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1 EFFECTS OF MEGALACR SUPPLEMENTATION ON MEASURES OF INFLAMMATION AND PERFORMANCE IN TRANSPORT STRESSED BEEF CALVES By DAVI BRITO DE ARAUJO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009
2 2009 Davi Brito de Araujo
3 To my mother Arlete, my father Dudu and my sister Mazinha Saudade existe para quem s abe ter...
4 ACKNOWLEDGMENTS I am deeply indebted to Dr. John D. Arthington, my supervisory committee chair. Dr. Arthington provided endless encouragement, opportunities, guidance, and financial support. I am proud of the chance that I had to work with him and I will always consider D r. Arthington as an excellent mentor and great friend. I appreciate his patience during my first steps trying to learn a new language and to live in a new country. Dr Arthingtons character, professionalism and attitudes are the best lessons I have learned to build my future. I thank all members of my committee who provided important support throughout my M.S. progr am. Dr. Matt Hersom gave me the opportunity to work with his group in two research projects which became essential to my professional developme nt. I also like to show my appreciation for Dr. Charles Staples for guidance and help during research trials in his laboratory. In addition, I would like to express appreciation to Dr. Bill Brown for his assistance during my first weeks in Gainesville, and his dedication for teaching the importance of the principles of the ruminant nutrition. I am very fortunate to have had Dr. Lynn Sollenberger, Dr. Joel Brendemuhl, Prof. Ashlee Mantooth, Dr. Adesogan Adegbola, Dr. Joel Yelich, Dr. William Thatcher, Dr. T im Olson, Dr. Goeffrey Dahl, Dr. Michael Fields, Dr. Dan Sharp and Dr. Cliff Lamb as professors. They have made a huge contribution on the improvement of my knowledge, criticism, and professionalism during my 2 years at the U. F. Special thanks go to Dr. G arry Hansen and all his family who treated me as a son during the 4 weeks I spent in Marianna, FL, and to Dr. Chad Chase Jr. for his assistance during my first experiment at the IFAS USDA STARS, Brooksville, FL. I extend my appreciation to Dr. Peter Ha ns en who more than a professor, became also a mentor and friend. Dr. Hansen is a great man, father and an excellent scientist.
5 Science is so much easy and pleasant with him. Thanks also to Dr. Richard Miles for his guidance during seminars, friendship, and jokes. I extend my utmost sincere appreciation to my parents who never stopped supporting me in any decision that I made in life. Their unconditional love, moral presence and courage have taught me to never quit, and always try to be happy. They always encouraged my sister and I to look towards the future and experience life. I thank them for always be ing there for me. I also would like to thank my beloved grandmother Eivany V Ni for her endless love and influence during my childhood on choosing the p rofession that I work in today. I am very appreciated for all company, friendship and moments which I have shared with my little sister Marlia. My sister was essential to make my life beautiful durin g the four months that she spent with me here in USA. I am very grateful to my girlfriend Mariele Pansani. She was very im portant in help ing me during the conclusion of this thesis. Her loveliness, serenity, amicability, attitude and intelligence have always encouraged and inspired me. After I met Mariele I hav e learned that even the most difficult things are not impossible to happen. Mariele has been a truly present in my life, because the distance between us is just a geographical detail. Thanks to all my relative s that have supported me in any way to fini sh this step in my journey. I really appreciate my grandparents Edmar V Mar and Maria Stela V Tela to be the roots of my background. I want to thank my aunts and uncles Lica, Eliane, Tet, Carla, D ema, Robertana and, specially Ulisses in memoriam, Sebastio, Tuti, Haid in memoriam, and Eliete who contributed to build my cowboy personality. Thanks also to my cousins Linho, Lucas Sequela, Ju Gorda, Z Kusseco Danilo, Daniel Tropeo, Fael, Gus, Vi, Thiago, Diego, Fer, Beco and Nana who ne ver stopped believing in our selves even when we are so far
6 from each other. Thanks to my godfather Edmundo Tito and his wife Paula, as well as my godmother Mayara Brait for all support and phone calls. Special thanks go to my physician and cousin Rodr igo Hoffmann and his wife Kelly who I have shared great moments in Miami and Orlando, and on telephone. I also want to show my appreciation to Dona Jlia, Cidinha and Genilda for the good food, hard work and friendship. I am very grateful to my friends an d staffs from the IFAS Range Cattle R. E. C., specially Andrea Dunlap and Tony Woods who were my every time help and contributed to improve my English skills In the station, I had the opportunity to meet true friendship and had a g reat work environment. Thanks to Mr. Alvin English, my buddies Austin Bateman and Joe Crazy Aldana, Mr. Terry Neels, Mr. Jeffery Steele, Mr. Dennis Kalich, Clay Newman, Joshua Sosa, Walt Beattie, Carly Althoff, Ms. Cindy Holley, Mrs. Kim Parks, Dr. Brent Sellers Ms. Shirley Searcy, Dr. Qiu and Mrs. Christina Markham, and Paulo Gustavo. I would like to thank my great friends Joo Joe Vendramini and Maria Lcia Mal Silveira for every conversation, advice, and the moments i n their house where I always fe l t like home. Spec ial thanks go to Reinaldo Anabol and Fl via Fuinha Cooke for all their help and friendship. Reinaldo was a tremendous contributor in my research. Thanks go to Harvey Standland, David Thomas, Todd Mathews in memoriam, and Don Jones for all their help during my research at the IFAS N. F. R. E. C., Marianna, FL. Special thanks also to Butch for his friendship, support and to make the work easier at the station. I would also like to thank my friend Herb and the Limestone Country Club for quenching me when it was necessary. I thank my friend and mentor Dr. Jos Z Eduardo Portela Santos as well as his family Christiane, Dudu and Laura for all care, amity and guidance. Z was responsib le for teach ing me
7 how to be a veterinarian, during my internship at the V. M. T. R. C. U. C. Davis in 2005. Today Z is teaching me how to be professional scientist. Special thanks go to Fbio Muzamba Lima a priceless friend, Helosa Xup Rut i gliano, Ralph Bruno and Daniele Resende for all assistance and help durin g my days in Tulare, CA. We had very great time when we were roommates in that small house. Very special thanks to my great friends Ronaldo Drac Mini ni nhoPreto Cerri and his wife Mayra Cruppe for all every time help, guidance, and friendship during the last 9 years. Ronaldo is important on contributing to build my character and professionalism. Heartfelt thanks go to my first professor and first advisor Jos Zequinha Luiz Moraes de Vasconcelos and his family Carla, Gabriel and Rafaela for all encourag ement, patience and support during my 5 years of Vet School. Zequinha open the U. S. doors for me to start my M. S. program. Zequinha is much more than a professor; he is also an estimated father, husband, friend, mentor and leader. Special appreciation go es to Ricarda Maria dos Santos who really showed me for the first time how good research really is made. Her friendship is precious. Thanks also to Gabriela Penada Perez Edmundo Pigarro Vilela and Nelson Perna Ferreira Jr. for all friendship, belief and guidance during my years of undergraduate. Thanks to the CONAPEC Jr. group where my dream started. Thanks to my friend Jackie Warhmund who gave me the opportunity to help on her research trial, Dr. Sam Kim for his valuable f riendship and for teaching me all the fatty acid analysis, and Serguei Sennikov and Joyce Hayen for their help during part of my laboratorial analysis. I thank Mrs. Glenda Tucker and Mrs. Joann Fisher for all the help I have asked them S pecial thanks go t o Mrs. Cora Webb for always taking car e of our department and for her kindness and every day smile.
8 Thanks to all the friends I made at the Department of Animal Science and in the U. S. that direct or indirectly helped in research or my s tay in Gainesvil le: Nick Lavieri and Luciano Silva very estimated buddies; my officemates Oscar Ataq Queiroz, Eduardo Cabrn Alava, Jamie Foster, Regina Esterman, Adriane Bell, Megan Thomas, Erin McKinniss, Ashley Hughes, Katie Arriola, and Aline Monari; I want to tha nk my friends Linconl Zotarelli and Vanessa Silva, Fr ank Z Lissoni, Tnia Broisler and their kids Joo e Antnio, Jeremy Block, Cristina Caldari Torres, Tara Felix, J. G. Vickers, Andr Pedroso and his family, Sylvia Morais, Alain Pozzo and Leanne Jepson, Franklin and Lidiane Behlau, Kathleen Pennington, Dillon Walker, Milerky Perdomo, Drew Cotton, Qien Yang, Jim Moss and his family, Rosana Seri kawa, Bruno Baiano and Sylvia Pedreira, Flvio Avila, Juliano and Juliana Laran, Beln Rabaglino, Carlos Can as, Brbara Loureiro, Luciano and Aline Bonila, Maria Pdua, Dr Alosio Bueno, Tibrio Saraiva and his family Robson Giglio, Marcelo Tamassia and Agda Barreirinhas, Cssio Ferrigno and Vanessa Castro, Miguel Castillo, Alex Oliveira, Beatriz Bernardo, Au g usto Guto e Camila Brito, Bry an Thompson and Rafael Bisinotto. Thanks to my French roommates Jeremy J Campfield and Christopher Chris Catalouppe. I really have good time living with those guys. I also want to thank my friends Lilian Oliveira and Izabella Thompson for helping me correcting and formatting this chapter. I want to thank my buddies Gabriel Soligo, T iago Ruiz and Gustavo Gu Mazon for all the great times in Jacksonville during the spring of 2008, mainly to Gu for all support, fellows hip and parties at the time we were roommates. Special thanks to my friends Bruno Caboclo do Amaral and Flvio Silvestre who contributed a lot with their researc h and opinions to the success and fulfillment of my thesis. In addition, I thank my classmate s Matthew Kemp, Heather Welsh,
9 Michelle Texier, Seth Jenkins, Karen Chandler, Erin Mullen and Kyle Fisk for making our classes so more enjoyable. I would like to expresses my appreciation to my good and old friends from my BNZ fraternity for always givin g me the pure friendship: Dona Teresa for everything and for every day, Fausto Tilango and his wife Thamara, Mrio Brakiara, Mauro Fudncio, Pedro Henrique K bo, Tiago Beudo, Ivan Cirola, Marcel Braxola, Vincius Zorba, Vagner K t to, Guilherme Pit Bull, Gilherme Japa, Pedro Torresmo, Rafael Corin, Marcelo Fofo, Natal Paraquat, Toms Panada, Eduador Dorrego, Ruy Genny and Gabriel Spantaio. Thanks to Aloysio Guaxo, Rafel Spaia and Tiago Tocinho for the help dur ing my experiments in Ona. I thank my best buddies Pietro Peta Micheri and Luciano Mazon for always being present even when absent. I would like to thank my sweet friends Leane K peta Oliveira, Juliana Vareta Pescara and Milena Inj Blanes for being in the same fight that I am. I would like to thank my good and old friend Guilherme Trxa Marquezini and his wife Maria Fernanda P a r do for their friendship and provision attended me. Thanks also to my unforgettable friend Tiziu in memoriam. L ast, but not least, I thank God for always bless ing and help ing me in any decision of my life.
