± 0.69ab 11.30 ± 0.70b 10.56 ± 0.67c 6.88 ± 0.55d <.0001 ∑ 22C HUFA4 20.82 ± 0.81 20.60 ± 0.61 20.94 ± 1.01 20.91 ± 0.99 21.23 ± 0.88 20.49 ± 0.79 20.27 ± 0.72 NS Retina ARA 9.48 ± 0.23c 9.70 ± 0.61bc 9.66 ± 0.32bc 9.81 ± 0.31b 9.96 ± 0.37b 9.95 ± 0.25b 10.74 ± 0.50a 0.0004 22:4n-6 1.66 ± 0.15e 1.72 ± 0.12de 1.77 ± 0.12d 1.81 ± 0.17d 1.98 ± 0.19c 2.13 ± 0.12b 2.98 ± 0.17a <.0001 22:5n-6 1.48 ± 0.30d 1.48 ± 0.32d 1.45 ± 0.16d 1.55 ± 0.30d 2.03 ± 0.64c 2.29 ± 0.26b 5.24 ± 0.39a <.0001 22:5n-3 0.64 ± 0.04a 0.60 ± 0.05b 0.63 ± 0.06ab 0.60 ± 0.03b 0.55 ± 0.03c 0.60 ± 0.05b 0.60 ± 0.05abc 0.0001 DHA 20.42 ± 1.20ab 20.70 ± 0.54a 20.07 ± 1.47ab 20.49 ± 1.31ab 19.50 ± 1.50bc 18.68 ± 0.96c 14.16 ± 1.37d <.0001 ∑ 22C HUFA 24.29 ± 1.34 24.61 ± 0.49 24.03 ± 1.49 24.55 ± 1.48 24.20 ± 1.31 23.81 ± 0.90 23.10 ± 0.98 NS Cerebellum ARA 10.40 ± 0.30c 10.76 ± 0.26bc 11.03 ± 0.31ab 10.44 ± 0.44c 10.95 ± 0.62ab 10.78 ± 0.43bc 11.48 ± 0.73a 0.007 22:4n-6 3.29 ± 0.41d 3.23 ± 0.38d 3.39 ± 0.32cd 3.43 ± 0.32cd 3.55 ± 0.19c 3.97 ± 0.25b 4.66 ± 0.31a <.0001 22:5n-6 2.12 ± 0.26cd 2.19 ± 0.34d 2.18 ± 0.19cd 2.17 ± 0.26d 2.70 ± 0.37bc 3.34 ± 1.09b 4.82 ± 0.23a <.0001 22:5n-3 0.35 ± 0.06bc 0.31 ± 0.05cd 0.32 ± 0.05cd 0.32 ± 0.05d 0.31 ± 0.02cd 0.36 ± 0.05b 0.47 ± 0.04a <.0001 DHA 11.29 ± 1.61a 11.08 ± 1.68a 11.29 ± 1.19a 11.19 ± 1.44a 10.37 ± 0.75ab 9.83 ± 0.76b 6.10 ± 0.28c <.0001 ∑ 22C HUFA 17.47 ± 1.79 17.19 ± 2.23 17.56 ± 1.51 17.47 ± 1.54 17.36 ± 0.70 17.88 ± 1.37 16.53 ± 0.30 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (%, FA/FA). 3∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 38 Table 2.5. FA composition of hippocampus, globus pallidus and inferior colliculus from piglets fed varying levels of AA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Hippocampus FA (% total FA) ARA 10.40 ± 0.89 10.78 ± 0.93 10.57 ± 1.09 10.61 ± 1.57 11.10 ± 0.97 10.85 ± 1.23 11.77 ± 0.96 NS 22:4n-6 4.57 ± 0.36 4.68 ± 0.43 4.63 ± 0.57 4.72 ± 0.63 4.78 ± 0.44 5.03 ± 0.51 5.37 ± 0.43 NS 22:5n-6 2.89 ± 0.22c 2.96 ± 0.39c 3.06 ± 0.39cd 2.68 ± 0.67d 3.71 ± 0.40b 3.31 ± 0.53bc 5.58 ± 0.60a <.0001 22:5n-3 0.24 ± 0.03ab 0.22 ± 0.05bc 0.22 ± 0.02bc 0.20 ± 0.05bc 0.19 ± 0.02c 0.24 ± 0.04ab 0.27 ± 0.04a 0.009 DHA 9.89 ± 0.26a 9.74 ± 1.43a 9.66 ± 0.68ab 9.63 ± 1.73a 9.44 ± 1.03a 8.27 ± 1.48b 6.12 ± 0.68c 0.0002 ∑ 22C HUFA4 17.20 ± 1.41 17.60 ± 1.87 17.13 ± 1.99 17.23 ± 2.89 18.12 ± 1.44 16.84 ± 2.23 17.34 ± 0.64 NS Globus pallidus ARA 10.33 ± 0.68 9.33 ± 1.28 10.07 ± 0.44 9.92 ± 0.76 10.02 ± 0.88 10.09 ± 0.96 10.73 ± 0.82 NS 22:4n-6 3.81 ± 0.32bc 3.59 ± 0.73b 4.25 ± 0.51ab 3.98 ± 0.87bc 3.72 ± 0.37bc 4.04 ± 0.58bc 4.78 ± 0.65a 0.02 22:5n-6 2.93 ± 0.37b 2.24 ± 0.77c 2.81 ± 0.40bc 2.64 ± 0.58bc 3.16 ± 0.73b 3.04 ± 0.78b 5.40 ± 0.92a <.0001 22:5n-3 0.15 ± 0.12 0.12 ± 0.04 0.13 ± 0.03 0.13 ± 0.02 0.11 ± 0.04 0.14 ± 0.06 0.19 ± 0.06 NS DHA 9.45 ± 0.97a 8.09 ± 2.61ab 8.80 ± 0.94ab 8.18 ± 1.36ab 7.91 ± 1.95b 7.66 ± 1.68bc 6.08 ± 0.35c 0.01 ∑ 22C HUFA 16.74 ± 1.12 14.49 ± 3.72 16.44 ± 1.37 15.41 ± 2.20 15.37 ± 2.68 15.34 ± 2.50 16.92 ± 1.43 NS Inferior colliculus ARA 8.58 ± 0.35 8.55 ± 0.75 8.85 ± 0.84 8.61 ± 0.52 8.63 ± 0.50 8.97 ± 0.73 9.45 ± 1.25 NS 22:4n-6 3.54 ± 0.21b 3.36 ± 0.19b 3.55 ± 0.31b 3.56 ± 0.29b 3.42 ± 0.30b 3.70 ± 0.35b 4.68 ± 1.16a <.0001 22:5n-6 1.58 ± 0.49bc 1.50 ± 0.35c 1.60 ± 0.45bc 1.62 ± 0.52bc 1.93 ± 0.53b 1.91 ± 0.32b 3.76 ± 0.45a <.0001 22:5n-3 0.19 ± 0.02ab 0.18 ± 0.04abc 0.19 ± 0.04ab 0.15 ± 0.02c 0.16 ± 0.03bc 0.18 ± 0.03bc 0.21 ± 0.06a 0.02 DHA 10.71 ± 0.94a 10.32 ± 1.88a 10.84 ± 1.50a 9.58 ± 1.21a 10.18 ± 1.27a 10.53 ± 0.85a 6.27 ± 1.14b <.0001 ∑ 22C HUFA 16.35 ± 1.10 15.66 ± 2.18 16.46 ± 2.03 15.28 ± 1.60 16.02 ± 1.46 16.61 ± 1.23 15.40 ± 1.74 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (%, FA/FA). 3∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 39 Heart ARA (% total FA) Brain ARA (% total FA) DHA content from 0.62 to 1.0% total FA (Diets d2 vs. d3) did not significantly increase heart or liver DHA levels. Cerebellum and hippocampus DHA levels also increased with dietary DHA content (P < 0.05), while globus pallidus and inferior colliculus DHA levels did not correlate with dietary DHA content (Figure S2, Appendix I). A 25 20 a 15 10 C 0.0 14 0.2 bc B d y = 2.22Ln(x) + 20.54 R2 = 0.86 P < 0.0001 0.4 0.6 0.8 1.0 D 12 10 y = 0.17x + 11.83 8 R2 = 0.02 P = NS 6 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) 1.0 Retina ARA (% total FA) Liver ARA (% total FA) 25 y = 1.18Ln(x) + 17.01 R2 = 0.57 20 P < 0.0001 a 15 b bc 10 0.0 0.2 0.4 0.6 0.8 14 y = 0.37x + 9.47 12 R2 = 0.09 P = NS 10 8 6 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) c 1.0 1.0 Figure 2.1. ARA tissue dose-response to dietary ARA with constant DHA (1.0% total FA). Dietary ARA content for a1, a2, a3 and a4 was 0.09, 0.53, 0.69 and 1.06% total FA, respectively. (A) Heart and (b) Liver responded significantly (P < 0.0001) while (C) Brain (cerebral cortex) and (D) Retina did not. Each point represents one animal (n = 32 per panel). P < 0.05 indicates significant correlation between dietary ARA content and tissue ARA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue ARA content; means not sharing a common letter are significantly different (P < 0.05). ARA and DHA competition for tissue incorporation Dietary ARA level had no effect on DHA accretion in the heart, liver or neural tissues (P > 0.05) (Figure 2.2; Figure S3, Appendix I). Conversely, there was an apparent competition between liver ARA and dietary-derived 40 Heart DHA (% total FA) DHA (Diets d1 – d3), with d3 pigs showing lower liver ARA levels than d1 pigs (P = 0.004) (Figure 2.3). This competition was not detected in the heart, brain or retina, indicating that the dietary DHA content has no effect on ARA levels in these tissues (Figure 2.3). A 6 B 14 Liver DHA (% total FA) 12 4 y = 0.14x + 4.88 R2 = 0.01 P = NS 2 10 8 y = 0.05x + 10.90 6 R2 = 0.00 P = NS 4 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 CD 15 25 Retina DHA (% total FA) 10 y = -0.28x + 12.10 R2 = 0.02 P = NS 5 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) 1.0 20 15 y = 0.04x + 20.42 R2 = 0.00 P = NS 10 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) 1.0 Figure 2.2. DHA tissue dose-response to dietary ARA content with constant DHA (1.0% total FA). Dietary ARA content for a1, a2, a3 and a4 was 0.09, 0.53, 0.69 and 1.06% total FA, respectively. Increasing ARA had no effect on DHA levels in (A) Heart, (B) Liver, (C) Brain (cerebral cortex) or (D) Retina. Each point represents one animal (n = 24 per panel). P < 0.05 indicates significant correlation between dietary ARA content and tissue DHA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue DHA content; means not sharing a common letter are significantly different (P < 0.05). Brain DHA (% total FA) 41 Heart ARA (% total FA) Liver ARA (% total FA) A 25 y = 0.45x + 19.20 R2 = 0.01 20 P = NS B 20 18 16 a b bc 15 14 y = -2.58x + 18.82 12 R2 = 0.32 P = 0.004 10 10 C 0.0 0.2 0.4 0.6 0.8 1.0 D 0.0 0.2 0.4 0.6 0.8 1.0 14 y = 0.05x + 11.80 R2 = 0.00 12 P = NS 14 y = -0.43x + 10.14 12 R2 = 0.14 P = NS 10 10 8 Retina ARA (% total FA) Brain AA (% total FA) 8 0.0 0.2 0.4 0.6 0.8 1.0 Dietary DHA (% total FA) 6 0.0 0.2 0.4 0.6 0.8 1.0 Dietary DHA (% total FA) Figure 2.3. ARA tissue dose-response to dietary DHA content with constant ARA (0.67% total FA). Dietary DHA content for d1, d2 and d3 was 0.32, 0.62 and 1.01% total FA, respectively. Increasing DHA reduced ARA levels in (B) Liver but not (A) Heart, (C) Brain (cerebral cortex) or (D) Retina. Each point represents one animal (n = 24 per panel). P < 0.05 indicates significant correlation between dietary DHA content and tissue ARA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue ARA content; means not sharing a common letter are significantly different (P < 0.05). Discussion The neonatal pig serves as a practical biomedical model of human infant development. Pigs are similar to humans in their metabolic responses, genetics of the FA desaturases and rates of perinatal brain growth (28,29). Their rapid postnatal growth enables facile collection of pre-clinically relevant data over a relatively short study duration. The sensitivity of heart ARA to the dietary ARA has been reported from time to time in pigs (19,21) and rats (17,18), yet it is poorly understood on a mechanistic basis and its clinical significance is unknown. In the present study, heart ARA levels reflected dietary ARA content and were significantly 42 different for pigs in all ARA groups including those with intermediate intakes, a2 (18.6 ± 0.7% total FA) and a3 (19.4 ± 1.0% total FA), indicating that heart ARA is limited by intake, at least at high DHA levels. These observations are particularly relevant to ARA recommendations for infant formula as 0.53% (a2) is slightly above the worldwide ARA mean in human breast milk and 0.67% (a3) is the level currently added in formula and is near the high end of breast milk values reported. ARA serves several known functions in cardiac physiology, such as providing precursor for eicosanoid signaling molecules and acting directly to modulate voltage-gated ion channel activity and cellular excitability (30). The degree to which heart ARA status influences cardiac physiology in the developing neonate, as well as the potential long term implications, remains to be determined. Biosynthesis of ARA and DHA from the nutritionally essential linoleic and linolenic acids occurs through a series of elongation and desaturation reactions catalyzed by a common set of enzymes (31). Studies with humans indicate that the rate of postnatal endogenous DHA synthesis is insufficient to support DHA accretion in neural tissues (32,33), and results in poorer visual and cognitive performance compared with infants consuming preformed DHA (3,5,6). These observations provide the basis for the addition of DHA to formula. Rates of endogenous synthesis of ARA from linoleic acid appear sufficient to maintain brain ARA levels in adult rats (34) but remain to be thoroughly investigated in growing animals. In the present study, higher levels of dietary ARA correlated with increased heart and liver ARA, while brain ARA was constant and unaffected by ARA intake. The trend for a modest increase in retina ARA with increasing dietary ARA was not supported by the linear regression analysis for the ARA dose-response. Liver lipids typically reflect 43 dietary FA content but are also influenced by the expression and regulation of enzymes involved in LCPUFA biosynthesis (31,35). The heart, too, expresses the FA desaturases and elongase enzymes (Elovl-1, -5 and -6) capable of synthesizing ARA, but is not generally considered a major site of LCPUFA biosynthesis (36). The sensitivity of the heart to dietary ARA level therefore suggests that the ARA content is influenced predominantly by preferential FA uptake and incorporation. Further, these data indicate that the rate of endogenous ARA biosynthesis may not be sufficient to match ARA accretion in the growing the heart. The choice to include an MR reference group provided a unique opportunity to investigate the contribution of sow milk, essentially devoid of DHA but not ARA, to the LCPUFA status of the developing offspring. This treatment is not unlike an extreme case of DHA-deficient breast milk that is reflective of few human populations worldwide (2,37). Because of this, we advise caution in extending results observed with the MR group, in particular comparing MR pigs with the FR groups fed higher levels of DHA, to breastfeeding practices in humans. The competition between n-3 and n-6 for tissue incorporation and the resulting effect on LCPUFA biosynthesis is well-studied. The relative availability of linoleic and linolenic acids, as well as their LCPUFA derivatives, drives the biosynthesis of n-3 and n-6 LCPUFA and modulates tissue levels of the two FA families in a reciprocal manner (20,38,39). Over the range of dietary ARA levels in the present study, tissue DHA was constant and unaffected by diet. On the other hand, we observed a specific reduction in liver ARA with the highest DHA intakes, consistent with previous reports in pigs (19) and baboon neonates (23) consuming preformed DHA. These 44 observations reinforce the concept that ARA and DHA in the liver are closely linked to diet because of the liver’s central role in LCPUFA biosynthesis, and are specific to function in the heart and neural tissue; all tissues being modulated by genetic and extra-dietary factors. The sensitivity of central nervous tissue DHA level has been previously investigated in numerous studies with term and preterm neonatal baboons (22,23,40). The brain consistently shows increased region-specific DHA accretion related to level and duration of preformed DHA feeding, and prematurity appears important. Here we observed higher levels of DHA in all tissues with increasing DHA content. Pigs in the high DHA group showed significantly higher levels of DHA in the cerebral cortex, retina and cerebellum compared with pigs in the low DHA group. Increases in mean DHA were 13%, 7% and 15%, respectively for these neural tissues. DHA levels in the hippocampus, globus pallidus and inferior colliculus were 17%, 15% and 3% higher in the high DHA group compared with the low DHA group; while these differences were not statistically significant they may be of biological importance. The relative insensitivity of basal ganglia, limbic and midbrain regions to postnatal dietary DHA consumption, compared with the cerebral cortex, is consistent with Hsieh et al. (23), and suggests that these deep brain regions may be influenced to a greater extent by the DHA status of the mother during gestation. Thus, these data support the recent dietary recommendations on total fat and fatty acids by the FAO/WHO for the provision of 200 mg/day DHA in the diets of pregnant and lactating women (41). Pigs that exhibited the lowest neural tissue DHA also showed enriched tissue 22:4n-6, 22:5n-6 and 22:5n-3, with greatest increases occurring in 45 22:5n-6. The increase in tissue 22:5n-6 in animals fed n-3-inadequate diets is well-characterized (42-44) and presumably occurs as an alternate substrate source for DHA-dependent functions in neural tissue. Recent examination of the frontal cortex from rat pups fed an n-3-deficient diet revealed a highly specific substitution of 22:5n-6, but not 22:4n-6 or ARA for DHA in brain phosphatidyl ethanolamine and phosphatidyl serine (45). Tight regulation of the “reciprocal replacement” of DHA and 22:5n-6 (42) among brain phospholipid molecular species illustrates the essential structure-function relationship between DHA and outcomes such as vision or cognition. Functionally, low DHA levels in neural tissue cannot be fully compensated by increased 22:5n-6 and manifests as poorer electroretinogram performance (46) and spatial learning (47). In the present study, the composition difference in neural tissue 22-carbon HUFA between diets d1 and d3 is of particular interest. Pigs in the low DHA group had significantly lower DHA and higher 22:5n-6 in cerebral cortex, cerebellum and retina. DHA at 0.32% total FA is the level currently added to infant formulas. Higher levels may provide further benefit for visual acuity and cognition, especially in target populations with endogenously low DHA status (5,9,11). Importantly, the d1-d3 groups held ARA at 0.67% which is at the highest levels found in human breast milk (2), and similar to the MR group which had extremely low DHA (Table 2.2). Changes in tissue FA composition could influence markers of formula safety and measures of overall development. The dietary ARA level (0.09 – 1.0% total FA) had a negligible effect on safety as assessed by clinical chemistry and measures of immune function in our piglets fed 1.0% DHA (Chapter One). Likewise, there were no differences in growth among them, and no reported differences in vigor by those observing piglets daily. 46 In summary, results from the present study demonstrate a tissue-specific responsiveness to the dietary ARA level in rapidly growing neonatal pigs. Whereas brain ARA remained unaffected by diet ARA, heart and liver ARA levels were responsive. Dietary ARA level had no effect on DHA accretion in heart, liver or neural tissue. The magnification of heart ARA levels with increasing dietary ARA content suggests that the growing heart specifically incorporates and concentrates ARA at a rate that may exceed the biosynthetic capacity for ARA. Further investigations are warranted to determine the biological implication of heart ARA status in the developing neonate. 47 REFERENCES 1. Rulis, A. M. (2001) Agency Response Letter GRAS Notice No. GRN 000080. CFSAN/Office of Premarket Approval. 2. Brenna, J. T., Varamini, B., Jensen, R. G., Diersen-Schade, D. 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Lim, S. Y., Hoshiba, J., Moriguchi, T. & Salem, N., Jr. (2005) N-3 fatty acid deficiency induced by a modified artificial rearing method leads to poorer performance in spatial learning tasks. Pediatr Res 58: 741-748. 