10 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................12 LIST OF FIGURES .......................................................................................................................13 LIST OF ABBREVIATIONS ........................................................................................................14 ABSTRACT ...................................................................................................................................17 CHAPTER 1 INTRODUCTION ..................................................................................................................19 2 LITERATURE REVIEW .......................................................................................................21 Lipids ......................................................................................................................................21 Definition of Dietary Lipids ............................................................................................21 Supplemental Fat Intake and Digestion ...........................................................................22 Fatty Acid Metabolism in the Rumen .............................................................................25 Fatty Acids Metabolism in Tissues .................................................................................26 Immune Function ....................................................................................................................29 The Acute Phase Reaction ..............................................................................................30 Physiological Stress and Growth .....................................................................................32 Supplemental Fat, Stress and Growth .............................................................................36 3 EFFECTS OF MEGALACR INCLUSION IN RECEIVING DIETS OF WEANED FEEDER STEERS ..................................................................................................................41 Materials and Methods ...........................................................................................................41 Animals and Facilities .....................................................................................................41 Diets .................................................................................................................................42 Sampling ..........................................................................................................................43 Blood analysis .................................................................................................................44 Statistical analysis ...........................................................................................................45 Results .....................................................................................................................................46 Measurements of Performance ........................................................................................46 Plasma Measurements and Coefficients of Correlation ..................................................47 Discussion ...............................................................................................................................48 Performance .....................................................................................................................48 Inflammatory Reaction ....................................................................................................50
11 4 EFFECTS OF MEGALACR SUPPLEMENTATION ON MEASURES OF PERFORMANCE AND ACUTE PHASE REACTION IN TRASPORTED BEEF HEIFERS ................................................................................................................................63 Material and Methods .............................................................................................................63 Animals and Facilities .....................................................................................................63 Diets .................................................................................................................................64 Sampling ..........................................................................................................................64 Blood Analysis ................................................................................................................65 Statistical Analys is ..........................................................................................................66 Results .....................................................................................................................................68 Measurements of Performance ........................................................................................68 Plasma Measurements and Coefficients of Correlation ..................................................68 Discussion ...............................................................................................................................69 Performance .....................................................................................................................69 Inflammatory Reaction ....................................................................................................69 5 GENERAL CONCLUSION ...................................................................................................83 LIST OF REFERENCES ...............................................................................................................84 BIOGRAPHICAL S KETCH .........................................................................................................96
12 LIST OF TABLES Table page 31 Ingredients and nutrient composition of grain based concentrate treatments fed to steers during pre and post shipping phase of the study. 1 .................................................54 32 Fatty acid profile of su pplemental fat sources used in the formulation of experimental diets. .............................................................................................................55 33 Nutrient composition of TMR fed to transport stressed steers during the post shipping phase of the study. ...............................................................................................56 34 Effect of supplemental fat source on plasma fatty acid concentrations on d 0 and d 29 of the study. ........................................................................................................................57 35 Correlations between plasma measurements, DMI and ADG of transport stressed steers duri ng post shipping phase of the study .................................................................59 41 Ingredient and nutrient composition of grainbased supplements fed heifers during the pre and post shipping phases of the study. .................................................................75 42 Fatty acid profile of supplemental fat source used in the formulation of experimenta l MG supplement ..................................................................................................................76 43 Nutrient composition of min eral and vitamin mix supplement .........................................77 44 Correlations between plasma measurements, DMI and ADG of transport stressed heifers during post shipping phase of the study. ...............................................................78
13 LIST OF FIGURES Figure page 31 Least squares means of DMI of steers during the post shipping phase of the study. ........60 32 Least squares means of covariately adjusted plasma fibrinogen concentrations of steers during the post shipping phase of the study.. ..........................................................61 33 Least squares means of plasma ceruloplasmin concentrations of steers during the post shipping phase of the study. .......................................................................................62 41 Least squares means for DMI of pen fed heifers during the post shipping phase of the study. ............................................................................................................................79 42 Post shipping concentrations of plasma ceruloplasmin of heifers fed grainbased supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping ..............................................................................................80 43 Post shipping concentrations of plasma cortisol of heifers fed grain based supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. .............................................................................................81 44 Plasma concentrations of haptoglobin of heifers fed grain based supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. ....................................................................................................................82
14 LIST OF ABBREVIATIONS ADG average daily gain APP acutephase proteins ARA arachidonic acid BH biohydrogenation BW body weight CCK cholecystokinin CLA conjugated linoleic acids CNS central nervous system CO control COX cyclo oxygenases Cp ceruloplasmin CP crude protein CSFA calcium salts of fatty acids CV coefficient of variation DGLA dihomolinolenic acid DHA docosahexaenoic acid DMI dry matter intake DPA docosapentaenoic acid EN Energy Booster 100 EPA eicosapentaenoic acid ETA eicosatetraenoic acid FA fatty acids Fb fibrinogen FL Florida
15 G:F gain to feed ratio GLNA linolenic acid HETE hydroxyl eicosatetraenoic acid HPETE hydroperoxy eicosatetraenoic acid Hp haptoglobin ID identification IGF 1 insulin like growth factor 1 IL 1 interleukin 1 IL 6 interleukin 6 LA linoleic acid LNA linolenic acid LPS lypopolysaccharide LSD least significant difference LT leukotrienes MG MegalacR MUFA monounsaturated fatty acids NDF neutral detergent fiber OM organic matter PG prostaglandins PUFA polyunsaturated fatty acids SEM standard error of the mean SFA saturated fatty acids SD standard deviation TDN total digestible nutrients TNF tumoral necrosis factor
16 TMR total mix ratio TX thromboxanes UFA unsaturated fatty acids VFA volatile fatty acids
17 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF MEGALACR SUPPLEMENTATION ON MEASURES OF INFLAMMATION AND PERFORMANCE IN TRANSPORT STRESSED BEEF CALVES By Davi Brito de Araujo August 2009 Chair: John David Arthington Major: Animal Sciences Two studies were conducted to evaluate measures of performance and inflammation in transport stressed beef calves supplemented with saturated or unsaturated fatty acids. In the first study, prior to transport (d 40 to 0), 64 weaned, Braford steers were stratified by initial BW and age, and randomly allocated to 2 pastures. Each pasture was randomly assigned to receive 1 of 2 treatments, which consis ted of grain based supplements with (EN) or without (CO) the inclusion of a prilled saturated fat source (Energy Booster 100). On d 0, steers were loaded onto a commercial trailer and transported for approximately 1,600 km over a 24 h period and delivered to a feedlot. Upon arrival (d 1), steers were stratified by pre shipping treatment and randomly re assigned in to receive EN, CO, or MG (grain based supplement containing MegalacR). Shrunk BW was recorded on d 40, 0 and 30 to determine ADG. Individual DMI was recorded daily during the post shipping period using the GrowSafe system (Model 4000E). Blood samples were collected on d 0, 1, 4, 8, 15, 22 and 29 for determination of fibrinogen and ceruloplasmin concentrations. No pre treatment effects or prex post shipping treatment interactions were observed. During the post shipping phase, steers fed MG had decreased (P < 0.05) ADG and lower mean DMI (P < 0.01) compared to CO fed steers (0.80 and 1.04 kg/d, and 2.37 and 2.80% of BW respectively). Steers f ed MG had poorer G:F (P < 0.05) compared to EN steers, and
18 tended (P = 0.10) to have decreased G:F compared to CO fed steers (0.37, 0.35 and 0.29 mean G:F for EN, CO and MG steers, respectively). In the second study, prior to shipping (d 30 to 0), 48 Bra hman crossbred heifers were stratified by initial BW and randomly allocated to 6 pastures. Each pasture was randomly assigned to receive 1 of 2 daily supplement treatments, consisting of a grainbased supplement, with (MG) or without (CO) the inclusion of MegalacR. On d 0, heifers were transported for approximately 1,600 km over a 24 hour period. Upon arrival (d 1), 24 of the 48 heifers were stratified by BW and assigned to individual feedlot pens. Pre shipping treatment allocation continued in the post s hipping phase. Shrunk BW was recorded on d 30, 1 and 28 to determine ADG. Individual voluntary hay intake was recorded daily from d 1 to 28. Blood samples were collected on d 0, 1, 4, 8, 15, 22 and 28 were used to determine plasma concentrations of cerulo plasmin, haptoglobin, and cortisol. A treatment x time interaction was detected for haptoglobin (P < 0.01) because MG fed heifers had decreased (P < 0.05) haptoglobin concentrations on d 1, 3 and 5, relative to transport, compared with CO fed heifers. The se data imply that that MegalacR supplementation appears to negatively affect performance of transport stressed beef calves ; decreasing ADG, DMI and G:F. In addition, the acutephase reaction following transport appears to be modulated when MegalacR is supplemented to beef calves at least 30 d prior shipping.
19 CHAPTER 1 INTRODUCTION In 2007, the beef cow herd of Florida ( FL ) was composed of 936,000 head and 68% of this herd was located in the Southern half of the state. The calves marketed from FL in 2007 totaled 721,000 head, over 80% of the calf crop (USDA NASS, 2008). Nationally, FL ranked 12th in beef cows and 18th in total cattle, and its beef industry consists basically of cow calf enterprises with high British Brahman crossbred genetic influence and grazed pasture as the major source of nutrition. Florida is the leading state in the United States for the number of large cow/calf operations (> 2,500 cows; USDA, 2002), with no commercial feedlot industry in FL, nearly all market steers are weaned and shipped outside of the state for further growing and finishing (Arthington et al., 2008). For example, Texas is the state which receives the majority of FL market calves, and it is located approximately 2,400 km to the west. This isolation results in important impacts on subsequent animal health and performance, caused mainly by stressors associated with weaning, weather changes and transportation. In the U.S., the calf morbidity and mortality associated with respiratory disease and shipping fever comple x is estimated to cost approximately $500 million to the beef industry (NASS, 1996). Many factors contribute to the cost of these diseases, such as pharmaceutical purchase, feed resources lost, increased labor, death and poor performance of animals (Loerch & Fluharty, 1999). Marketing processes at feedlot arrival are crucial events causing a considerable amount of stress for cattle. Most of the health problems with newly arrived calves occur within the first 2 weeks due to the impact of stress on feed intake and immunocompetency (Fluharty & Loerch, 1997). Previous studies have evaluated the effects of weaning management and transportation on the acutephase reaction and performance of beef calves (Arthington et al., 2003 and 2005), and
20 indicate that plasma c oncentrations of acute phase proteins ( APP ) are significantly affected by these procedures, and may be used as an indicator of stress and performance of these animals. The inclusion of polyunsaturated fatty acids ( PUFA ) into diets has been shown to modulate immune responses (Calder et al., 2002). The majority of PUFA originating from common feedstuffs are extensively modified in the rumen ( Palmquist & Jenkins, 1980), and the addition of calcium soaps of fatty acids into diets may provide protection of these PUFA from the rumen microorganisms (Ngidi et al., 1990). Being a rumen inert technology, the supplementation of MegalacR (Church & Dwight Co., Inc. Princeton) might be a management option for increasing the delivery of PUFA to the small intestine providing essential precursors for modulating the immune system. For these reasons, two experiments were conducted to evaluate the effect of supplemental MegalacR on measures of performance and physiological responses of growing cattle following transportation.
21 CHAPTER 2 LITERATURE REVIEW Lipids Definition of Dietary Lipids Lipids are a chemically diverse group of compounds defined commonly by their insolubility in water, but are generally soluble in organic solvents. Dietary lipids of significant importance include fatty acids ( FA ), triglycerides, cholesterol and esters of cholesterol, and fat soluble vitamins (Spallho lz et al., 1999). Lipids have multiple functions including supplying dietary energy serving as a source of heat, insulation and protection for the animal body, providing essential FA, and serving as a carrier for absorption of fat soluble vitamins (Jurgens, 2002). The fats and oils used almost universally as stored f orms of energy in living organism are deriv ates of fatty acids (Nelson & Cox, 2005). Although fat usually comprises less than 5% of the ruminant diet, ruminants depend more on nonglucose metabolites for energy metabolism than nonruminants (Palmquist & Jenkins, 1980). Traditionally, in beef cattle grazing systems, pasture is the primary feed source and the range of FA content in forages varies widely from 1.6 to 10% (Clapham, 2005). In green plants, FA are predominantly in the form of glycolipids and phospholipids. G lycolipids account for 70 to 80% of ethe r extract in plant leaves whereas the concentration of triacylglycerol is negligible in the leaf (Harfoot, 1978). In contrast, triacylglycerols are the primary lipid in grains and whole seeds, and triacylgly cerol are the major lipid class in the diet of cows fed in confinement management programs, unless fat sources containing free FA are included as dietary components of the rati on (Doreau & Ferlay, 2004). Linoleic acid ( LA ) is the predominant FA in most oi linolenic acid ( LNA ) is usually greatest. Linoleic acid and LNA are considered essential FA
22 because they cannot be synthesized by mammals and rumina l microorganisms (Sprecher, 1981 ), and both LA and LNA are considered PUF A because they contain more than one point of unsaturation, or double bond. Fatty acids are classified and abbreviated according to the length of the acyl chain, number of unsaturations, and location and configuration of the unsaturation. By convention, numbering system, which begins numbering carbons starting at the methyl end of the FA. For 6 family, a lso called n 6, because the first of its double bonds is localized at the sixth carbon from the methyl end. Processing of FA in one family can only generate fatty acids of the same family. For example, FA of the n 3 family cannot be converted into a member of n 6 family and vice versa (Mattos et al., 2000). Supplemental Fat Intake and Digestion Beef cattle production systems are traditionally classified into two broad categories: grazing and feedlot. In addition to the lipids provide d by the forages, grazin g cattle can obtain triacylglycerol and other FA through supplements. Most feedlot diets are grainbased to increase their energy concentration, which typically improves the efficiency and cost of gain (Gibb, 2004). Although containing less energy than gra in, sources of fiber are often included in diets to help maintain rumen function (Bull et al., 1965) and animal health (Cheng et al., 1998). In confinement, lipids are often fed to increase energy density without decreasing fiber of the diet (Gibb, 2004). The inclusion of fat to cattle diets can affect dry matter intake ( DMI ; Jerred et al., 1990). The type of fat fed, and the type and amount of forage offered, also have an effect on the extent to which DMI is affected (Allen, 2000). The mechanisms involved with the FA induced depression of DMI are unclear, but may include alterations in palatability, gut motility, brain
23 signaling satiety centers and fiber digestion. The palatability of a fat can vary according to type (saturated or unsaturated) source (oil or whole seeds) and concentration in the diet (Miller et al., 1958). Grummer et al. (1990) stated that adaptation to supplements improved fat acceptability, and the differences in acceptability among fats can be minimized by mixing fats with other dietar y ingredients. Chemical nature and other factors, such as odor, physical form, and appearance also influence fat acceptability (Grummer et al., 1990) Fat is a potent stimulator of cholecystokinin ( CCK ) release and evidence exists that CCK contributes to satiety control (Reidelberger, 1994). It is probable that the CCK suppresses feed intake by inhibit ing gastric emptying (Moran, 199 2). Choi and Palmquist (1996) observed decreased DMI following decreased postprandial plasma concentrations of insulin and increased postprandial plasma concentrations of CCK by feeding fat to lac ta ting dairy cows. Also, an intravenous injection of exogenous CCK depressed feed intake of sheep (Grovum, 1981). Reidelbe rger et al. (1994) suggested that peripheral action of gut CCK may activate vagal and splanchnic afferent neurons that inhibit the brain satiety centers, and an increased rate of oxidation of FA in the liver can alter signals generated by hepatic vagal afferent ner ves to brain satiety centers (Allen, 2000). In addition, gut motility may be decreased by the presence of PUFA in the small intestine, which could decrease DMI (Drackley et al.,1992). Devendra and Lewis (1974) summarized four theories to explain the negati ve effect of supplemental fat on fiber digestibility; 1) Physical coating of the fiber with fat, thus preventing microbial interaction; 2) Modification of the ruminal microbial population from possible toxic effects of fat on certain microorganisms; 3) Inhibition of microbial activity from surface active effects of fatty acids on the cell membrane; and 4) Reduction of cation availability to key ruminal microbes resulting from formation of insoluble complexes with long chain fatty acids.
24 The substitution of nonfiber carbohydrate with fat sources can reduce microbial protein production since carbohydrates are the primary energy source for ruminal microbes. Further, excessive unsaturated FA ( UFA ) supplementation may lead to depressed fiber digestion due to the toxic effects of UFA on ruminal microorg anisms (Jenkins & Jenny, 1992). To avoid the inhibitory effects of free FA on ruminal bacteria, the calcium soaps technology was developed (Jenkins & Palmquist, 1982; Sukhija & Palmquist, 1990). Calcium salts of FA ( CSFA ) should be inert in the rumen, dissociating in the abomasum at low pH (Wu & Palmquist, 1991, Wu et al., 1991), thus becoming available in the small intestine for further absorption ( Jenkins, 1993). Pal mquist and Jenkins (1980) report ed beneficial effects of fat added to diets of high producing dairy cows. Cows fed fat from 3 to 5% of the total diet increased energy intake without negative effects on fiber digestion. Feedlot finishing diets containing 2 to 5% of total fat may stimulate weight gain of beef cattle (Moore et al. 1986), although apparent digestibility increased, whereas true digestibility decreased when fat was added up to 8% of diet ary DM (Haaland et al., 1981). Grummer (1988) report ed no effect on nutrient digestion and ruminal fermentation i n dry Holstein cows fed CSFA or prilled fat when supplemented at 3.5% or less of the total ration DM, but cows fed prilled fat consumed a greater amount of fat than those fed CSFA due to the greater fat content in the prilled fat. Dif ferently, Chalupa et al. (1986) report ed reduced feed intake an d lower milk yields from low producing dairy cows fed prilled fat at 6 or 9% of the total ration DM compared to prilled fat fed cows at 0 and 3%. The effects of fat supplementation on digestibility and performance were reported by several authors. Garcia et al. (2003) fed whole sunflower seeds ( containing approximately 70% LA ) at 5% of dietary DM and report ed no differences in the live weight, average daily gain ( ADG), or carcass weight of beef heifers compared to those fed no dietary lipid supplement.
25 Atkinson et al. (2006) supplemented sheep with increasing amounts of highlinol eate safflower oil (0, 3, 6, or 9% of dietary DM) and sugest ed no effects on apparent ruminal digestibility of organi c matter, neutral detergent fiber ( NDF ), and nitrogen. Elliot et al. (1997) demonstrated a decrease in ruminal organic matter ( OM ) digestibility but no difference in postruminal OM digestibility when tallow was supplemented to beef steers consuming a 60% f orage diet. Brokaw et al. (2002) sugest ed that total tract OM digestibility decr eased when soybean oil (2.25% of dietary DM ) was supplemented to ruminants consuming high forage diets, and Brokaw et al. (2000) noted that heifers supplemented with highoil c orn at 0.5 % of body weight ( BW ) selected forage that was less digestible than forage selected by heifers fed conventi onal corn. However OM intake was not affected by feeding supplemental high oil corn at 1.5% to 1.74% of diet ary DM (Brokaw et al, 2001). Additionally, forage intake and diet digestibility were not affected in steers offered switchgrass hay and canola seeds to provide approximately 4% of diet ary DM as crude fat (Leupp et al., 2006). Hess et al. (2008), in a review of summarized results, indicated that an optimal inclusion rate for supplemental fat is less than 3% of dietary DM if the goal is to maximize the use of forage based diets, and that supplemental fat should be limited to 2% of DM or less if the goal is to pr event substitution of forage with intake of supplemental fat. However, ruminants fed high concentrate diets may receive up to 6% supplemental fat in the diet without ill effects on utilization of other components (Kucuk et al., 2004; Atkinson et al., 2006; and Hess et al., 2008). Fatty Acid Metabolism in the Rumen The first step in lipid metabolism in the rumen is hydrolysis of ester bonds via microbial lipases (Dawson et al., 1977). The end products produced by ruminal hydrolysis are free FA, glycerol and galactose, which are converted to volatile fatty acids ( VFA ), mainly propionate and butyrate (H azlewood & Dawson, 1975). The extent of hydrolysis ranges from 85 to 95% for
26 most unprotected lipids, and this percentage is greater for diets rich in fats than for conventional diets, in which most of the lipids are in the cellular structure (Bauchart et al., 1990). Lipolysis is a prerequisite to biohyrogenation ( BH ), which provides the free carboxyl group from UFA (Cha lupa & Kutches, 1968). The microbial BH only occurs on free FA, released from triacylglycerols, adsorbed on feed particles or microbial cells (Doreau & Ferlay, 1994). The BH process involves a series of microbial enzymes called isomerases and reductases that initially isomerizes UFA and sequentiall y hydrogenate the double bonds (Kepler et al., 1966). Because the BH of PUFA is very extensive, 92% for LNA and 80% for LA (Doreau & Ferlay, 1994), most of the dietary PUFA is modified and therefore not absorbed as such in the small intestine. Consequently the composition of absorbed FA does not reflect the same composition of FA intake from the diet. Gul ati et al. (2005) evaluated BH of prilled fat, CSFA, extruded oilseeds, formalintreated oilseeds and untreated oilseeds. Formalin treatment provide d the most protection from BH at around 90%, followed by CSFA at about 60% and then prilled fat or extruded fat at 30%. Therefore, CSFA can be used as one of the alternatives to increase delivery of PUFA postruminally. This method of protection of FA promotes a chemical linkage between the free carboxyl group and a molecule of calcium, making the FA carboxyl group unavailable for microbial enzymes (Wu et al., 1991), avoiding reducing ruminal BH (Chalupa & Kutches, 1968). MegalacR is a commercial example of CSFA composed of a mixture of palm and soybean oils, which contains about 39% LA and 3% LNA (Church & Dwight Co, Princeton, NJ ). Fatty Acids Metabolism in Tissues The amount of each FA incorporated into organs and tissues depends on the amount of precursors p resent in the diet (Mattos et al., 2000) Lessard et al. ( 2003) showed an increased concentration of blood FA in Holstein cows fed whole flaxseed, micronized soybean and CS of
27 soybean oil prior to and during the breeding period. Cows fed CS of soybean oil presented a lesser blood concentration of LNA on d 5 and 21 after calving; and on d 0 and 20 after first artificial insemination when compared to cows fed fl axseed. Cows fed flaxseed had a blood n 6 to n3 ratio three times less than cows fed C S of soybean oil. Increased milk fat content and oleic acid concentration were observed when lactating dairy cows were supplemented with CSFA (Megalac) at 3 and 6% of the total diet DM compared t o cows not fed CSFA (Schauff & Clark, 1992). In contrast Harrison et al (1995) reporte d a decreased concentration of oleic acid and an increased concentration of LA in milk when lactating dairy cows were fed CSFA (Megalac; 2.7% of dietary DM ) during the 3 months after calving compared to cows fed saturated fat source Biohydrogenation of dietary LA and oleic acid in lactating dairy cows decreased slightly when soybean oil was fed to cows as CSFA (Lundy III, et al., 2004). The term conjugated linoleic acids ( CLA ) refers generically to a class of positional and geometri c conjugated dienoic isomers of LA, two of which (cis 9,trans 11 and trans 10,cis 12 CLA) are known to posses biological activity (Pariza et al., 2001). These two types of CLA are produced in the rumen of cattle and other ruminant animals during an incompl ete microbial BH of LA and LNA (Pariza et al., 2000), thus their principal dietary sources for humans are dairy products, beef and other foods derived from ruminants (Dhiman et al., 1999). Changes in substrate supply and extent of BH will affect the supply of intermediate and end products of BH resulting in an altered LA, LNA and CLA content of milk and meat (Kelly et al., 1998). The CLA are primarily recognized for their anticarcinogenic and lipolytic effects (Park et al., 1997), but CLA also influence im munity (Pariza, 2001) and reproduction (Garcia, 2003) affecting the synthesis of eicosanoids, cytokines and steroid hormones.