52 CHAPTER THREE EVALUATION OF BIOEQUIVALENCY AND TOXICOLOGICAL EFFECTS OF THREE SOURCES OF ARACHIDONIC ACID (ARA) IN DOMESTIC PIGLETS* Abstract Arachidonic acid (ARA) is routinely added to infant formula to support growth and development. We evaluated the safety and bioequivalence of three oils providing ARA for potential use in infant formula using the neonatal pig model. On d 3 of age, domestic pigs were matched for weight and gender and assigned to 1 of 3 formula diets, each containing ARA at 0.62 – 0.67% total fatty acids. Long chain polyunsaturated fatty acids (LCPUFA) in the Control diet were provided by ARASCO and DHASCO (Martek Biosciences, Corp.) in a formulation similar to Enfamil; ARA in experimental diets (a1 and a2) came from proprietary blends of oils. Blood collection was performed weekly and body weight measurements taken every 2-3 d. Pigs were sacrificed on d 22 and organs sampled. Intake and growth was similar across all diets. Few differences in clinical chemistry and hemogram parameters were detected, with aspartate aminotransferase and creatine kinase levels highest in the Control. Liver sections had no signs of histopathological abnormality. ARA and docosahexaenoic acid (DHA) levels in cerebral cortex, retina and heart were not different across all diets. In the liver, ARA levels *Tyburczy, C., Brenna, M. E., Kothapalli, K. S. D., Blank, B. S., Valentine, H., McDonough, S. P., Banavara, D., Diersen-Schade, D. A., Brenna, J. T. (2010) Food Chem Toxicol, submitted. 53 were 2% lower in a1 pigs compared with the Controls (P = 0.002) while DHA levels in the a1 pigs averaged 7% higher than the Control and a2 groups (P < 0.05). We conclude that the experimental ARA oils are safe and bioequivalent to a commercially available ARA source in the domestic piglet. Introduction Arachidonic acid (ARA) is a long chain polyunsaturated fatty acid (LCPUFA) that is routinely added to infant formula along with docosahexaenoic acid (DHA). Both are natural components of breast milk and are important for growth and development during the perinatal period (1-3). ARA and DHA serve as the major LCPUFA in central nervous tissue and in the heart ARA comprises upwards of 25% of total fatty acids (FA) (Chapter Two). Tissue ARA and DHA accretion during growth occurs via the uptake of preformed LCPUFA from breast milk or formula or through the endogenous biosynthesis of LCPUFA from the dietary essential FA, linoleic and linolenic acids. Rates of endogenous ARA and DHA biosynthesis appear to be a limiting factor in the accretion of LCPUFA in tissues (4-6)(Chapter Two), indicating a requirement for these LCPUFA in the diets of human infants. In 1994, the FAO/WHO set recommendations for ARA and DHA in the formula of term infants at 0.66% and 0.33% of total FA, respectively (7). These levels are based largely on mean worldwide breast milk levels (8,9) and supported by the evidence for benefit in vision and cognitive performance in clinical studies with human infants (10,11). Single-cell oils serve as a convenient vehicle for adding target levels of LCPUFA to commercial infant formula. In the U.S., two main sources of ARA have been determined to be Generally Regarded as Safe (GRAS) for use in 54 infant formula, ARASCO (Martek Biosciences, Corp., Columbia, MD) (12,13) and SUNTGA40S (Suntory, Ltd., Osaka, Japan) (14). Both are triglyceride oils derived from the fungus Mortierella alpina (M. alpina) and contain ARA at approximately 40% total FA. A third oil, “refined arachidonic acid-rich oil” (RAO; Cargill, Inc., Wuhan, China) also derived from M. alpina is currently pending FDA approval for use in preterm and term infant formulas (15). These ARA oils may be derived from a common fungal source but differences in the manufacturing process inevitably lead to subtle variations in the final product. Of particular importance here are the levels of trace chemical and fat soluble components, and potentially bioactive microbiological constituents. While an ARA oil may comprise less than 2% of the total fat in formula, the extended consumption of these oils by the vulnerable, growing infant population may exaggerate any toxicological or allergenic effect caused by trace or undetected contaminants (16,17). Thus, preclinical feeding studies with animals are essential to determine the safety of novel formulations for use in the diets of human infants. The present study sought to determine the safety and bioequivalence of two experimental, proprietary ARA oils for potential use in infant formula compared with the commercially available ARASCO using the neonatal pig model. Piglets were fed one of three ready-to-use formulas that provided ARA at 0.64% and DHA at 0.34% of total FA from day 3 to 22 of life, upon which tissues were analyzed for ARA and DHA accretion and livers examined for pathohistological changes. We hypothesized that the experimental ARA oils would be safe, nutritionally bioequivalent to ARASCO and produce no toxicological effects in our neonatal pigs. 55 Materials and methods Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Cornell University. Domestic piglets were selected for gender and weight from 5 sows at the swine facility at Cornell University. One day before the scheduled, term farrowing date, pregnant sows were injected with 2 cc of Lutalyse (Pfizer Animal Health, Kalamazoo, MI) to induce farrowing and obtain a block of piglets with the same birth date. Newborn piglets were processed according to standard facility practices (i.e. intramuscular injection of iron dextran (100 mg) and penicillin G (½ cc), tooth clip, tail dock, ear notch) and remained with the sow until day 3 of age, upon which they were matched for weight and assigned to one of three experimental formula diets (n=8 per diet). Piglets were then transported to the campus large animal research facility where they were housed individually in raised metal cages, provided appropriate enrichment and maintained on a 16/8 hr light/dark cycle. Diets Experimental formulas were designed to meet or exceed nutrient requirements for growing pigs 3 – 5 kg in body weight (18). Milk replacer diets were prepared as ready-to-use formulas that provided 1.0 kcal/ml with a target nutrient composition as follows (% wt/wt): protein, 4.9; fat, 5.5; ash, 1.2. The detailed composition and nutrient analysis for the experimental formulas is presented in Table 3.1. Target specifications for ARA and DHA were 35.8 and 17.9 mg/100 kcal. LCPUFA in the Control diet were supplied by ARASCO and DHASCO (Martek Biosciences, Corp., Columbia, MD) in a formulation similar 56 Table 3.1. Nutrient composition of experimental diets Nutrient (% wt/wt)1 Control a1 a2 Fat 5.5 5.6 5.6 Protein 4.9 4.9 4.8 Carbohydrate 6.4 6.1 6.2 Ash 1.2 1.2 1.2 Total solids 18.1 17.7 17.8 Mineral (mg/100 kcal) Na 59.3 62.6 60.6 Mg 15.0 14.5 14.6 P 146.0 149.7 146.3 K 306.3 301.9 304.4 Ca 217.5 214.8 208.8 Vitamin (units/100 kcal) Vitamin E (IU) 1.6 1.6 1.6 Vitamin K (mcg) 16.7 18.3 17.5 Vitamin D (IU) 13.4 13.2 13.5 Vitamin C (mg) 6.3 6.7 6.3 Vitamin A (IU) 83.0 88.0 83.0 Thiamine HCl (mcg) 82.8 67.1 66.8 Riboflavin (mcg) 204.5 185.7 188.3 Vitamin B6 (mcg) 65.6 60.5 69.4 Vitamin B12 (mcg) 0.5 0.4 0.5 1Composition taken as percent of total weight (wt). 57 Table 3.2. FA composition of experimental diets1 Diet Control a1 a2 FA (% total FA) 10:0 0.70 ± 0.02 0.74 ± 0.02 0.46 ± 12:0 8.19 ± 0.08 8.12 ± 0.09 7.37 ± 14:0 4.21 ± 0.04 4.24 ± 0.03 4.20 ± 16:0 21.76 ± 0.05 21.77 ± 0.08 22.07 ± 16:1 0.18 ± 0.01 0.18 ± 0.01 0.19 ± 18:0 4.58 ± 0.02 4.50 ± 0.02 4.53 ± 18:1 38.14 ± 0.12 37.96 ± 0.15 38.59 ± 18:2n-6 17.74 ± 0.03 17.86 ± 0.06 17.96 ± Conjugated 18:2 0.29 ± 0.03 0.31 ± 0.01 0.25 ± 18:3n-6 0.10 ± 0.01 0.11 ± 0.00 0.10 ± 18:3n-3 2.00 ± 0.03 2.01 ± 0.04 2.01 ± 20:0 0.35 ± 0.01 0.35 ± 0.00 0.36 ± 20:1 0.21 ± 0.01 0.21 ± 0.01 0.21 ± 20:2n-6 0.03 ± 0.00 0.01 ± 0.01 0.03 ± 20:3n-6 0.07 ± 0.00 0.08 ± 0.01 0.08 ± ARA 0.67 ± 0.01 0.62 ± 0.00 0.67 ± 22:0 0.27 ± 0.01 0.30 ± 0.01 0.32 ± DHA 0.35 ± 0.01 0.35 ± 0.01 0.34 ± 23:0 0.04 ± 0.01 0.05 ± 0.01 0.05 ± 24:0 ∑ SFA2 0.12 ± 0.00 0.21 ± 0.01 0.22 ± 40.18 ± 0.15 40.22 ± 0.20 39.53 ± ∑ MUFA 38.53 ± 0.13 38.36 ± 0.15 38.99 ± ∑ n-6 18.62 ± 0.03 18.67 ± 0.06 18.83 ± ∑ n-3 2.34 ± 0.04 2.36 ± 0.03 2.35 ± 18:2n-6/18:3n-3 8.89 ± 0.15 8.91 ± 0.18 8.94 ± 0.01 0.07 0.03 0.09 0.02 0.04 0.15 0.04 0.03 0.01 0.03 0.00 0.01 0.00 0.00 0.00 0.01 0.01 0.00 0.01 0.17 0.14 0.04 0.04 0.16 1Values represent means ± SD, n = 3 extractions per diet. 2SFA, saturated FA; MUFA, monounsaturated FA. 58 to that currently used in the commercial human infant formula Enfamil (MeadJohnson Nutrition, Evansville, IN). LCPUFA in the