28 Gassman et al. (2000) fed CS of CLA to finishing steers, with diets containing 0, 1 and 2.5% of CLA (DM basis) and reported decreased feed intake and BW gain as the intake of CLA increased. Additionally, increased CLA concentrations in adipos e and lean tissue were suggeste d. In contra st, Gillis et al. (2004) reporte d an increased ADG when finishing steers were supplemented with CS of CLA (2% of dietary DM ) when compar ed to steers fed corn oil (4% of dietary DM) or no oil supplement Haddad & Younis (2004) also observed decreased DMI when Awassi lambs were supp lemented with CS of CLA added at 2.5 and 5% of total dietary DM. Park et al (1997) fed CLA to mice at 0 or 0.5% of total diet ary DM in 2 experiments and no differences in DMI and ADG were reporte d between CLA fed group and the control group, although the concentration of CLA was 2.5 times greater in the body of mice fed CLA in dicating that greater dietary CLA increases CLA content in body tissues. Cholesterol is a precursor of several reproductive hormones such as steroids and prostaglandins (Mattos et al., 2000). Dietary fat supplementation in cows consistently increases plas ma choles terol concentration (Grummer & Carrol l 1991) and the greater availability of cholesterol can result in increased secretion of progesterone (Staples et al., 1998). Oldick el al. (1997) report ed an increased plasma concentration of nonesterified F A, triglyceride and cholesterol when ruminally cannulated dairy cows received abomasal infusion of tallow or yellow grease (0.45 kg/d) compared to cows which received an infusion of glucose (1 k g/d). Lloyd et al. (2002) suggest ed increased serum cholestero l and triglyceride concentrations when CSFA (Megalac; 0.113g/heifer daily ) were supplemented to pubertal Angus heifers prior to breeding. These data agree wit h Lucy et al. (1991), who suggest ed increased plasma concentrations of cholesterol and basal LH i n early postpartum dairy cows receiving CSFA (Megalac).
29 Dietary supplementation of LNA reduced the in vitro synthesis of prostaglandin ( PG ) F, one important eicosanoid responsible for luteolysis. Feeding fish meal ( Mattos et al., 2004) or abomasally infusing a fat source rich in LA (Oldick et al., 1997) resulted in an attenuation of the induced PGFM (a measurable metabolite of PGF) response in peripheral plasma compared with control animals. These results indicate that high concentrations of PUFA ca n decrease the endometrial secretion of prostaglandins resulting in greater chances of pregnancy esta blishment (Thatcher et al., 1997). Conclusively, feeding fats and targeting of FA to reproductive tissues may be a potential strategy to integrate nutritio nal management to improve animal p roductivity (Santos et al. 2008). Among these multiple biological functions, dietary PUFA also appear to impact multiple immunological functions. Lessard et al. (2003) indentified that blood concentration of PGE2 was redu ced in cows fed flaxseed compared with those fed CS of soybean oil or micronized soybean, while progesterone concentrations were increased in cows fed flaxseed compared w ith those fed CSFA during the breed period. Essential FA, such as LA and LNA may modulate immune reactions and inflammatory responses, by influencing biological membrane fluidity (Schmitz & Ecker, 2008), and activating cellular communication by stimulating eicosanoids biosynthesis (Calder & Grimble 2002 ; Yaqoob, 2004). Immune Function Th e s pecific immune response can be classified into two types, humoral and cell mediated. Humoral immunity is mediated by antibodies that are released by B lymphocytes into the bloodstream and are responsible for specific recognition and elimination of antigens. Cell mediated immunity involves specific antige n recognition by T lymphocytes Miles & Calder, 1998). According to Calder et al. (1996) and Miles & Calder (1998) the l eucocytes are the principal group of cells of the immune system, consisting of: B lym phocytes, T lymphocytes,
30 dendritic cells, natural killer cells, mononuclear phagocytes ( incl uding monocytes and macrophages), and granulocytes ( including neutrophils, eosinophils and basophils) The AcutePhase Reaction The acute phase reaction involves an organisms response to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth, or immunological disorders (Heinrich et al., 1990). It consist of an early and local reaction at the site of injury characterized by an accumulation and activation of leukocytes, mainly granulocytes and mononuclear cells, fibroblasts and endothelial cells, which in turn release acute phase glycoprotein mediators called cytokines. The acutephase reaction may be beneficial to the injured organism with t he objective of restoring the disturbed homeostasis (Pepys & Balyz, 1983). Cytokines act on specific receptors in several organs and tissues, leading to a systemic reaction characterized mainly by fever, leukocytosis, increase of adrenocorticotropic hormon e and glucocorticoids secretion, activation of the clotting cascade, and dramatic changes in the plasma concentration of some liver derived proteins called APP (Heinrich, 1990). Macrophages and monocytes secrete three cytokines that have a profound metabol ic effect on the organism: interleukin 1 ( IL 1), interleukin6 ( IL 6 ), and tumor necrosis factor TNF ). Because macrophages and monocytes represent the first line of defense in the immune system, collectively these three immune mediators are recognized as pro inflammatory cytokines (Johnson, 1997). Tumor necrosis factor bacterial endotoxin, followed by IL 1 and IL 6, and TNF 1 and IL 6 (Peck, 1994). The analysis of plasma concentrations for APP is commonly used as a sensitive indicator of inflammation in mammals (Breazile, 1996). In addition to their secondary response to an activated immune system, the APP also have other vital functions in the organism, specifi c of
31 each protein. Ceruloplasmin ( Cp ), also known as ferroxidase, is an enzyme containing 6 atoms of carbon in its structure. It is the major copper carrying protein in the blood and participates in iron homeostasis. Concentrations of Cp may increase due t o inflammation. Haptoglobin ( Hp ) is responsible to bind free hemoglobin in the blood, forming the Hp hemoglobin complex. Also, it prevents the loss of body iron and its concentrations are normally undetectable in bovine blood unless there is tissue damage. Fibrinogen ( Fb ) is involved in the clotting cascade and in the formation of the fibrin matrix for tissue repair. Increased Fb concentrations are detected during internal hemorrhage or tissue damage. According to Johnson (1997), the proinflammatory cytokines are large, hydrophilic molecules (17 to 26 kDa) and therefore incapable of cross ing the bloodbrainbarrier. The mechanism by which cytokines communicate with the brain is through accessing circumventricular organs devoid of a bloodbrain barrier, follo wing production of secondary signals, such as prostaglandins. Receptors for IL 1 have been observed in the hippocampus and choroid plexus (Farrar et al., 1991) and binding sites for TNF been identified also in the brainstem, cortex, cerebellum, thal amus, and basal ganglia of rats brain (Kinouchi et al., 1991). In cattle, the APP reaction following transport has been characterized by Arthington et al. (2003). The magnitude of this response may be a key indicator of subsequent productivity in the feedlot, especially during the initial receiving period (Qiu et al., 2007; Arthington et al., 2005). Arthington et al. (2005) observed increased plasma concentrations of Cp and Hp in steers following 24 hours of transportation, and the concentration of these APP (Cp and Hp) was greater in calves weaned normally were compared with calves which were weaned 211 d prior to transport. In another experiment, Arthington et al. (2008) evaluated performance and the acute phase reaction following transportation of steer s submitted to 4 weaning management strategies.
32 During the 29d receiving period, plasma Cp concentrations were decreased for the pre weaned group when compared to control or creepfed steers. Also, ADG, DMI and feed efficiency were greater for pre weaned steers compared with control steers. These data are supported by Dhuyvetter et al. (2005), who indicated that preconditioning of calves before marketing helped to avoid depression of performance and health upon feedlot arrival. Weaning and diet management prior to shipment can become an important practice to decrease morbidity and mortality rates of transported beef cattle. Physiological Stress and Growth In terms of the marketing process, weaning is likely the greatest stress imposed to cattle, and accor ding to Loerch & Fluharty (1999) many other factors are able to cause stress in calves. For example, weaning breaks the bond between dam and calf and causes prolonged vocalization. Marketing and transportation causes depravation of water and feed, which is a common occurance in cattle shipments from Florida to other states. Weather changes, over crowding, unexpected and loud noises, poor air quality and poor sanitization are extra stressors often experienced by the calves during the weaning and transport process. Upon arrival at the feedlot, calves are exposed to new diets and unfamiliar fee ders and waterers. I n the new feedlot environment, calves also may have to acclimate to a new social dominance and new pathogens. Processing procedures, such as vaccinat ion, castration, commingling and dehorning are stressors commonly found in feedlot facilities. The effects of weaning and transportation on blood serum components have been studied by several groups. Large increases in blood concentrations of cortisol, epi nephrine and norepinephrine have been observed in steers after weaning and transportation (Cole et al. 1988; Lefcourt & Elsasser, 1995), although corticosteroids w ere most responsive to transport stress.
33 Agnes (1990) compared isolated metabolic effects o f stress caused by transport, loading and noise; all three caused rapid increases in cortisol. Growth is a genetically programmed sequence of events in the young animal (Klasing & Korver, 1997), and when this sequence is disrupted by stress, several physiological changes contribute to favor a process of reallocation of animal resources important in survival. Conceptually, the status of the immune system (immunosuppression versus immunopotentiation) will depend upon the net effect of these changes (Khansari et al. 1990). Domestic food animals with clinical and subclinical infections eat less, grow slower, and convert feed to body tissues and products in an inefficient manner (Johnson, 1998), suggesting that a suppressed immune status can interact with the cen tral nervous system and modulate feed intake. Summarily, the immune system is able to use cytokines to delivery information to other systems, including the central nervous system, regarding the level of immunologic activity. Sely e (1976) stated that biological stress is the non specific response of the body to any demand, and when animals are exposed to stress, they react in a three step process name d General Adaptative Syndrome. The first answer to this syndrome is called Alarm Reaction and it is cha racterized by vocalization, hypothalamic pituitary adrenal axis response and catabolism. The second stage is called Resistance which is characterized by anabolism and increased feed intake. When resistance is not successful, the animals start the third and last step of the syndrome called Exhaustion. In this stage the adaptative capability of the animal is limited, re sulting in exhaustion before it adapts to the stressor. Survival and/or recovery of stressed animals are dependent on the level of stress they were exposed to prior, during, and after the marketing process.
34 Changes in the ruminal environment are normally observed in cattle after a long period of water and feed depravation. Cole & Hutcheson (1985) reported that fermentative capacity of ruminal micorbes was reduced by 75% following a 48h period without feed as well as total reduction in bacteria l number s by 10 to 25% of baseline (Baldwin, 1967). Animals returned to control levels by 7 d after re alimentation. According to Loerch & Fl uharty (1999), DMI, ruminal volume, and weight of ruminal contents decreased as duration of feed and water absence increased; however, 4 d after arrival, there were no longer differences in DMI or weight of ruminal contents. These results may indicate that fermentative and digestive capacities play a minor role in low feed intakes during the first two weeks after arrival into the feedlot. Probably, the most important impact of stress on feed intake is due to its negative effects on animal immunocompetency. The inflammatory response is marked by accelerated muscle degradation and increased hepatic APP synthesis. At least 60% of the amino acids liberated by body protein degradation are utilized by the liver as fuel for production of APP (Johnson, 1997). The cy tokines IL 1 and TNF 6 induces in vitro (Richards et al., 1991). Muscle degradation is mediate d by IL 1, IL 6, and TNF 1 has been shown to inhibit the anabolic effects of i nsulin on skeletal muscle (Klas ing & Johnstone, 1991). Increased plasma concentration of nonesterified FA an d hypergly ceridemia is associated with a variety of infections (Grunfeld et al., 1989). Tumor necrosis factor by increasing h epatic FA synthesis, inhibiting lipase activity, and stimulating lipolysis (Memon et al., 1994). High cortisol and plasma urea nitrogen concentrations have been observed in pigs ex posed to environments that provide high imm unological challenges (Williams 1993).
35 Ban et al. (1992) proposed a neural link between the periphery and CNS In this association, peripheral cytokines are able to stimulate vagal afferent nerves in the viscera, avoiding the necessity of an elevated concentration of peripheral cytokine s to activate afferent nerves which lead to cytokine synthesis within direct CNS involvement. It is concluded, therefore, that inflammatory stimuli in the periphery induces synthesis of proinflammatory cytokines in the brain. Peripheral and central injec tion of recombinant IL 1 induced anorexia as well as a number of profound behavioral alterations that are reminiscent of sickness in rats (Dantzer & Kelley, 1989). This reduction in food intake is attributed to a reduction in both meal size and meal durati on. Peripheral intra venous and intra parenteral administration of recombinant human TNF Sonti et al. (1996) reported synergistic effects of IL 1 and TNF when these cytokines were administered intravenous or intra cerebroventricular. It is known that pro inflammatory cytokines suppress appetite and f eed intake by acting directly on the CNS, although the same cytokines can regulate appetite in immune challenged a nimals indirectly. Tu mor necrosis factor stimulated leptin secretion by acting directly on adipocytes (Finck et al., 1998). Both peripheral and central injection of leptin reduce d food intake, increased energy expenditure, and deplete adipose tissue in l ean mice (Hallas et al., 1995). Interleukin1 induces a decrease in the plasma concentrations of thyroxin and an even larger decrease in triidothyronine concentrations (Klasing & Korver 1997), and in combination with TNF d concentr ations of growth hormone (Els asser et al., 1988) and i nsulinlike growth factor 1( IGF 1) in the circulation, liver, skeletal muscle, and pituitary (Klasing & Kover, 1997), which presumably contributes to impaired growth. In addition, the release of growth hormone releasing hormone from medi al basal hypothalamic explants was
36 decreased by IL 1, whereas somatost atin release was s increased (Spurlock, 1997). In conclusion, pro inflammatory cytokines can act on a number of targets that are likely to contribute to poor intake and growth of immunologically challenged animals (Klasing, 1988). The immunological stress is characterized by the direct and indirect impacts of cytokine s on CNS and other organ systems (Johnson, 1997). Supplemental Fat, Stress and Growth Ei cosanoids are a group of chemical messengers synthesized from 3 types of 20carbon chain PUFA: dihomolinolenic acid ( DGLA ; 20:3n6), arachidonic acid ( ARA; 20:4n6) and eicosapentaenoic acid ( EPA ; 20:5n3). Eicosanoids include mainly PG, thromboxanes ( T X ), leukotrienes ( LT ), and other inflammatory mediators. The PUFA precursors for eicosanoid synthesis are stored in an esterified form in cell and organelle membrane phospholipids or in cytoplasmatic lipid bodies bound to glycerides and phospholipids at the cytosolic surface (Schmitz & Elker, 2008). They are mobilized and re hydrolized usually by the action of phospholipase A2 activation in response to a cellular stimulus (Calder et al., 2002). Because the majority of cell membranes contain predominately AR A compared to EPA and DGLA, ARA is the key precursor for eicosanoid biosynthesis. linolenic acid ( GLNA; 18:3n6) by action of delta 6desaturase. A specific enzyme called el ongase 5 converts GLNA into DGL A which is converte d to the key intermediate ARA by action of delta 5 desaturase. Arachidonic acid is further metabolized to docosapentaenoic acid ( DPA; 20:5n6) and eicosanoids. The n3 LNA is converted to stearidonic acid (18:4n3) and to eicosatetraenoic acid ( ETA ; 20:4n3) to form EPA using the same series of enzymes as those used to synthesize ARA. The EPA is further metabolized to docosahexaenoic acid ( DHA, 22:6n3) or eicosanoids.
37 The n 6 ARA can be converted to eicosanoids of the PG2, TX2, LT, derivates of HPE TE and HETE, lipoxin A4 by action of COX and lipo oxygenases. In contrast, EPA is converted to PG3, TX3, and LT5. Resolvins, docosatrienes, and neuroprotectins are synthesized from DHA. For example, PGE2 and PGI2 are pro arrhythmic while PGE3 and PGI3 are anti ar rhythmic; TXA2 is a platelet activator and TXA3 is platelet inhibitor; TXB2 causes vasoconstriction and the TXB3 causes vasodilatation; LTB4 is pro inflammatory an d HPETE and HETE are involved in inflammation processes, in contrast to LTB5, resolvin E1, re solvin D and neuroprotectin D1 which promote anti inflammatory response s According to Schmitz & Ecker (2008), the main difference between n 6 and n3 FA derived eicosanoids is that most of the mediators formed from ARA are pro inflammatory whereas those formed from EPA and DHA are anti inflammatory. Several studies have investigated the effects of the amount and type of fat in the diet on immune reactions (Yaqoob & Calder, 1993; Jolly et al, 1997; Cullens, 2005), and the inclusion of PUFA into diets have been shown to modulate immune cell function (Calder et al., 2002). Th e mechanism by which FA might modulate immune functions is not yet understood, but apparently PUFA alter the production of mediators involved in communication between cells of the immune system through the synthesis of eicosanoids, cytokines and nitric oxide (Miles & Calder, 1998), and to alter the expression of adhesion molecules which are involved in direct cell to c ell contact (Calder, 1999). Farran et al. (2008) fed flaxseed, rolled f ull fat soybeans and tallow at 13, 20 and 4% of dietary DM to beef heifers respectively Heifers supplemented with flaxseed and full fat soybeans showed greater ADG than heifers fed tallow. Also, the flaxseedfed heifers had increased plasma concentration s of total n 3 PUFA, whereas, full fat soybean fed heifers had
38 increased plasma concentration s of total n 6 PUFA. In another experiment, the same authors fed flaxseed (12.9% of dietary DM), rolled full fat soybeans, and tallow (both at 20% of dietary DM) to lipopolysaccharide challenged ( LPS ) heifers. After LPS challenge, rectal temperatures were less for flaxseed and soybean fed heifers than those fed for tallow ; and concentrations of plasma TNF was greater in heifers supplemented with soybean than tallow. In agreement, Chang et al. (1992) observed increased serum TNF for LPS challenged mice fed n 3enriched fish oil compared with those fed corn, coconut oil, or a low fat diet. Po mposelli et al. (1989) reported that diets containing fish oil reduced fever response in guinea pigs. Flaxseed is rich in LNA while fish oil is abundant in EPA and DHA, all members of the n3 FA family. Because many studies have demonstrated the anti inflammatory effects of n 3 FA these results may explain the decreased fever an d serum conc entrations of TNF. Calder et al. (2002) stated that DHA and LNA can be converted to EPA in animal cells. Additionally, EPA, by virtue of its ability to compete with ARA receptors, can competitively inhibit production of eicosanoids such as the 2series PG and 4 series LT from ARA, thereby red ucing inflammation (Calder, 1999). Silvestre (2009) observed a lesser n 6 to n 3 FA ratio in neutrophils of dairy cows fed CS of fat enriched fish oil compar ed with those fed CS of palm oil. In addition, me an concentration of TNF were lower for cows fed CS of fish oil. Further, the neutrophil concentration of EPA, DPA and DHA was greater in cows fed CS of fish oil than cows fed CS of palm oil. Do Amaral (2008) report e d greater plasma concentrations of acid soluble prote ins after calving when Holstein cows were fed CS of trans C18:1 (55% C18:1 trans) starting five wk prepartum than cows fed sunflower oil (80% C18:1 cis ). In another experiment using PUFA supplementation prior to calving in heifers and mature cows, the same author
39 observed an effect of fat source and parity on plasma concentration of APP. Plasma concentration of Cp were greater in heifers fed n6 FA (CS of sunflower oil) when compared with heifers fed n 3 FA (CS of palm and fish oils), although heifers fed n3 FA had greater plasma concentrations of Fb compared to heifers fed n6 FA. Cows and heifers fed n3 FA from linseed oil had lower concentration s of blood neutrophils than these fed n6 FA from Megala cR (Do Amaral, 2008). Lower postpartum Fb concentration s were reporte d by Cullens (2005) when primiparous lactating cows were fed CSFA (MegalacR; 2% of dietary DM ) during the prepartum period compared to those not fed fat In the same experiment, mult iparous cows fed CSFA had greater plasma PGFM concentrations at approximately d 5, 6, and 7 postpartum compared with those not fed fat prepartum Juchem et al. (2008) sugested d greater plasma concentrations of PGFM at d 1 postpartum when primiparous cows where fed prepartum with CS of fat enriched in LA and trans octadecenoic acids. This response was associated with a lesser incidence of uterine infection compared to cows fed C S of palm oil. Polyunsaturated FA such as EPA and DHA, inhibited production of IL ocytes (Purasiri et al., 1994). These results agree with Far ran et al. (2008) and Ca ughey et al. (1996), who demonstrated that diets enriched with f laxseed and fish oil inhibited IL 1 and TNF Palm and coconut oils provide mainly saturated FA (SFA). MegalacR, sunflower oil, and soybean oil a re sources of n6 FA. Opposite to effects of the n 3 FA n6 FA are associated with proinflammatory and inflammatory responses, observed b y the stimulation of cytokines and other physiological mediators, such as PGE2 and LTB4. Greater concentrations of blood neutr ophil in n6 FA fed cows suggested by Do Amaral (2008) may indic ate that n 6 FA stimulate neut rophil activity during the peri partum period, which may explain the lesser post -
40 partum uterine infection and greater serum concentrations of PGFM observed by Juchem (2008) in primiparous cows fed CS of fats enriched in trans C 18:1 and LA. Conclusively, dietary FA participate in immunomodulatory effects in mammals, and the role of lipids in immunity is focused on PUFA, especially the n3 and n 6 families. The nature of the difference between these two families on immunomodul atio n is not certain, although speculations about changes in lipid sources and their interactions on physiol ogical responses of immune challenged animals have been made.