experimental diets, a1 and a2 were provided by a proprietary blend of oils that may include SUNTGA40S, RAO or a novel ARA source. The detailed FA composition of the experimental formulas is presented in Table 3.2. Fresh formula was provided three times per day in stainless steel bowls fixed into the cage doors and water was offered ad libitum. Piglets were fed at 80% ad libitum intake, based on pilot data, and formula intakes were recorded daily. Sampling Body weights measurements were taken every other day during the first week of life and thereon every third day for the remainder of the study. Nonfasted blood samples were collected from the anterior vena cava into EDTA-containing vacutainer tubes (BD, Franklin Lakes, NJ) on days 3, 7, 14 and 21 of age. Whole blood was separated into plasma, red blood cell (RBC) and white cell fractions using Leucosep tubes (Grenier Bio-One, Monroe, NC) with lymphocyte separation media (Mediatech, Inc., Manassas, VA) according to manufacturer’s directions. Piglets were sacrificed on day 22 of age via an intravenous injection of Fatal Plus (1 ml/4.54 kg body weight; Vortech Pharmaceuticals, Dearborn, MI, USA) followed by exsanguination. Organs were harvested, weighed and samples flash frozen in liquid nitrogen within 10 minutes of cessation of heart beat. Clinical chemistry and hemogram analysis Plasma and EDTA-blood from piglets on day 21 of age were delivered promptly to the Animal Health Diagnostic Center (College of Veterinary 59 Medicine, Cornell University) for analysis of clinical chemistry and hemogram parameters. Liver histopathology Freshly harvested samples from the right central lobe (~ 1 g) were fixed in 10% neutral buffered formalin and submitted to the Pathohistology Laboratory (College of Veterinary Medicine, Cornell University) where they were embedded in wax, sectioned and stained with hematoxylin and eosin. Sections were examined under light microscope by a veterinary pathologist for determination of histopathological abnormalities. Fatty acid analysis FA methyl esters (FAME) were prepared from approximately 50 mg tissue and 50 μl of plasma, RBC and formula according to the One-step hydrolysis, extraction and methylation procedure (19) with modifications (20). A detailed protocol for the One-step method is presented in Appendix II. Retinas, which were suspended in saline at necropsy, were first dried using a Savant SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA). FAME were quantified on a 5890 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a BPX70 fused silica column (25 m x 0.22 mm i.d. x 0.25 μm film; SGE Incorportated, Austin, TX) and integrated using PeakSimple 3.78 software (SRI Instruments, Torrance, CA). An equal weight FAME mixture was used daily to verify response factors. FAME were structurally identified by covalent-adduct chemical ionization mass spectrometry on a Saturn 2000 mass spectrometer (Varian, Inc., Walnut Creek, CA) attached to a Varian Star 3400 gas chromatograph (21). 60 Statistical analysis Statistical analysis was carried out using the Fit Model platform of JMP (2008 SAS Institute, 8.0) to fit mixed models. Fixed effects were diet, gender, day 3 of age body weight and the full factorial of interactions. Interaction effects were considered significant at P < 0.10 and fixed effects at P < 0.05. For each parameter analyzed, effects not considered significant were removed from the final model. Random effects were litter and animal nested within litter for repeated measures of body weight and RBC FA content. Significance of pairwise comparisons was determined using the Student’s t-test. Values are reported as means ± SD. Linear regression analysis was performed to determine the relationship between RBC LCPUFA content and postnatal age. Results Formula composition Total formula intakes averaged 29.6 ± 1.7 L and were similar for all three dietary treatment groups. The experimental formulas provided 1 kcal/ml, corresponding to a mean total energy intake of 29,600 ± 1,700 kcal per pig and a mean daily intake of 1.5 L or 1,500 kcal. Mean total intake of ARA was 10.60 ± 0.59 g, while the mean total intake of DHA was 5.30 ± 0.30 g. Clinical observations and growth All 24 piglets remained on the study until day 21 of age, and very few health ailments were noted during the course of the study. Loose stools during the first week of life were the greatest concern, although by the second week of life, the occurrence of loose stools had cleared in most piglets. One piglet was given an intramuscular injection of penicillin G benzathine (75,000 61 IU) by the attending veterinarian on day 11 of age for an undiagnosed, yet persistent disinterest in consuming formula. That animal grew significantly less than all other pigs starting on day 13 of age, as determined using Grubb’s test for statistical outliers, and was removed from the body weight analysis starting at this time point. The temporal pattern of body weight gain during the study is presented in Figure 3.1. Piglets weighed 2.1 ± 0.2 kg at the start of the study (day 3 of age) and rapidly grew to 8.4 ± 0.4 kg by day 22 of age. Mean body weights were similar among the three dietary treatments at every time point measured. Organ weights at necropsy, both relative and absolute, were similar among all dietary treatment groups. Figure 3.2 presents the summary of relative organ weights (organ weight as a percentage of body weight at necropsy) for piglets sacrificed on day 22 of age. Hemogram and clinical chemistry A summary of mean hemogram and clinical chemistry parameters is presented in Table 3.3. Only two parameters, aspartate aminotransferase (AST) and creatine kinase, were determined to be significantly influenced by diet. Control pigs had significantly higher AST (37 ± 13 U/L) than a1 (28 ± 7 U/L) and a2 (25 ± 7 U/L) pigs (P = 0.01), and higher creatine kinase (895 ± 520 U/L) than the a1 (541 ± 227 U/L) and a2 (520 ± 228 U/L) pigs (P = 0.001). AST and creatine kinase levels were similar between the a1 and a2 groups. Additional parameters that were unaffected by diet were mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, red cell distribution width, segmented neutrophils, monocytes, eosinophils, basophils, 62 Weight (kg) large unstained cells, mean platelet volume, creatinine, phosphate and total bilirubin (data not presented). 10 8 6 4 Control a1 2 a2 0 0 5 10 15 20 25 Age (days) Figure 3.1. Temporal pattern of body weight gain in piglets from day 3 to 22 of age. Values represent means ± SD, n = 8/diet. One piglet from Diet a1 was not included in body weight measurements on days 13 – 22 due to a slower rate of growth caused by an undiagnosed, yet persistent disinterest in formula consumption. Body weights were similar among all dietary treatment groups at every time point measured. Liver histopathology Histological diagnosis: Normal. “Comment: All samples were within normal histopathological limits. The lobular architecture was intact, the portal regions were unremarkable and there was no evidence of cholestasis. Hepatocytes were uniform in size and shape, replete with glycogen and contained no discernable lipid. No mitrotic figures were observed and only rare binucleate hepatocytes were noted. Occasional Kupffer cells were present but no hepatic stellate (Ito) cells could be identified. Variable numbers of small foci of extramedullary hematopoietic tissue were randomly dispersed 63 throughout the parenchyma (incidental). No histopathological changes were present in the sections examined. In particular, no changes similar to those described in rodent studies of peroxisome proliferator exposure were detected.” Weight (% body weight) 3.5 3.0 Control 2.5 a1 a2 2.0 1.5 1.0 0.5 0.0 Brain Liver Heart Lung Kidney Spleen Organ Figure 3.2. Relative organ weights from piglets on day 22 of age. Values represent means ± SD, n = 8 for Control and Diet a2; n = 7 for Diet a1. Pairwise comparisons determined using Student’s t-test. Organ weights were similar for all dietary treatment groups. Tissue LCPUFA accretion The FA composition of heart, liver, brain (cerebral cortex) and retina, harvested from pigs on day 22 of age is presented in Tables 3.4 and 3.5. Mean ARA levels in the brain, retina and heart were 10.97 ± 0.36%, 10.50 ± 0.43%, 20.38 ± 0.82% of total FA, respectively, and were similar for all three dietary treatment groups. ARA levels in the liver were 2% lower in pigs fed Diet a1 (17.33 ± 0.78% FA) compared with the Control (17.66 ± 0.49% FA), while the a2 pigs showed an intermediary liver ARA content (17.38 ± 0.57% 64 