41 CHAPTER 3 EFFECTS OF MEGALACR INCLUSION IN RECEI V ING DIETS OF WEANED FEEDER STEER S Mat erials and Methods This experiment was conducted from July to September 2006, and was divided into a pre shipping (d 40 to 0) and a post shipping phase (d 1 to 30). The pre shipping phase was conducted at the University of Florida IFAS, Range Cattle Res earch and Education Center, Ona, and the post shipping phase at the University of Florida IFAS, North Florida Research and Education Center, Marianna. The animals utilized in these experiments were cared for in accordance with acceptable practices as ou tlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Animals and Facilities Sixty four weaned Braford steers (BW SD = 218 23 kg; age SD = 226 27 d) were utilized in this experiment. For the pre shipping phase (d 40) steers were stratified by initial BW and age and randomly allocated to two bahiagrass ( Paspalum notatum ) pastures (32 steers /pasture) of 2.04 ha Each pasture was randomly assigned to one of the two following supplement ation treatments: 1) No fat control (CO) or 2) Saturated Fat (EN). On d 0, all steers were loaded into a commercial livestock trailer and transported 1,600 km from Ona, FL to a research feedlot facility in Marianna, FL Steers remained in the truck for 24 h, before being received into the feedlot. Upon arrival, steers were stratified by pre shipping treatment and current BW, received electronic ear ID tags (Allflex USA, Inc., Dallas Ft. Worth, TX) for the measurement of individual feed intake with the Grow Safe System (Model 4000 E, GrowSafe Systems Ltd., Airdrie, AB, Canada), and re allocated into three feedlot pens (104.6 m2) during the first 7 d. Each pen was concrete floor, covered, and provided of two feed bunks
42 and one waterer. Animal s were then rand omly assigned, in a 2 x 3 factorial arrangement, to one of three supplemental treatments: 1) CO, 2) EN, or 3) MegalacR supplementation (MG). On d 8, three more feedlot pens were added (two pens/treatment; eleven animals/pen). Diets Pasture qua lity during the pre shipping phase was estimated to be 54.0% TDN and 9.6% CP (DM basis) from hand plucked samples collected at the beginning of the trial and analyzed by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). Samples were taken at 30 locations per pasture according to procedures determinated by Vendramini et al. (2006). The pastures utilized in this experiment were not fertilized prior to or during the experimental period. Treatments consisted of two grain based supplements (Tables 3 1 and 3 2) with (EN) or without (CO ) the inclusion of a prilled saturated fat source (Energy Booster 100; MSC Co, Carpentersville, IL ) Suppl ement intake was limited (4.1 kg/d) with the EN providing 5.91% of dietary fat/steer daily (DM basis) For the post shipping phase, diets were prepared and fed as a TMR in ad libitum a mounts daily, except the first four days when steers were offer ed daily with 5 kg of Tifton 85 bermudagrass ( Cynodon dactylon) hay (as fed) and a 7 0:30 mixture of concentrate:cott onseed hulls, separately Following this initial period, steers were offered in ad libitum amounts a 60:25:15 mixture of concentrate :cottonse ed hulls:bermuda grass hay from d 5 to 12, and followed by a 65:28:7 mixture of the same ingredients in ad libitum amounts from d 13 to the end of the experiment (d 30) The EN and CO concentrate ingredients were similar to the pre shipping phase, where as the MG treatment consisted of a grain based supplement with the inclusion of a source rumen inert PUFA (MegalacR ; Tables 3 1, 32) providing approximately 5.0% of
43 dietary fat/steer daily (DM basis; T able 33). Water was offered ad libitum throughout all phases of the experiment s Sampling During the pre shipping phase, steer shrunk BW (16 h of feed restriction) was recorded on d 40, whereas full BW was recorded on d 25, 11, and immediately prior to shipping on d 0. During the post shipping phase, steer shrunk BW was recorded on d 1 (immediately following arrival at feedlot facility) and at the end of the experimen t (d 30). Full BW was recorded on d 4, 8, 15, 22 and 29. Only s hrunk BW values were utilized to determine ADG during the pre shipping and post shipping phases. Individual feed intake was recorded daily during the post sh ipping phase using the GrowSafe feed intake s ystem. The GrowSafe system is a technology which continuously measures individual feed consumption. Representative samples of all feed stuffs were obt ained weekly during each phase. Samples were dried after collection for 96 h at 60oC in a fo rced air oven to calculate concentration of DM. The weekly samples were ground through a 1mm Wiley mill screen (Model 4, A. H. Thomas, Philadelphia, PA) to be analyzed for nutrient composition according to analytical procedures of a commercial feed labora tory ( Dairy One Forage Laboratory, Ithaca, NY). Fol l owing transport, blood samples were collected on d 0, 1, 4, 8, 15, 22, and 29 for determination of fibrinogen and ceruloplasmin concentrations. Plasma samples used for determination of fatty acid composit ion were collected on d 0 and 29.
44 Blood analysis B lood samples were collected via jugular venipuncture into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin. Samples were placed immediately on ice and centrifuged at 2,000 g at 5oC for 30 min (GPR Centrifuge, Model 349702; Beckman Instruments Inc., Fullerton, CA) for plasma separation and collection. Plasma was frozen at 20C on the same day of collection. A Coagulation Analyzer (Fibrometer, Rankin Biomedical Corp., Holly, MI) was used to determine plasma fibrinogen concentration from a standard curve using a human reference (Sigma procedure No. 880; Sigma Diagnostics, St. Louis, MO) A spectrophotometer (ThermoSpectroni c Genesys 20; Thermo Fisher Scientific Inc., Waltham, MA) was used to determine plasma ceruloplasmin concentration. The p lasma ceruloplasmin oxidase activity was measured in duplicate samples using colorimetric procedures described by Demetriou et al. (1 974). Ceruloplasmin concentrations were expressed as mg/dL, as described by King (1965). The intra and interassay CV were 4.8 and 6.4% for fibrinogen and 3.2 and 6.7 for ceruloplasmin, respectively. Plasma fatty acid extraction and methylation were deter mined using procedures describe d by Kramer et al. (1997). The fatty acid methyl esters were determined using a gas liquid chromatography (GLC; CR3800 Gas Chromatograph, Varian, Inc. Corparate Headquaters, Palo Alto, CA) equipped with autosampler (Varian CP 8400), flame ionization detector, and Varian capillary column (CP Sil 88, 100 m x 0.25 mm x 0.2 m). The peak was indentified and calculated based on the retention time and peak area of known standards.
45 Statistical analysis Performance data from the pre shipping phase were not statistically analyzed and are reported as mean SD because steers were allocated to a single pasture per treatment and supplements were groupfed, therefore the experimental unit (pasture) was not replicated. For the post shipping phase, performance and physiological data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001). The statistical model used for plasma measurements and DMI was: Yijkl = + TRTAi + TRTBj + CALFk (ij) + Dl + TRTADik + TRTBDjk + TRTABDijk + + Eijkl where Y = response variable = mean TRTA = fixed effect of pre shipping treatment TRTB = fixed effect of post shipping treatment CALF = random effect of steer within pre x post shipping treatment D = fixed effect of d ay TRTAD = eff ect due to interaction of pre shipping treatment and day TRTBD = effect due to interaction of post shipping treatment and day TRTABD = effect due to interaction between preand post shipping treatment and d E = residual error The statistical model used fo r ADG analysis was: Yij = + TRT Ai + TRTBj + CALFk(i) + Eij where Y = response variable = mean
46 TRTA = fixed effect of pre shipping treatment TRTB = fixed effect of post shipping treatment CALF = random effect of steer within pre x post shipping treatme nt E = residual error Plasma measurements were analyzed using values from d 0 as a covariate. Results are reported as least square means. Means were separated using LSD. Significance was determined at P ly significant interactions were reported. Pearson correlation coefficients among plasma measurements, ADG and DMI were generated using the CORR procedure of SAS (SAS, 2001). Results Measurements of Performance The ADG observed during the pre shipping phase of the study was 0.35 0.18 kg/d for EN fed steers and 0.34 0.20 kg/d for CO fed steers. N o pre shipping or pre shipping x post shipping treatment effects were detect ed, therefore, all results reported here in are derived from the post shipping treatments in the postshipping phase of the study. Steers fed MG had decreased ( P < 0.05) ADG when compared with CO but not EN fed steers (1.04, 0.98, and 0.80 kg/d for CO, EN, and MG treatm ents, respectively; SEM = 0.08). Treatment x day interactions was observed ( P < 0.01) for DMI (Figure 3 1). Steers fed MG had less overall DMI compared to CO fed steers ( P < 0.01), but not compared to EN fed steers (2.37, 2.80, and 2.55 % of BW for MG, CO, and EN, respectively; SEM = 0.10). Steers fed MG had decreased ( P < 0.05) G:F when compared to EN fed steers, and tended ( P < 0.10) to have less G:F compared to CO fed steers (0.29, 0.37, and 0.35 kg/kg of G:F for MG, EN, and CO, re spectively; SEM = 0.026).
47 Plasma Measurements and Coefficients of Correlation No differences were observed among treatments for covariately adjusted mean plasma fibrinogen concentrations (331.0, 351.9, and 378.9 mg/dL for CO, EN, and MG, respectively; SEM = 27.0) and mean plasma cer uloplasmin concentrations (18.0, 17.7, and 18.9 mg/dL for CO, EN, and MG, re spectively, SEM = 0.69). A d ay effect ( P < 0.01) for fibrinogen and ceruloplasmin was observed (Figure 3 2 a nd 33). Pearson correlation coefficient s a mong ceruloplasmin, fibrinogen, DMI and ADG a re presented in T able 3 7. Significant positive correlations were observed between ceruloplasmin and fibrinogen concentrations ( P < 0.05), and between ADG and DMI ( P < 0.01). Fibrinogen and ceruloplasmin concentrations were both negatively correlated with ADG and DMI ( P < 0.05). Plasma FA concentrations from d 0 and 29 are presented i n Table 3 6. Concentrations of C16:0, C17:0, C18:0, C18:1c9, SFA, MUFA total n 6 and SFA/UFA were less ( P < 0.03) for CO compared with EN fed steers on d 0 (feedlot entry). Also on d 0, steers suppl emented with CO tended to have decreased d ( P < 0.09) concentrations of LNA, ARA and total FA when compared to EN fed steers. On d 29, CO fed steers had decreas ed ( P < 0.05) plasma concentrations of C14:0, C16:0, C17:0, LA, SFA, PUFA, total n6 and total FA than EN fed steers. When compared to MG fed calves, concentrations on d 29 of C16:0, LA, LNA, PUFA, total n 3, total n6 and total FA were less ( P < 0.04) and tende d to be less ( P < 0.08) for DPA and SFA in CO fed steers. In contrast CO fed steers tended to have a greater ( P = 0.08) SFA to UFA ratio than MG fed steers. On d 29, MG fed steers had less C14:0, C17:0 ( P = 0.05) than EN fed steers, and tended to have grea ter ( P < 0.09) concentrations of EPA, total n3 and n3 to n6 ratio.