Table 3.3. Summary of hemogram and clinical chemistry parameters from piglets on day 21 of age1-2 Test Control a1 a2 P Hematocrit (%) 43 ± 3 46 ± 5 42 ± 2 NS Hemoglobin (g/dL) 13.2 ± 0.9 13.8 ± 1.3 12.9 ± 0.5 NS RBC (mill/μL) 6.3 ± 0.4 6.5 ± 0.5 6.3 ± 0.2 NS WBC (thou/μL) 10.5 ± 2.2 10.9 ± 1.3 9.9 ± 2.6 NS Lymphocytes (thou/μL) 6.1 ± 1.1 6.4 ± 1.1 5.7 ± 1.3 NS Platelet Count (thou/μL) 709 ± 76 697 ± 169 667 ± 136 NS Urea (mg/dL) 8±2 8±1 7±2 NS Creatinine (mg/dL) 0.7 ± 0.2 0.7 ± 0.2 0.7 ± 0.1 NS Total Protein (g/dL) 4.6 ± 0.3 4.7 ± 0.2 4.5 ± 0.2 NS Albumin (g/dL) 3.6 ± 0.3 3.5 ± 0.2 3.5 ± 0.1 NS Globulin (g/dL) 1.0 ± 0.3 1.1 ± 0.3 1.1 ± 0.1 NS Glucose (mg/dL) 178 ± 37 165 ± 31 166 ± 33 NS AST (U/L) 37 ± 13a 28 ± 7b 25 ± 7b 0.01 GGT (U/L) 23 ± 11 24 ± 14 24 ± 16 NS Creatine Kinase (U/L) 895 ± 520a 541 ± 227b 520 ± 228b 0.001 1Abbreviations: RBC, red blood cell count; WBC, white blood cell count; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase. 2Values represent means ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 65 Table 3.4. FA composition of heart and liver from piglets fed one of three sources of ARA1 Diet Control a1 a2 P Heart Σ SFA2 FA (% total FA) 31.92 ± 0.29 32.15 ± 0.49 32.37 ± 0.39 NS Σ MUFA 18.53 ± 0.75 18.35 ± 0.77 18.09 ± 0.74 NS ARA 20.67 ± 0.56 19.93 ± 0.79 20.57 ± 0.93 NS 22:4n-6 0.92 ± 0.10 0.86 ± 0.09 0.83 ± 0.13 NS 22:5n-6 tr. tr. tr. n/a Σ n-6 43.40 ± 0.86 43.44 ± 1.12 43.59 ± 1.00 NS 20:5n-3 0.42 ± 0.04 0.39 ± 0.03 0.39 ± 0.04 NS 22:5n-3 0.76 ± 0.08 0.71 ± 0.04 0.73 ± 0.05 NS DHA 4.16 ± 0.39 4.04 ± 0.28 3.98 ± 0.35 NS Σ n-3 6.05 ± 0.40 5.95 ± 0.31 5.85 ± 0.40 NS ARA/DHA 5.00 ± 0.38 4.96 ± 0.40 5.22 ± 0.63 NS Σ 22C HUFA3 5.84 ± 0.37 5.61 ± 0.34 5.53 ± 0.37 NS Liver Σ SFA 42.15 ± 0.91 42.46 ± 0.57 41.89 ± 0.74 NS Σ MUFA 13.64 ± 1.17 13.50 ± 0.71 13.89 ± 1.24 NS ARA 17.66 ± 0.49a 17.33 ± 0.78b 17.38 ± 0.57ab 0.01 22:4n-6 0.64 ± 0.07 0.63 ± 0.10 0.65 ± 0.08 NS 22:5n-6 0.06 ± 0.01 0.06 ± 0.01 0.05 ± 0.02 NS Σ n-6 34.10 ± 0.69 33.49 ± 0.59 34.17 ± 0.55 NS 20:5n-3 0.33 ± 0.04 0.28 ± 0.08 0.31 ± 0.05 NS 22:5n-3 1.03 ± 0.18 1.04 ± 0.13 1.04 ± 0.06 NS DHA 7.70 ± 0.47b 8.23 ± 0.38a 7.62 ± 0.62b 0.046 Σ n-3 9.80 ± 0.47 10.21 ± 0.43 9.70 ± 0.56 NS ARA/DHA 2.30 ± 0.12 2.11 ± 0.12 2.29 ± 0.15 NS Σ 22C HUFA 9.43 ± 0.52 9.96 ± 0.33 9.37 ± 0.68 NS 1Values represent means ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2SFA, saturated FA; MUFA, monounsaturated FA. 3∑ 22C HUFA = ∑ 22:4n-6, 22:5n-6, 22:5n-3, DHA. 66 Table 3.5. FA composition of brain (cerebral cortex) and retina from piglets fed one of three sources of ARA1 Diet Control a1 a2 P Cerebral cortex ∑ SFA2 FA (% total FA) 44.49 ± 0.58 44.82 ± 0.74 44.60 ± 0.42 NS ∑ MUFA 20.63 ± 0.90 20.16 ± 1.06 20.28 ± 1.21 NS 22:3n-9 0.31 ± 0.05 0.31 ± 0.08 0.28 ± 0.04 NS ARA 10.83 ± 0.34 11.04 ± 0.42 11.04 ± 0.33 NS 22:4n-6 3.99 ± 0.34 4.12 ± 0.38 4.20 ± 0.36 NS 22:5n-6 5.90 ± 0.50 5.82 ± 0.70 5.58 ± 0.57 NS ∑ n-6 23.32 ± 0.73 23.62 ± 0.50 23.53 ± 0.80 NS 20:5n-3 0.12 ± 0.03 0.10 ± 0.04 0.12 ± 0.04 NS 22:5n-3 0.31 ± 0.04 0.31 ± 0.05 0.31 ± 0.04 NS DHA 10.17 ± 0.71 9.97 ± 0.79 10.24 ± 0.39 NS ∑ n-3 10.67 ± 0.70 10.48 ± 0.78 10.76 ± 0.41 NS ARA/DHA 1.07 ± 0.08 1.11 ± 0.08 1.08 ± 0.04 NS ∑ 22C HUFA3 20.67 ± 0.52 20.54 ± 0.60 20.62 ± 0.64 NS Retina ∑ SFA 41.92 ± 0.92 42.65 ± 0.90 42.13 ± 1.19 NS ∑ MUFA 17.64 ± 0.37 17.71 ± 0.52 17.83 ± 0.60 NS 22:3n-9 0.09 0.02 0.08 0.02 0.08 0.03 NS ARA 10.45 ± 0.42 10.42 ± 0.43 10.63 ± 0.45 NS 22:4n-6 2.51 ± 0.19 2.45 ± 0.16 2.43 ± 0.11 NS 22:5n-6 2.58 0.41 2.41 0.56 2.36 0.41 NS ∑ n-6 19.06 ± 0.85 18.90 ± 0.92 18.88 ± 0.68 NS 20:5n-3 0.46 ± 0.07 0.48 ± 0.09 0.40 ± 0.12 NS 22:5n-3 0.62 ± 0.07 0.64 ± 0.07 0.62 ± 0.06 NS DHA 19.87 ± 0.65 19.20 ± 0.83 19.75 ± 1.66 NS ∑ n-3 21.02 ± 0.69 20.38 ± 0.85 20.83 ± 1.76 NS ARA/DHA 0.53 ± 0.02 0.54 ± 0.03 0.54 ± 0.05 NS ∑ 22C HUFA 25.58 ± 0.90 24.70 ± 1.06 25.16 ± 1.58 NS 1Values represent means ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2SFA, saturated FA; MUFA, monounsaturated FA. 3∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 67 FA; P = 0.009). Experimental diets equally supported DHA accretion in the brain, retina and heart, while in the liver, DHA levels were 7% higher in a1 pigs (8.23 ± 0.38) compared with the Control (7.70 ± 0.47% FA) and a2 (7.62 ± 0.62% FA) groups (P = 0.046). No other statistically significant differences in tissue FA accretion were observed among the dietary treatments. Mean ARA levels in the RBC fraction were similar among all dietary treatment groups at every time point examined (days 3, 7, 14 and 21; Table 3.6). RBC DHA values were similar among dietary treatment groups on days 3 and 21 of age, averaging 1.74 ± 0.32% and 2.57 ± 0.27% of total FA, respectively, but were significantly affected by diet on days 7 and 14 (Figure 3.3). On day 7 of age, RBC DHA levels were significantly higher in the a1 pigs (1.86 ± 0.19% FA) compared with a2 group (1.69 ± 0.26% FA), while Control pigs had intermediary levels (1.81 ± 0.27% FA; P = 0.03). On day 14 of age, RBC DHA levels were the highest in the a1 pigs (2.85 ± 0.31% FA) and were similar between the Control (2.59 ± 0.37% FA) and a2 (2.49 ± 0.14% FA) groups (P = 0.02). Mean RBC ARA, 20:5n-3, 22:5n-3, 22:4n-6 and 22:5n-6 were similar for all dietary treatment groups at every time point examined. Because of this, treatment groups were pooled and linear regression analysis was performed to determine the temporal pattern of LCPUFA content in RBC. Over the course of the experimental period, mean RBC ARA levels decreased from 6.98 ± 0.60% of total FA on day 3 of age to 4.62 ± 0.32% FA on day 21 of age (P < 0.0001; Figure 3.3). Oneway analysis of RBC ARA content by day of age confirmed this temporal decline, with each incremental increase in age corresponding to a significant decrease in RBC ARA (P < 0.0001). Mean RBC 68 Table 3.6. LCPUFA composition of RBC collected from piglets on days 3, 7, 14 and 21 of life1 Diet Control a1 a2 P Day 3 ARA 7.06 ± 0.65 FA (% total FA) 7.04 ± 0.67 6.84 ± 0.52 NS 22:4n-6 0.97 ± 0.17 0.99 ± 0.14 1.00 ± 0.20 NS 22:5n-6 1.96 ± 0.30 1.92 ± 0.12 1.84 ± 0.26 NS 20:5n-3 0.16 ± 0.08 0.17 ± 0.11 0.14 ± 0.03 NS 22:5n-3 0.49 ± 0.08 0.53 ± 0.19 0.49 ± 0.14 NS DHA 1.78 ± 0.37 1.70 ± 0.25 1.73 ± 0.36 NS Day 7 ARA 5.94 ± 0.42 6.09 ± 0.47 5.76 ± 0.27 NS 22:4n-6 0.72 ± 0.07 0.77 ± 0.08 0.69 ± 0.08 NS 22:5n-6 1.09 ± 0.25 1.23 ± 0.10 1.03 ± 0.18 NS 20:5n-3 0.16 ± 0.06 0.16 ± 0.06 0.14 ± 0.03 NS 22:5n-3 DHA 0.50 ± 0.07 1.81 ± 0.27ab 0.52 ± 0.08 1.86 ± 0.19a 0.48 ± 0.07 1.69 ± 0.26b NS 0.03 Day 14 ARA 22:4n-6 22:5n-6 20:5n-3 22:5n-3 DHA 5.04 ± 0.48 0.60 ± 0.08 0.50 ± 0.05 0.22 ± 0.06 0.50 ± 0.06 2.59 ± 0.37b 5.32 ± 0.37 0.61 ± 0.06 0.54 ± 0.12 0.22 ± 0.06 0.55 ± 0.05 2.85 ± 0.31a 4.89 ± 0.22 0.57 ± 0.03 0.46 ± 0.08 0.21 ± 0.03 0.51 ± 0.04 2.49 ± 0.14b NS NS NS NS NS 0.02 Day 21 ARA 22:4n-6 22:5n-6 20:5n-3 4.71 ± 0.39 0.48 ± 0.06 0.24 ± 0.05 0.20 ± 0.09 4.55 ± 0.49 0.50 ± 0.07 0.25 ± 0.06 0.18 ± 0.08 4.59 ± 0.23 0.50 ± 0.07 0.24 ± 0.05 0.15 ± 0.09 NS NS NS NS 22:5n-3 0.48 ± 0.05 0.47 ± 0.05 0.48 ± 0.04 NS DHA 2.61 ± 0.29 2.59 ± 0.26 2.52 ± 0.27 NS 1Values represent means ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 69 RBC ARA (% total FA) A 10 8 a b 6 4 y = -0.13x + 7.08 2 R2 = 0.75 P < 0.0001 0 26 B4 3 2 Control a1 a2 c 10 14 Age (days) ab a b bab RBC DHA (% total FA) 1 0 Day 3 Day 7 Day 14 d 18 22 Day 21 Figure 3.3. Temporal pattern of ARA (A) and DHA (B) content in RBC collected from piglets on days 3, 7, 14 and 21 of life. (A) represents linear regression analysis of RBC ARA content by age; each point corresponds to one pig. In (B), values represent means ± SD, n = 8 per diet. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 70 22:4n-6 (P < 0.0001) and 22:5n-6 (P < 0.0001) also decreased during the study, while 20:5n-3 and 22:5n-3 levels remained constant. Discussion Preclinical studies with animals provide an invaluable tool for determining the safety of novel food additives for use in the diets of humans. In the present study, we evaluated two proprietary ARA oils for potential use in infant formula for toxicological effects and nutritional bioequivalence compared with a commercially available ARA source. Domestic piglets were used because of their rapid rates of growth and similarities to humans in terms of brain development and metabolism of lipids and LCPUFA (22,23). Growth performance is a well-accepted measure of health and vitality in developing infants. In the present study, the two experimental ARA formulas were readily consumed and equally supported growth in the neonatal pigs compared with the Control formula. Body weights were similar among the three dietary treatment groups at every time point and no differences were observed for organ weights upon sacrifice. The 2000 Center for Disease Control growth charts show that human infants typically double their birth weight by 4 – 5 months of life and grow to 4 times their birth weight by 34 – 36 months (24). In comparison, our piglets grew to 4 times their initial body weight by day 22 of age. This rapid rate of growth and concomitant high demand for formula suggests that any trace toxicological constituent of the formulas could have manifested as poorer growth or suboptimal clinical conditions during this short study duration. Rather, pigs in this study appeared healthy and presented with few health ailments. Loose stools in a number of piglets were the greatest concern during the first week of the study. This was 71 likely caused by stresses from the transition period and adaptation to a new diet and environment (25), as opposed to a specific diet or dietary component. Mean values for clinical chemistry and hemogram parameters were consistent with previous studies in neonatal pigs (26-28), and very few differences were observed among the three dietary treatment groups. Control pigs showed elevated plasma AST and creatine kinase compared with pigs fed either of the experimental formulas. For AST, a biomarker of tissue inflammation, Control values were within limits previously observed in nursery pigs (26) and weaned growing pigs (29), suggesting that the numerical difference among diets is likely biologically insignificant. Creatine kinase levels were highest in the Control pigs compared with the two experimental ARA groups, although all treatment means were at the low end of the normal range for commercial pigs (30) and were comparable to values previously observed in a similar neonatal pig study (Chapter One). In all pigs, liver sections were determined to be normal following pathohistological examination and showed no indication of gross abnormalities or toxicological changes. These results are consistent with other studies in pigs showing that the provision of an ARA-rich oil in the milk replacer formula up to 5x the level currently used in formula is safe and produces no adverse effect on serum clinical indicators or liver histology (27,31). Thus, results from the present study indicate that the two experimental ARA oils produce no pathohistological abnormalities in the liver and have no adverse effect on clinical chemistry or hemogram parameters in rapidly growing piglets compared with the Control formula containing ARASCO. The three ARA oils equally supported ARA accretion in the brain, retina and heart. On the other hand, liver ARA levels showed a remarkable 72 sensitivity to the dietary ARA content with the Control pigs having significantly higher ARA levels than the a1 group. This discrepancy in liver ARA levels is consistent with the a1 diet providing 8% less ARA (0.62% vs. 0.67% total FA) than the Control and a2 diets, a difference that may arise from normal variability across product lots and over time during in the manufacturing process. However, liver DHA levels also appeared responsive to the difference in dietary ARA level, with the a1 pigs having the highest DHA content. Numerous studies have demonstrated a sensitivity of liver LCPUFA to dietary FA composition (32,33)(Chapter Two) that occurs primarily as a result of dietary FA composition but also due to the regulation of LCPUFA biosynthesis by dietary LCPUFA (34,35). Overall, these results demonstrate that the experimental ARA oils are nutritionally bioequivalent to ARASCO and that subtle variations in total formula ARA content may influence liver ARA and DHA levels and endogenous LCPUFA biosynthesis. Studies with human infants are limited by the information they provide, especially with regard to the ethical limitations and potential risks associated with frequent blood sampling as well as the general unavailability of tissue from healthy individuals (36). This study provided a unique opportunity to examine acute changes in RBC LCPUFA levels that may have been otherwise overlooked in clinical studies with infants. The lack of significant differences among dietary treatment groups permitted pooling of sample means to determine the temporal pattern of RBC LCPUFA in pigs fed 0.64% ARA and 0.32%. Here, we observed significant decreases in RBC ARA, 22:4n-6 and 22:5n-6 and an increase in RBC DHA compared with baseline values. The rapid and dramatic decline in RBC ARA has not been previously documented in neonatal animals and the cause remains unclear. Human infants that 73 consume breast milk or formula with added LCPUFA show a modest decline in RBC ARA during the first year of life (37). At the same time, the infant brain rapidly accumulates ARA (4), and in Chapter Two, we demonstrated that the growing heart specifically incorporates and concentrates ARA, and that ARA accretion is limited by dietary ARA content. It is probable that the temporal decline in RBC ARA we observed in the present study occurred as a result of rapid tissue ARA accretion. With regard to DHA, we observed a general increase in RBC DHA that appeared to reach plateau by day 14 of age. The low levels of RBC DHA may be explained by the negligible content of DHA in the sow milk previously analyzed from this herd (Chapters One and Two), while the content of RBC DHA on days 14 and 21 are consistent with most (38-41), but not all (42) studies of rapidly growing, neonatal pigs consuming low DHA. In 2001, ARASCO was determined to be GRAS for use in term infant formula up to 1.88% of total fat and providing ARA up to 0.75% of total fat (12). More recently, SUNTGA40S was determined to be GRAS for preterm and term infants when providing ARA up to 0.40% of total fat (14), and a third ARA-rich oil, RAO is currently pending FDA approval for use in infant formula (15). It is possible that the experimental oils used in this study are SUNTGA40S, evaluated at a higher formula content than is currently approved for use in formula, and RAO which has not been reported in preclinical studies with neonatal pigs. However, we cannot exclude the possibility that the experimental ARA oils are of a novel, proprietary formulation that may or may not be derived from M. alpina and for which little safety data is known. Overall, this study evaluated the safety and bioefficacy of three ARA-rich oils for potential use in commercial infant formula. Oils provided 74 ARA at 0.64% of total FA, were added with DHASCO to provide 0.34% DHA and resulted in no other major alterations in the formula FA composition. We conclude that the two experimental ARA sources were nutritionally bioequivalent to ARASCO, each equally supporting ARA accretion in the brain, retina and heart, and produced no toxicological effects in neonatal pigs. Results further demonstrate that ARA-rich oils that deliver equivalent levels of ARA equally support the content of ARA in formula when prepared using a common manufacturing process. 75 REFERNCES 1. Carlson, S. E. (2001) Docosahexaenoic acid and arachidonic acid in infant development. Semin Neonatol 6: 437-449. 2. Innis, S. M. (2005) Essential fatty acid transfer and fetal development. Placenta 26 Suppl A: S70-75. 3. Anonymous (2007) Human milk: maternal dietary lipids and infant development. Proc Nutr Soc 66: 397-404. 4. Makrides, M., Neumann, M. A., Byard, R. W., Simmer, K. & Gibson, R. A. 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J Nutr 137: 2049-2055. 79 Heart DHA (% total FA) Brain DHA (% total FA) APPENDIX I SUPPLEMENTAL FIGURES AND TABLES FOR CHAPTER TWO A 14 y = 4.34x + 6.37 12 R2 = 0.57 10 P < 0.0001 a 8 6 4 C 0.0 0.2 0.4 15 a b 0.6 0.8 b 10 y = 2.08x + 9.91 R2 = 0.44 P = 0.0005 5 0.0 0.2 0.4 0.6 0.8 Dietary DHA (% total FA) Liver DHA (% total FA) B bc 6 4 ab bc a 2 0 1.0 D 0.0 c 25 20 0.2 0.4 a y = 2.62x + 2.28 R2 = 0.66 P < 0.0001 0.6 0.8 1.0 bc Retina DHA (% total FA) 15 y = 2.04x + 18.08 R2 = 0.17 P = 0.045 10 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Dietary DHA (% total FA) Supplemental Figure 1. DHA tissue dose-response to dietary DHA with constant ARA (0.67% total FA). Dietary DHA content for d1, d2 and d3 was 0.32, 0.62 and 1.01% total FA, respectively. Tissue DHA rises with dietary DHA in all tissues presented, (A) Heart, (B) Liver, (C) Brain (Cerebral cortex) and (D) Retina. Each point represents one animal (n = 24 per panel). P < 0.05 indicates significant correlation between dietary DHA content and tissue DHA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue DHA content; means not sharing a common letter are significantly different (P < 0.05). 