48 Discussion Performance Growth can be disrupted by many factors including the pattern of DMI and immunological status (Baumann & Gauldie, 1994) Decreased DMI and BW gain as a result of supplemental fat has been reported previously in cattle (Pavan et al., 2007; Gibb et al., 2004; Harris on et al., 1995; Sklan et al., 1991; Beam & Buttler, 1998). Among the several types of fat sources used as supplements, the CSFA appears to have the grea test impact for inhibiting intake (Bateman et al., 1996; Simas et al., 1995; Ngidi et al., 1990), which might help describe the differences in ADG observed between the fat supplements evaluated in the current experiment. In the feedlot MG fed steers had d ecreased less overall ADG compared with CO and EN fed, and decreased less overall DMI when compared with CO fed steers. In addition, MG fed steers had reduced (P < 0.08) feed efficiency (G:F) compared with EN and CO fed steers. These results a re in agre ement with Allen (2000) who proposed that feed sources of PUFA, mainly CS (i.e. MegalacR) are more detrimental to intake than prilled fats or other sources of SFA. Hess et al. (2008) stated that an optimal inclusion rate for supplemental fat should be less than 3% of dietary DM if the goal is to maximize the use of forage based diets, and supplemental fat should be limited to less than 2% of dietary DM if the goal is to prevent substitution of forage with intake of supplemental fat. The maximum fat cont ent observed in the CO diet was 3.6% (DM basis). In this expe riment a TMR was utilized to feed the treatment supplement with hay and cottonseed hulls. MegalacR and EN contributed 1.4 to 1.7% fat to the final diets which contained a total fat content of 4.9 and 4.8%, respectively. According to Hess et al. (2008), these values indicate that the diets offered during this experiment were formulated to avoid potential negative effects on DMI and fiber digestibility. Further, there were no differences for DMI and ADG between ENand CO fed calves, suggesting that the dietary fat concentration in the diets
49 likely did not inhibit intake, but the type of fat utilized may have contributed to decreased DMI due to lower acceptability of CSFA provided by MG, than the prilled fat provided by EN. According to Grummer et al. (1990), Energy Booster 100 is a source of prilled long chain SFA, which is more acceptable to dairy cows than CSFA (i.e. MegalacR). The authors results showed that the CSFA acceptability was less than other commercial fat sources even if CSFA had been offered alone, in a topdressing, or mixed (TMR) into other dietary components. Zinn (1988 ) and Zinn ( 1989) recommended that fat s hould be introduce d into diets gradually, facilitating the animals adaption for the new feed ingredient. This ex periment was developed as a 2 x 3 factorial design. During the pre shipping phase, steers received EN or no fat supplement (CO) for a period of 40 d prior to transport. In the feedlot, steers allocated to the MG treatment received this fat source for the first time. The absence of gradual adaptation in addition to the lower acceptability of MG, may contribute to the reduced performance measures observed in MG fed steers compared to EN and CO fed steers. Unsaturated long chain FAs are potentially more detrimental on r umi nal fermentation (Sch auff & Clark, 1989) because UFA are more soluble, and therefore are more likely to adsorb onto bacteria ( Chalupa et al., 1984) In other words, impaired ruminal fermentation is more likely with diets containing UFA compared SFA. Calcium salts of FA are classified as rumen inert and may decrease BH rate of PUFA and may increase absorption of PUFA in the small intestine (Wu et al., 1991). Juchem (2007) demonstrated that more than 70% of the LA and more than 85% of the LNA fed to lactating cows were BH in the rumen when fed as unprotected oils or as CS of long chain FA According to the manufact urer, the SFA to PUFA ratio of MG is 0.4 and of EN is 1.2, which means that MG has a greater PUFA proportion content compared to EN. It is suggested,
50 therefore, that MG may more negatively affect ruminal fermentation This negative effect of MG on ruminal fermentation mig ht decrease microbial synthesis and then decrease the availability of amino acids for protein synthesis in other tissues ( Palmquist and Moser, 1981), which might have affected the growth of MG fed steers in the current experiment. This observation is in ag reement with Palmquist (1994) who suggested that feeding more PUFA may negatively affect microbial production in the rumen. Additionally, Schauff & Clark ( 1989) have reported that prilled saturated fat appears to depress the acetate to propionate ratio com pared to CSFA, and therefore, potentially improve animal performance. Inflammatory Reaction In response to immunological stress, the liver will produce the acute phase proteins Cp and Fb (Baum an n & Gauldie, 1994). It is unclear whether increased concentrations of APP are due to greater stress (indicating an adverse state) or due to a greater immune response (indicating a healthier state). In this study the plasma concentration of Fb and Cp ranged from 63.5 to 922.9 mg/dL and 4.8 to 27.1 mg/dL, respecti vely. According to The Merck Veterinary Manual (1997), normal values of APP in cattle range from 100 to 600 mg/dL for Fb and 16.8 to 34.2 mg/dL for Cp. In this study no differences among treatments were observed for mean plasma Cp and covariately adjusted Fb concentrations during the first 29 d post shi pping. I mmune reactions have been reported to be modulated by the diet; including the PUFA composition of the diet (Yacoob & Calder, 1993; Miles & Calder, 1998; Pamposelli et al., 1989). The mechanisms involved in this regulation are not yet understood, but evidences exist that n3 and n6 FA composition in the diet may influence cellular activation through the synthesis of eicosanoids, steroid hormones and cytokines (Calder et al. 2002). Several authors have reported immunological and physiological changes in animals provided diets containing PUFA during immune challenge (Calder et al. 2002; Farran et al., 2008; Cullens, 2005; Silvestre 2009, Do
51 Amaral, 2008), but these studies mainly examined inflam matory processes caused by parturition or LPS challenge, and not by transport. Lessard et al. (2004) evaluated the cellular immune function of dairy cows fed supplemental CS of palm oil, flaxseed and micronized soybeans from 6 wk pre partum to 6 wk postpar tum. The authors concluded that cellular immune function was modulated around parturition; however, feeding diets rich in n3 or n6 FA did not have a major impact on immune function. Cullens (2005) report ed that mean plasma concentrations of Fb during the first 27 d postpartum tended to be greater for control cows than for cows fed CS of long chain FA (MegalacR). In addition, the author suggest ed that the initiation of PUFA supplementation before parturition can affect the immune status and physiological response of mature cows after parturition. In these studies, PUFA supplementation commonly started at least 3 wk prior to immune challeng e and continues at least 3 wk after. The authors suggest that a preliminary period of supplementation is necessary to observe the effects of PUFA on immunomodulation, and the duration of this supplementation period must be long enough to potentiate these effects. Continual feeding of CS of a mixture enriched with fish oil increased concentrations of EPA and DHA in endome trium, liver, mammary, muscle, subcutaneous and internal adipose tissues of dairy cows (Bilby et al., 2006), which may indicate that daily feeding CS of FA is a practical approach to manipulate tissue FA composition (Silvestre, 2009). In the current experi ment, the steers began MG consumption immediately after shipping. Because MG supplementation did not occur during the pre shipping phase of the experiment, chan ges on immune responses by MG could not be expected during the first d after shipping. Further, steers provided EN in the pre shipping phase experienced an APP reaction to shipping, which did not differ from CO fed steers. Energy Booster 100 is a supplemental fat source that is rich in SFA and low in PUFA. In a review of research i nvestigating eff ects of SFA compared
52 to PUFA on measures of immune function, it was concluded that SFA impacts immune competence to a lesser degree compared to PUFA ( Miles & Calder, 1998). Similarly, Farran et al. (2008) observed no difference in concentrations of plasma TNF challenged steers provided tallow (rich in SFA and MUFA) or no supplemental fat (control). Ruminal BH can influence the amount of PUFA reachin g the small intestine, although according to Juchem (20 07), the continual feeding of PUFA, regardless the source, can increase the concentration of PUFA in cells and tissues despite BH It is in agreement with Mattos et al. (2000) who stated that the amount of each FA incorporated into organs and tissues depend on the amount of precursors present in the diet. According to the manufacturer, EN contains 60% SFA and MUFA and 36% PUFA. MegalacR contains 55% PUFA, and LA and LNA represents 87% and 9% of this PUFA. It is suggested that feeding steers with EN or MG may a ffect concentration and composition of FA in the blood differently. Total plasma FA concentration was increased after fat supplementation during pre and post shipping phases of the current experiment. On d 29, MG fed steers tended to have increased EPA and DPA and total n3 FA compared to EN fed steers. Also, MG fed steers had greater LNA and lesser SFA to UFA ratio compared to CO fed steers. It is likely that MG increased plasma concentrations of n 3 FA such as LNA, EPA and DPA due to the greater amount of n 3 FA in this treatment. These results agree with Lessard et al. (2003) who observed no differences in plasma LA concentrations in dairy cows supplemented with Megalac or flaxseed during the first 21 d of the feeding period, like ly due to the similar amounts of LA provided by the Megalac and flaxseed diets. However, greater plasma n 3 FA concentrations were observed in flaxseed fed cows due to a greater concentration of LNA in this source of supplemental fat. In addition, Petit (2002) fed die ts containing Megalac and micronized
53 soybean to lact ating dairy cows during 16 wk post partum. On d 70, there were no treatment differences in plasma concentrations of LNA and EPA; however, greater plasma n 6 FA concentrations were suggested in micronized soybean fed cows because of greater concentration of LA in micronized soybeans compared to Megalac. A negative relationship among plasma APP concentrations and ADG and DMI was observed in this study. Similar findings have been reported in weaned, transported calves (Arthington et al., 2005). These results are supportive of the link between inflammatory processes and feed intake and performance (Johnson, 1998). Growing evidence linking dietary PUFA to immune function, especially modulation of infl ammatory processes (Calder et al. 2002), strengthens the concept for using dietary fats to modulate the acute phase reaction and improve livestock performance. Further studies are required to better understand the effects of PUFA on inflammatory response s of transport stressed beef steers.
54 Table 3 1. Ingredients and nutrient composition of grainbased concentrate treatment s fed to steers during pre and post shipping phase of the study. 1 Treatments 2 CO EN MG Ingredients ( % as fed ) Wheat middlings 30.63 29.97 29.85 Cracked corn 20.00 19.58 19.50 Ground corn 20.00 19.58 19.50 Cottonseed meal 11.85 11.60 11.55 Cottonseed hulls 10.00 9.80 9.75 Cane molasses 6.25 6.12 6.10 Calcium carbonate 1.25 1.22 1.22 Mineral and vitamin mix 3 0.03 0.03 0.03 Energy Booster 100 2.10 ... Megalac R ... ... 2.50 Nutrient (DM basis) NE g Mcal/kg 1.17 1.24 1.24 TDN, % 72.97 75.42 75.37 CP, % 17.31 16.94 16.87 NDF, % 26.26 25.72 25.60 EE, % 3.93 5.91 6.02 Ca, % 0.68 0.67 0.90 P, % 0.64 0.62 0.62 1 Steers were provided supplement daily (4.1 kg/steer) for a period of 40 d while grazing bahiagrass pastures (54.0 and 9.6% TDN and CP, respectively). 2 CO = grain based supplement nonfortified with a rumen inert fat source; EN = grain based supplement fortified with Energy Booster 100 (MSC Co, Carpentersville, IL), MG = grain based supplement fortified with MegalacR (Church & Dwight C o, Priceton, NJ). 3 Cattle Select (Lakeland Animal Nutrition; Lakeland, FL); Ca (14%), P (9%), NaCl (64%), K (0.2%), Mg (0.3%), S (0.3%), Co (50 ppm), Cu (1,500 ppm), I (210 ppm), Mn (500 ppm), Se (40 ppm), Zn (3,000 ppm), F (800 ppm), Fe (800 ppm), Vitamin A (360,000 ppm).
55 Table 3 2. Fatty acid profile of supplemental fat sources used in the formulation of experimental diets (% of fatty acids) .1 Fat Supplements 2 EN MG C12:0 0.1 0.1 C14:0 2.7 0.9 C16:0 41.5 36.3 C16:1 1.4 0.2 C18:0 38.7 3.9 cis C18:1 12.6 26.7 trans C18:1 0.1 01 C18:2 1.7 28.5 C18:3 0.1 3.0 Others 3 1.1 0.3 1 C12:0 = Lauric acid; C14:0 = Myristic acid; C16:0 = Palmitic Acid; C16:1 = Palmitoleic acid; C18:0 = Ste a ric Acid; cis C18:1 = O leic acid; trans C18:1 = Vaccenic acid; C18:2 = Linoleic Linolenic acid. 2 The fatty acid profile of the fat supplement was determined according to the manufacturer; EN = grain based diet fortified with Energy Booster 100 (MSC Co, Carpentersville, IL); MG = MegalacR (Church & Dwight Co, Priceton, NJ). 3 Others = Not detected.