80 Cerebellum DHA (% total FA) A 14 12 a 10 8 6 y = 2.16x + 9.08 4 R2 = 0.32 2 P = 0.004 0 0.0 0.2 0.4 C ab 0.6 0.8 Globus pallidus DHA (% total FA) 14 y = 1.69x + 7.02 12 R2 = 0.09 10 P = NS 8 6 4 2 0 0.0 0.2 0.4 0.6 0.8 Dietary DHA (% total FA) Hippocampus DHA (% total FA) B b 14 y = 1.40x + 8.06 12 R2 = 0.09 b 10 P = 0.02 8 6 4 2 0 1.0 0.0 0.2 0.4 D a 0.6 0.8 Inferior colliculus DHA (% total FA) 14 12 10 8 6 y = 0.46x + 10.21 4 R2 = 0.01 2 P = NS 0 1.0 0.0 0.2 0.4 0.6 0.8 Dietary DHA (% total FA) ab 1.0 1.0 Supplemental Figure 2. Neural tissue DHA dose-response to dietary DHA with constant ARA (0.67% total FA). Dietary DHA content for d1, d2 and d3 was 0.32, 0.62 and 1.01% total FA, respectively. Tissue DHA rises with dietary DHA in all tissues presented, (A) Cerebellum, (B) Hippocampus, (C) Globus pallidus and (D) Inferior colliculus. Mean DHA levels in Hippocampus, Globus pallidus and Inferior colliculus were 17%, 15% and 3% higher in d3 pigs compared with d1 pigs, although these differences were not statistically significant. Each point represents one animal (n = 24 per panel). P < 0.05 indicates significant correlation between dietary DHA content and tissue DHA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue DHA content; means not sharing a common letter are significantly different (P < 0.05). 81 Globus pallidus DHA (% total FA) Cerebellum DHA (% total FA) A 14 12 10 8 6 y = 0.00x + 11.23 4 R2 = 0.00 2 P = NS 0 0.0 0.2 0.4 0.6 0.8 C 14 12 10 8 6 y = -1.35x + 9.33 4 R2 = 0.08 2 P = NS 0 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) B 1.0 D 1.0 Inferior colliculus DHA (% total FA) Hippocampus DHA (% total FA) 14 12 10 8 6 y = -0.40x + 9.92 4 R2 = 0.01 2 P = NS 0 0.0 0.2 0.4 0.6 0.8 14 12 10 8 6 y = -0.84x + 10.87 4 R2 = 0.04 2 P = NS 0 0.0 0.2 0.4 0.6 0.8 Dietary ARA (% total FA) 1.0 1.0 Supplemental Figure 3. Neural tissue DHA dose-response to dietary ARA content with constant DHA (1.0% total FA). Dietary ARA content for a1, a2, a3 and a4 was 0.09, 0.53, 0.69 and 1.06% total FA, respectively. Increasing ARA had no effect on DHA levels in (A) Cerebellum, (B) Hippocampus, (C) Globus pallidus or (D) Inferior colliculus. Each point represents one animal (n = 24 per panel). P < 0.05 indicates significant correlation between dietary ARA content and tissue DHA level; NS, not significant. Student’s t-test used to determine significance of pairwise comparisons of mean tissue DHA content; means not sharing a common letter are significantly different (P < 0.05). 82 Supplemental Table 1. FA composition of heart from piglets fed varying levels of ARA and DHA from days 3 - 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Σ SFA 32.81 ± 1.00bc 32.16 ± 0.55c FA (% total FA) 32.63 ± 0.69bc 32.62 ± 0.62bc 32.64 ± 0.61ab 33.36 ± 0.90a 32.60 ± 0.66abc 0.006 Σ MUFA ARA 22:4n-6 22:5n-6 Σ n-6 20:5n-3 22:5n-3 DHA Σ n-3 ARA/DHA ∑ 22C HUFA4 16.78 ± 1.05 17.74 ± 0.99 17.35 ± 1.05 17.50 ± 0.50 17.10 ± 0.88 17.59 ± 0.94 17.17 ± 0.75 14.76 ± 1.07d 18.57 ± 0.67c 19.41 ± 1.00b 20.90 ± 0.93a 19.98 ± 1.38ab 19.09 ± 1.88bc 20.26 ± 1.20ab 0.63 ± 0.09e 0.71 ± 0.09e 0.71 ± 0.06de 0.82 ± 0.09cd 0.88 ± 0.13bc 0.96 ± 0.12b 1.33 ± 0.13a 0.00 ± 0.01b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.02 ± 0.03b 0.08 ± 0.01a 43.29 ± 1.09bc 42.95 ± 0.55c 43.45 ± 0.96bc 43.19 ± 0.77bc 44.12 ± 0.88b 44.06 ± 1.20b 46.85 ± 0.97a 0.75 ± 0.09a 0.54 ± 0.09b 0.49 ± 0.04b 0.41 ± 0.04c 0.42 ± 0.04c 0.32 ± 0.03d 0.28 ± 0.06d 0.59 ± 0.07d 0.59 ± 0.08d 0.57 ± 0.04d 0.57 ± 0.04d 0.71 ± 0.07c 0.83 ± 0.13b 1.24 ± 0.07a 4.81 ± 0.24b 5.20 ± 0.49a 4.78 ± 0.43b 5.01 ± 0.25ab 4.20 ± 0.64c 3.00 ± 0.50d 0.75 ± 0.08e 6.58 ± 0.21ab 6.73 ± 0.57a 6.24 ± 0.42b 6.37 ± 0.31ab 5.71 ± 0.67c 4.57 ± 0.51d 2.48 ± 0.11e 3.07 ± 0.19e 3.60 ± 0.39de 4.08 ± 0.34cd 4.18 ± 0.22cd 4.83 ± 0.58c 6.46 ± 0.78b 27.26 ± 3.17a 6.04 ± 0.25bc 6.50 ± 0.51a 6.06 ± 0.4abc 6.40 ± 0.23ab 5.78 ± 0.72c 4.81 ± 0.59d 3.40 ± 0.15e NS <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:4n-6, 22:5n-6, 22:5n-3, DHA. 83 Supplemental Table 2. FA composition of liver from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P ∑ SFA ∑ MUFA 20:4 (n-6) 22:4 (n-6) 22:5 (n-6) ∑ (n-6) 20:5 (n-3) 22:5 (n-3) 22:6 (n-3) ∑ (n-3) ARA/DHA ∑ 22C HUFA4 FA (% total FA) 42.89 ± 1.78 42.11 ± 0.44 42.88 ± 1.88 42.47 ± 2.23 42.29 ± 0.77 42.71 ± 1.62 39.97 ± 1.50 NS 13.40 ± 1.08b 12.77 ± 1.11b 12.75 ± 0.83b 13.17 ± 0.99b 13.12 ± 0.77b 13.21 ± 0.68b 19.51 ± 3.80a <.0001 13.92 ± 1.49d 16.14 ± 0.87c 16.12 ± 1.10bc 16.88 ± 1.50b 17.36 ± 0.63b 17.87 ± 1.46a 17.00 ± 1.36abc <.0001 1.08 ± 0.29d 1.15 ± 0.22cd 1.27 ± 0.22c 1.36 ± 0.22bc 1.28 ± 0.20bc 1.50 ± 0.10b 2.27 ± 0.35a <.0001 0.03 ± 0.06d 0.06 ± 0.05d 0.04 ± 0.04d 0.06 ± 0.07d 0.20 ± 0.09c 0.37 ± 0.11b 1.89 ± 0.53a <.0001 30.25 ± 1.10d 31.34 ± 0.58cd 31.57 ± 1.01bc 31.70 ± 1.34bc 32.58 ± 0.64b 34.05 ± 1.39a 35.16 ± 1.66a <.0001 0.61 ± 0.14a 0.42 ± 0.15bc 0.45 ± 0.10b 0.36 ± 0.11cd 0.34 ± 0.05cd 0.31 ± 0.04d 0.18 ± 0.06e <.0001 0.94 ± 0.11c 0.91 ± 0.09c 0.94 ± 0.12c 0.94 ± 0.14c 1.01 ± 0.07c 1.11 ± 0.16b 1.35 ± 0.20a <.0001 10.77 ± 1.77a 11.43 ± 1.22a 10.49 ± 1.41a 10.45 ± 1.79a 9.65 ± 1.01b 7.52 ± 0.69c 2.37 ± 0.30d <.0001 12.67 ± 1.79a 13.08 ± 1.15a 12.22 ± 1.57a 12.11 ± 1.90a 11.34 ± 1.10b 9.29 ± 0.76c 4.13 ± 0.49d <.0001 1.31 ± 0.17f 1.43 ± 0.19ef 1.55 ± 0.16de 1.65 ± 0.24cd 1.82 ± 0.21c 2.39 ± 0.20b 7.21 ± 0.42a <.0001 12.82 ± 1.86a 13.55 ± 1.26a 12.74 ± 1.44ab 12.81 ± 1.90ab 12.14 ± 1.21b 10.51 ± 0.81c 7.88 ± 1.22d <.0001 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:4n-6, 22:5n-6, 22:5n-3, DHA. 84 Supplemental Table 3. FA composition of cerebral cortex from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P ∑ SFA ∑ MUFA 22:3n-9 20:4n-6 45.67 ± 0.53ab 45.77 ± 0.81ab 46.10 ± 0.90a 18.55 ± 0.53bc 18.87 ± 0.75bc 18.34 ± 0.67c 0.23 ± 0.06b 0.21 ± 0.03b 0.21 ± 0.02b 11.95 ± 0.29 11.82 ± 0.60 11.80 ± 0.35 FA (% total FA) 46.04 ± 1.10a 45.27 ± 0.74bc 45.93 ± 0.79a 44.14 ± 0.64c 18.27 ± 0.77c 18.70 ± 0.44bc 18.95 ± 0.76b 19.97 ± 0.93a 0.22 ± 0.06b 0.23 ± 0.04b 0.19 ± 0.03b 0.31 ± 0.00a 12.04 ± 0.63 11.93 ± 0.40 11.77 ± 0.68 12.33 ± 0.00 0.005 0.006 0.009 NS 22:4n-6 22:5n-6 ∑ n-6 20:5n-3 22:5n-3 22:6n-3 ∑ n-3 ARA/DHA ∑ 22C HUFA4 4.03 ± 0.52 4.49 ± 0.40c 22.58 ± 0.77d 0.06 ± 0.01b 0.20 ± 0.02c 12.10 ± 0.83a 12.36 ± 0.84a 0.99 ± 0.07c 20.82 ± 0.81 4.06 ± 0.16 4.09 ± 0.35 4.21 ± 0.60 4.21 ± 0.32 4.19 ± 0.26 4.81 ± 0.30 NS 4.44 ± 0.47c 4.68 ± 0.29c 4.61 ± 0.56c 5.52 ± 0.64b 5.49 ± 0.25b 8.26 ± 0.18a <.0001 22.36 ± 1.03d 22.54 ± 0.92d 22.78 ± 1.40cd 23.65 ± 0.85bc 23.56 ± 0.80b 27.62 ± 0.50a <.0001 0.06 ± 0.02b 0.06 ± 0.02b 0.07 ± 0.01b 0.05 ± 0.02b 0.06 ± 0.02b 0.11 ± 0.03a 0.005 0.19 ± 0.02c 0.19 ± 0.04c 0.19 ± 0.03c 0.20 ± 0.02c 0.25 ± 0.05b 0.32 ± 0.03a <.0001 11.91 ± 0.74ab 11.98 ± 0.69ab 11.90 ± 0.69ab 11.30 ± 0.70b 10.56 ± 0.67c 6.88 ± 0.55d <.0001 12.16 ± 0.76ab 12.23 ± 0.71ab 12.16 ± 0.69ab 11.55 ± 0.72b 10.87 ± 0.67c 7.31 ± 0.55d <.0001 1.00 ± 0.09c 0.99 ± 0.05c 1.02 ± 0.10bc 1.06 ± 0.09bc 1.12 ± 0.11b 1.80 ± 0.17a <.0001 20.60 ± 0.61 20.94 ± 1.01 20.91 ± 0.99 21.23 ± 0.88 20.49 ± 0.79 20.27 ± 0.72 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 85 Supplemental Table 4. FA composition of retina from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Σ SFA 44.70 ± 0.83 44.39 ± 0.51 44.89 ± 0.97 FA (% total FA) 44.63 ± 0.91 44.40 ± 1.13 44.57 ± 0.69 43.61 ± 0.32 NS Σ MUFA 18.40 ± 0.59 18.38 ± 0.37 18.48 ± 0.57 18.24 ± 0.65 18.63 ± 0.71 18.72 ± 0.43 19.50 ± 0.70 NS 22:3n-9 20:4n-6 22:4n-6 22:5n-6 Σ n-6 20:5n-3 22:5n-3 22:6n-3 Σ n-3 ARA/DHA ∑ 22C HUFA4 0.10 ± 0.03 0.11 ± 0.02 0.10 ± 0.02 0.10 ± 0.10 0.13 ± 0.02 0.11 ± 0.03 0.12 ± 0.02 NS 9.48 ± 0.23c 9.70 ± 0.61bc 9.66 ± 0.32bc 9.81 ± 0.31b 9.96 ± 0.37b 9.95 ± 0.25b 10.74 ± 0.50a 0.0004 1.66 ± 0.15e 1.72 ± 0.12de 1.77 ± 0.12d 1.81 ± 0.17d 1.98 ± 0.19c 2.13 ± 0.12b 2.98 ± 0.17a <.0001 1.48 ± 0.30d 1.48 ± 0.32d 1.45 ± 0.16d 1.55 ± 0.30d 2.03 ± 0.64c 2.29 ± 0.26b 5.24 ± 0.39a <.0001 15.31 ± 0.54d 15.42 ± 0.89d 15.41 ± 0.47d 15.57 ± 0.70d 16.37 ± 0.91c 16.98 ± 0.24b 21.65 ± 0.96a <.0001 0.18 ± 0.05ab 0.15 ± 0.04b 0.18 ± 0.05a 0.13 ± 0.02c 0.10 ± 0.04c 0.08 ± 0.02d 0.02 ± 0.01e <.0001 0.64 ± 0.04a 0.60 ± 0.05b 0.63 ± 0.06ab 0.60 ± 0.03b 0.55 ± 0.03c 0.60 ± 0.05b 0.60 ± 0.05abc 0.0001 20.42 ± 1.20ab 20.70 ± 0.54a 20.07 ± 1.47ab 20.49 ± 1.31ab 19.50 ± 1.50bc 18.68 ± 0.96c 14.16 ± 1.37d <.0001 21.24 ± 1.22ab 21.44 ± 0.57a 20.89 ± 1.48ab 21.22 ± 1.34ab 20.15 ± 1.55bc 19.36 ± 0.99c 14.78 ± 1.36d <.0001 0.47 ± 0.03c 0.47 ± 0.04c 0.48 ± 0.04c 0.48 ± 0.04bc 0.51 ± 0.04bc 0.53 ± 0.03b 0.77 ± 0.11a <.0001 24.29 ± 1.34 24.61 ± 0.49 24.03 ± 1.49 24.55 ± 1.48 24.20 ± 1.31 23.81 ± 0.90 23.10 ± 0.98 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 86 Supplemental Table 5. FA composition of cerebellum from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P Σ SFA 41.83 ± 1.91 42.29 ± 2.00 42.72 ± 1.43 FA (% total FA) 42.12 ± 1.93 42.41 ± 1.66 42.40 ± 2.31 41.57 ± 0.85 NS Σ MUFA 22:3n-9 20:4n-6 22:4n-6 22:5n-6 Σ n-6 27.22 ± 0.99 26.93 ± 1.50 25.95 ± 1.04 27.07 ± 1.30 26.35 ± 1.60 25.98 ± 2.60 27.20 ± 1.43 NS 0.42 ± 0.07abc 0.38 ± 0.05c 0.37 ± 0.04c 0.37 ± 0.04c 0.44 ± 0.04ab 0.38 ± 0.07bc 0.48 ± 0.06a 0.009 10.40 ± 0.30c 10.76 ± 0.26bc 11.03 ± 0.31ab 10.44 ± 0.44c 10.95 ± 0.62ab 10.78 ± 0.43bc 11.48 ± 0.73a 0.007 3.29 ± 0.41d 3.23 ± 0.38d 3.39 ± 0.32cd 3.43 ± 0.32cd 3.55 ± 0.19c 3.97 ± 0.25b 4.66 ± 0.31a <.0001 2.12 ± 0.26cd 2.19 ± 0.34d 2.18 ± 0.19cd 2.17 ± 0.26d 2.70 ± 0.37bc 3.34 ± 1.09b 4.82 ± 0.23a <.0001 17.98 ± 1.09d 18.15 ± 1.17d 18.47 ± 0.84cd 18.07 ± 0.64d 19.22 ± 0.65c 20.27 ± 1.12b 23.26 ± 0.92a <.0001 20:5n-3 22:5n-3 22:6n-3 Σ n-3 ARA/DHA ∑ 22C HUFA4 0.21 ± 0.03 0.35 ± 0.06bc 11.29 ± 1.61a 11.85 ± 1.67a 0.91 ± 0.09d 17.47 ± 1.79 0.22 ± 0.04 0.20 ± 0.05 0.22 ± 0.05 0.20 ± 0.06 0.20 ± 0.07 0.31 ± 0.05cd 0.32 ± 0.05cd 0.32 ± 0.05d 0.31 ± 0.02cd 0.36 ± 0.05b 11.08 ± 1.68a 11.29 ± 1.19a 11.19 ± 1.44a 10.37 ± 0.75ab 9.83 ± 0.76b 11.61 ± 1.71a 11.82 ± 1.21a 11.73 ± 1.47a 10.88 ± 0.79ab 10.39 ± 0.81b 0.96 ± 0.11d 0.97 ± 0.07cd 0.95 ± 0.11cd 1.06 ± 0.12bc 1.10 ± 0.09b 17.19 ± 2.23 17.56 ± 1.51 17.47 ± 1.54 17.36 ± 0.70 17.88 ± 1.37 0.21 ± 0.05 NS 0.47 ± 0.04a <.0001 6.10 ± 0.28c <.0001 6.77 ± 0.28c <.0001 1.89 ± 0.20a <.0001 16.53 ± 0.30 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 87 Supplemental Table 6. FA composition of hippocampus from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P ∑ SFA 41.80 ± 1.32abc 42.33 ± 1.12ab 41.48 ± 1.62c FA (% total FA) 41.13 ± 1.76bc 42.51 ± 0.94ab 41.53 ± 1.34bc 43.67 ± 0.520a 0.046 ∑ MUFA 26.65 ± 3.10 25.62 ± 3.24 27.02 ± 4.01 27.44 ± 5.57 24.75 ± 2.42 26.92 ± 3.89 23.67 ± 1.48 NS 22:3n-9 20:4n-6 0.48 ± 0.08 10.40 ± 0.89 0.46 ± 0.12 10.78 ± 0.93 0.49 ± 0.12 10.57 ± 1.09 0.45 ± 0.09 10.61 ± 1.57 0.46 ± 0.10 11.10 ± 0.97 0.52 ± 0.15 10.85 ± 1.23 0.47 ± 0.12 11.77 ± 0.96 NS NS 22:4n-6 22:5n-6 ∑ n-6 4.57 ± 0.36 2.89 ± 0.22c 20.27 ± 1.21c 4.68 ± 0.43 4.63 ± 0.57 4.72 ± 0.63 4.78 ± 0.44 2.96 ± 0.39c 3.06 ± 0.39cd 2.68 ± 0.67d 3.71 ± 0.40b 20.62 ± 1.26bc 20.46 ± 1.85c 20.09 ± 2.66bc 21.64 ± 1.40b 5.03 ± 0.51 5.37 ± 0.43 3.31 ± 0.53bc 5.58 ± 0.60a 21.47 ± 1.94b 24.83 ± 0.99a NS <.0001 0.0001 20:5n-3 22:5n-3 22:6n-3 ∑ n-3 ARA/DHA ∑ 22C HUFA4 0.30 ± 0.14 0.24 ± 0.03ab 9.89 ± 0.26a 9.74 ± 1.15ab 1.10 ± 0.09c 17.20 ± 1.41 0.26 ± 0.11 0.32 ± 0.15 0.32 ± 0.21 0.25 ± 0.10 0.29 ± 0.11 0.21 ± 0.05 0.22 ± 0.05bc 0.22 ± 0.02bc 0.20 ± 0.05bc 0.19 ± 0.02c 0.24 ± 0.04ab 0.27 ± 0.04a 9.74 ± 1.43a 9.66 ± 0.68ab 9.63 ± 1.73a 9.44 ± 1.03a 8.27 ± 1.48b 6.12 ± 0.68c 9.96 ± 1.45ab 9.45 ± 1.36bc 9.83 ± 1.78a 9.64 ± 1.05ab 8.50 ± 1.50c 6.39 ± 0.70d 1.12 ± 0.13c 1.16 ± 0.18c 1.11 ± 0.09c 1.18 ± 0.07c 1.33 ± 0.14b 1.93 ± 0.18a 17.60 ± 1.87 17.13 ± 1.99 17.23 ± 2.89 18.12 ± 1.44 16.84 ± 2.23 17.34 ± 0.64 NS 0.009 0.0002 <.0001 <.0001 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 88 Supplemental Table 7. FA composition of globus pallidus from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P ∑ SFA 42.71 ± 1.41 41.00 ± 2.51 42.24 ± 1.05 FA (% total FA) 41.98 ± 1.42 41.56 ± 1.54 41.65 ± 2.00 42.67 ± 1.93 NS ∑ MUFA 27.27 ± 2.77 31.99 ± 6.70 28.36 ± 2.42 29.79 ± 3.98 30.05 ± 4.63 29.86 ± 4.61 26.74 ± 3.75 NS 22:3n-9 20:4n-6 22:4n-6 22:5n-6 ∑ n-6 0.42 ± 0.08 0.47 ± 0.11 10.33 ± 0.68 9.33 ± 1.28 3.81 ± 0.32bc 3.59 ± 0.73b 2.93 ± 0.37b 2.24 ± 0.77c 18.93 ± 1.02b 17.00 ± 2.41c 0.47 ± 0.06 0.50 ± 0.10 10.07 ± 0.44 9.92 ± 0.76 4.25 ± 0.51ab 3.98 ± 0.87bc 2.81 ± 0.40bc 2.64 ± 0.58bc 18.82 ± 1.15bc 18.18 ± 1.93bc 0.50 ± 0.09 0.48 ± 0.11 0.50 ± 0.09 NS 10.02 ± 0.88 10.09 ± 0.96 10.73 ± 0.82 NS 3.72 ± 0.37bc 4.04 ± 0.58bc 4.78 ± 0.65a 0.02 3.16 ± 0.73b 3.04 ± 0.78b 5.40 ± 0.92a <.0001 18.59 ± 1.65bc 18.99 ± 1.88b 22.72 ± 2.22a 0.0002 20:5n-3 0.35 ± 0.11 0.54 ± 0.31 0.39 ± 0.15 0.45 ± 0.17 0.48 ± 0.20 0.44 ± 0.19 0.33 ± 0.15 NS 22:5n-3 22:6n-3 ∑ n-3 ARA/DHA ∑ 22C HUFA4 0.15 ± 0.12 9.45 ± 0.97a 9.96 ± 0.89a 1.10 ± 0.07d 16.74 ± 1.12 0.12 ± 0.04 0.13 ± 0.03 0.13 ± 0.02 0.11 ± 0.04 0.14 ± 0.06 0.19 ± 0.06 NS 8.09 ± 2.61ab 8.80 ± 0.94ab 8.18 ± 1.36ab 7.91 ± 1.95b 7.66 ± 1.68bc 6.08 ± 0.35c 0.01 8.75 ± 2.34ab 9.33 ± 0.88ab 8.76 ± 1.23ab 8.50 ± 1.79b 8.25 ± 1.57b 6.60 ± 0.29c 0.005 1.24 ± 0.30bcd 1.15 ± 0.11cd 1.23 ± 0.15bcd 1.32 ± 0.26bc 1.37 ± 0.30b 1.77 ± 0.15a <.0001 14.49 ± 3.72 16.44 ± 1.37 15.41 ± 2.20 15.37 ± 2.68 15.34 ± 2.50 16.92 ± 1.43 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 89 Supplemental Table 8. FA composition of inferior colliculus from piglets fed varying levels of ARA and DHA from days 3 – 28 of age1 Diet a1 a2 a3-d3 a4 d2 d1 MR2 ARA/DHA3 0.09/1.00 0.53/1.02 0.69/1.01 1.06/1.04 0.67/0.62 0.66/0.33 0.74/0.01 P ∑ SFA 40.88 ± 1.24 41.85 ± 1.15 41.59 ± 1.62 FA (% total FA) 40.66 ± 1.38 41.45 ± 1.16 41.52 ± 1.28 40.94 ± 1.87 NS ∑ MUFA 22:3n-9 20:4n-6 22:4n-6 22:5n-6 ∑ n-6 30.94 ± 2.12 30.92 ± 3.15 0.33 ± 0.04bc 0.30 ± 0.04c 8.58 ± 0.35 8.55 ± 0.75 3.54 ± 0.21b 3.36 ± 0.19b 1.58 ± 0.49bc 1.50 ± 0.35c 15.97 ± 0.56b 15.53 ± 1.04b 30.08 ± 3.70 32.32 ± 2.74 30.81 ± 1.93 29.83 ± 2.49 0.29 ± 0.06c 0.37 ± 0.07b 0.32 ± 0.07bc 0.29 ± 0.04c 8.85 ± 0.84 8.61 ± 0.52 8.63 ± 0.50 8.97 ± 0.73 3.55 ± 0.31b 3.56 ± 0.29b 3.42 ± 0.30b 3.70 ± 0.35b 1.60 ± 0.45bc 1.62 ± 0.52bc 1.93 ± 0.53b 1.91 ± 0.32b 16.12 ± 1.24b 15.86 ± 0.93b 16.14 ± 1.03b 16.77 ± 1.13b 31.02 ± 4.44 NS 0.48 ± 0.10a <.0001 9.45 ± 1.25 NS 4.68 ± 1.16a <.0001 3.76 ± 0.45a <.0001 19.93 ± 2.37a <.0001 20:5n-3 22:5n-3 22:6n-3 ∑ n-3 ARA/DHA ∑ 22C HUFA4 0.39 ± 0.07 0.35 ± 0.12 0.34 ± 0.11 0.43 ± 0.09 0.19 ± 0.02ab 0.18 ± 0.04abc 0.19 ± 0.04ab 0.15 ± 0.02c 10.71 ± 0.94a 10.32 ± 1.88a 10.84 ± 1.50a 9.58 ± 1.21a 11.29 ± 0.92a 10.85 ± 1.83a 11.37 ± 1.46a 10.17 ± 1.15a 0.81 ± 0.06b 0.84 ± 0.11b 0.82 ± 0.08b 0.91 ± 0.10b 16.35 ± 1.10 15.66 ± 2.18 16.46 ± 2.03 15.28 ± 1.60 0.36 ± 0.05 0.33 ± 0.07 0.39 ± 0.14 NS 0.16 ± 0.03bc 0.18 ± 0.03bc 0.21 ± 0.06a 0.02 10.18 ± 1.27a 10.53 ± 0.85a 6.27 ± 1.14b <.0001 10.70 ± 1.25a 11.04 ± 0.82a 6.87 ± 1.07b <.0001 0.86 ± 0.09b 0.85 ± 0.06b 1.55 ± 0.32a 0.0001 16.02 ± 1.46 16.61 ± 1.23 15.40 ± 1.74 NS 1Values represent mean ± SD, n = 8 per diet. P < 0.05 indicates that all means are not equal; NS, not significant. Pairwise comparisons determined using Student’s t-test; means not sharing a common superscript are significantly different (P < 0.05). 2ARA/DHA, dietary content of ARA/DHA (% FA/FA). 3SFA, saturated FA, MUFA, monounsaturated FA. 4∑ 22C HUFA = ∑ 22:3n-9, 22:4n-6, 22:5n-6, 22:5n-3, DHA. 90 APPENDIX II ONE-STEP HYDROLYSIS, EXTRACTION AND METHYLATION PROCEDURE FOR THE PREPARATION OF FATTY ACID METHYL ESTERS FROM VERTEBRATE SOFT TISSUE This procedure is adapted from the One-step procedure of Garces and Mancha (1993). Reagents: Aqueous Reagent: Methanol 2,2-Dimethoxypropane Concentrated sulfuric acid (85:11:4 by volume) Organic Reagent: Heptane Toluene (63:37 by volume) Heptane Saturated sodium chloride Di-17:0 phosphatidylcholine, 5.65 ug/ul concentration for internal standard 91 Protocol: 1. Weigh out sample (~ 50mg)1 in a 16 x 125 mm test tube. 2. Add internal standard, total amount based on sample type. 3. Add 2 mL heptane. 4. Add 1.4 mL of the aqueous reagent and 1.6 mL of the organic reagent with a glass pipette. 5. Seal cap inside and out with Teflon tape and vortex for 1 min. 6. Place tubes in a heating block at 80°C for two hours. 7. Vortex tubes until they come to room temperature (~10 min). 8. Add 2 mL of saturated sodium chloride and vortex. 9. Centrifuge for 10 min at 3500 rpm. 10. Transfer top layer to a clean test tube (to be dried down). 11. Add 2 mL of heptane to sample tube. 12. Vortex for 1 min followed by centrifugation for 10 min at 3500 rpm. 92 13. Transfer top layer to the clean tube (to be dried down). 14. Evaporate fatty acid methyl esters (FAME) + heptane (in clean tube) under nitrogen. 15. Resolubilize FAME in 200 μL helptane. 16. Transfer FAME to GC vial for storage. 150 mg of tissue is preferred weight. Alternatively, up to 50 μL of aqueous sample (e.g. plasma, milk, red blood cells) may be used. Samples suspended in a greater volume of aqueous solution must first be dried using a Speed Vac concentrator. 93