56 Table 3 3. Nutrient composition of TMR fed to transport stressed steers dur ing the post shipping phase of the study. Treatments 1 Nutrient ( DM basis ) 2 CO EN MG D 1 to 4 3 CP % 14.1 13.9 13.9 NDF % 58.6 58.4 58.4 EE % 2.7 3.4 3.4 TDN % 62.9 63.8 63.7 NE g Mcal/kg 0.8 0.8 0.8 Ca % 0.5 0.5 0.5 P % 0.4 0.4 0.4 D 5 to 12 4 CP % 14.8 14.6 14.5 NDF % 46.5 46.2 46.1 EE % 3.4 4.6 4.7 TDN % 62.8 64.2 64.2 NE g Mcal/kg 0.8 0.9 0.9 Ca % 0.6 0.5 0.7 P % 0.5 0.5 0.5 D 13 to 29 5 CP % 14.9 14.7 14.6 NDF % 44.1 43.8 43.7 EE % 3.6 4.8 4.9 TDN % 62.5 64.1 64.1 NE g Mcal/kg 0.8 0.9 0.9 Ca % 0.6 0.6 0.7 P % 0.5 0.5 0.5 1 CO = grain based diet nonfortified with a rumen inert fat source; EN = grain based diet fortified with Energy Booster 100 (MSC Co, Carpentersville, IL); MG = grainbased diet fortified with MegalacR (Church & Dwight Co, Priceton, NJ). 2 Except NEg which unit is Mcal/kg of DM. 3 D 1 to 4 = free choice of hay and 70:30 mixture of treatment concentrate:CSH6. 4 D 5 to 13 = free choice of 60:25:15 mixture of treatment concentrate:CSH:hay. 5 D 12 to 29 = free choice of 65:28:7 mixture of treatment concentrate:CSH:hay. 6 CSH = Cottonseed hulls.
57 Table 3 4. Effect of supplemental fat source on plasma fatty acid concentrations on d 0 and d 29 of the study1. d 0 d 29 P value 3 SEM 4 Fatty acid 2 CO EN CO EN MG a b c d x y ---------------------------mg/mL --------------------------C14:0 0.0064 0.0073 0.0054 0.0067 0.0054 0.18 0.05 0.92 0.05 0.0006 0.0006 C16:0 0.0880 0.1050 0.1301 0.1611 0.1621 0.01 0.02 0.01 0.93 0.0064 0.0135 C16:1 0.0036 0.0035 0.0034 0.0035 0.0035 0.88 0.82 0.85 0.96 0.0003 0.0006 C17:0 0.0052 0.0061 0.0073 0.0089 0.0074 0.03 0.04 0.91 0.05 0.0004 0.0007 C18:0 0.1061 0.1367 0.1969 0.2344 0.2307 0.01 0.11 0.14 0.87 0.0100 0.0220 C18:1t 0.0080 0.0067 0.0087 0.0109 0.0113 0.11 0.29 0.21 0.85 0.0008 0.0021 C18:1c 0.0695 0.0849 0.0750 0.0850 0.0850 0.01 0.16 0.15 0.97 0.0054 0.0068 C18:2n 6 0.2226 0.2422 0.4422 0.5557 0.5656 0.45 0.04 0.03 0.85 0.0260 0.0550 C18:3n 3 0.0102 0.0130 0.0060 0.0073 0.0090 0.07 0.22 0.01 0.12 0.0010 0.0010 CLAc 0.0009 0.0009 0.0001 0.0001 0.0001 0.98 0.70 0.73 0.97 0.0001 0.0001 CLAt 0.0001 0.0002 0.0002 0.0002 0.0003 0.25 0.99 0.78 0.78 0.0001 0.0001 C20:4n 6 0.0143 0.0122 0.0177 0.0208 0.0183 0.07 0.20 0.79 0.29 0.0010 0.0020 C20:5n 3 0.0029 0.0033 0.0010 0.0010 0.0017 0.26 0.98 0.11 0.09 0.0004 0.0004 C22:5n 3 0.0048 0.0047 0.0019 0.0022 0.0036 0.70 0.74 0.08 0.15 0.0004 0.0010 C22:6n3 0.0021 0.0018 0.0006 0.0005 0.0005 0.19 0.67 0.66 0.99 0.0001 0.0003 SFA 5 0.2056 0.2547 0.3396 0.4110 0.4056 0.01 0.05 0.07 0.88 0.0200 0.0350 MUFA 6 0.0810 0.0951 0.0870 0.0991 0.0997 0.02 0.15 0.12 0.94 0.0059 0.0080 PUFA 7 0.2581 0.2783 0.4697 0.5879 0.5991 0.48 0.05 0.03 0.84 0.0280 0.0580 Total n 3 8 0.0200 0.0230 0.0095 0.0110 0.0150 0.20 0.47 0.01 0.07 0.0022 0.0020 Total n 6 9 0.2370 0.2540 0.4600 0.5800 0.5800 0.03 0.05 0.03 0.89 0.5100 0.0600 Total CLA 10 0.0010 0.0010 0.0003 0.0003 0.0004 0.99 0.86 0.72 0.85 0.0001 0.0002 Total FA 11 0.5447 0.6282 0.8963 1.0980 1.1044 0.09 0.05 0.04 0.94 0.0500 0.1000 Ratios SFA/UFA 12 0.6185 0.7036 0.6240 0.6020 0.5840 0.01 0.33 0.08 0.44 0.0260 0.0225 n3/n 6 13 0.0870 0.0950 0.0200 0.0200 0.0250 0.18 0.83 0.13 0.07 0.0050 0.0030 1 CO = grain based diet non fortified with a rumen inert fat source. EN = grain based diet fortified with Energy Booster (MSC Co, Carpentersville, IL); MG = grain based diet fortified with MegalacR (Church & Dwight Co, Priceton, NJ).
58 2 C12:0 = Lauric acid; C14:0 = Myristic acid; C16:0 = Palmitic Acid; C16:1 = Palmitoleic acid; C1 8:0 = Stearic Acid; C18:1t = Vaccenic; C18:1c9 = Oleic acid ; C18:2n6 = Linoleic acid; C18:3nLinolenic acid; CLAc = cis 9, tra ns 11CLA; CLAt = CLA trans 10, cis 12; C20:4n6 = Arachidonic acid; C20:5n3 = eicosapentaenoic acid; C22:5n 3 = decosapentaenoic acid ; C22:6 = docosahexaenoic acid 3 a = difference of LS means of CO x EN on d 0; b = difference of LS means of CO x EN on d 29; c = difference of LS means of CO x MG on d 29; d = difference of LS means of EN x MG on d 29. 4 x = standard error of measurement on d 0; y = standard error of me asurement on d 29. 5 SFA = C14:0 + C16:0 + C17:0 + C18:0. 6 MUFA = C16:1 + C18:1c + C18:1t. 7 PUFA = C18:2n6 + C18:3n3 + CLAc + CLAt + C20:4n 6 + C20:5n3 + C22:5n3 + C22:6n3. 8 Total n 3 = C18:3n3 + C20:5n3 + C22:5n3 + C22:6n3. 9 Total n 6 = C18:2n6 + C20:4n6. 10 Total CLA = CLAc + CLAt 11 Total FA = Sum of all indentified fatty acids. 12 SFA/UFA ratio = (C14:0 + C16:0 + C17:0 + C18:0) / (C16:1 + C18:1c + C18:1t + C18:2n6 + C18:3n3 + CLAc + CLAt + C20:4n 6 + C20:5n3 + C22:5n3 + C22:6n3). 13 n3/n6 ratio = (C18:3n3 + C20:5n3 + C22:5n3 + C2 2:6n3) / (C18:2n6 + C20:4n6) .
59 Table 3 5. Correlations between plasma measurements, DMI and ADG of transport stressed steers during post shipping phase of the study .1 Item Ceruloplasmin Fibrinogen ADG Fibrinogen 0.25 0.05 ADG 0.26 0.26 < 0.05 < 0.05 DMI 0.39 0.24 0.76 < 0.01 0.05 < 0.01 1 Upper row = correlation coefficients ; Lower row = P values.
60 Figure 3 1. Least squares means of DMI of steers during the post shipping phase of the study. Steers were fed diets containing MegalacR (MG), Energy Booster 100 (EN), or no supplemental fat (CO). The asterisks indicate when CO fed steers had greater (P fed steers (treatment x d ay interaction; P ay effect was observed (P < 0.001). * 0.0 1.0 2.0 3.0 4.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 DMI (% of BW)Days after shipping CO EN MG ** ** **
61 Figure 3 2. Least squares means of covariately adjusted plasma fibrinogen concentrations of steers during the post shipping phase of the study. Steers were fed diets containing MegalacR (MG), Energy Booster 100 (EN), or no supplemental fat (CO). Steers arrived in the feedlot on d 1. A day effect was observed (P < 0.01). 150.0 250.0 350.0 450.0 550.0 1 4 8 15 22 29 Fibrinogen (mg/dL)Days after shipping CO EN MG
62 Figure 3 3. Least squares means of plasma ceruloplasmin concentrations of steers during the post shipping phase of the study. Steers were fed diets containing MegalacR (MG), Energy Booster 100 (EN), or no supplemental fat (CO). Steers arrived in the feedlot on d 1. A day effect was observed (P < 0.01). 10.0 15.0 20.0 25.0 1 4 8 15 22 29 Ceruloplasmin (mg/dL)Days after shipping CO EN MG
63 CHAPTER 4 EFFECTS OF MEGALACR SUPPLEMENTATION ON MEASURES OF PERFORMANCE AND ACUTE PHASE REACTION IN TRASPORTED BEEF HEIF ERS Material and Methods This experiment was conducted from September to November 2007 at the University of Florida IFAS, Range Cattle and Education Center, Ona in 2 phases: pre shipping (d 30 to 0), and post shipping (d 1 to 29). The animals utilized in this experiment were cared for in accordance with acceptable practices outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Animals and Facilities Fort y eight Brahman crossbred heifers (BW SD = 276 32 kg; age SD = 330 17 d) were utilized for this experiment. Prior to the start of the study, heifers were separated from dams and allowe d to acclimate for a period of seven days from the stress of weaning. F ollowing acclimation, heifers were stratified by BW and age, and randomly assigned to two supplementation treatments (pre shipping phase). Treatments consisted of a 1) grain based supplement with MegalacR (MG ), or 2) grain based suppl ement without MegalacR (CO ). Heifers were randomly allocated into six bahiagrass pastures (three pastures/treatment; eight heifers/pasture) and fed for a period of 30 days. On d 0 the heifers were loaded onto a commercial livestock trailer and transported 1,600 km for a period of 24 h. Post shipping, heifers w e re stratified by shrunk BW and assigned to one of the following two housing systems; 1) pasture based system identical to the pre shipping model (n = 24; three pastures / treatment; four heifers / pasture) or 2) individual feedlot facility (24 pens; twelve pens / t reatment; one heifer / pen). Preshipping treatment allo cations were continue d in the 29d post shipping phase
64 Pastures areas used during the pre and the post shipping phase were 1.07 ha in size. Individual pens used during the post shipping phase had concrete floors and were 48 m2 in size. Each pen contained a water source and two f eed bunks (one for hay; one for concentrate). Diets Two grainbased supplement s were utilized for this experiment, one with and one without the inclusion of Megalac R (150 g/heifer daily; Tables 4 1 and 4 2). Supplements were limitf ed at a r ate of 3.0 and 2.5 kg of DM/ heifer daily for CO and MG, respectively during pre and post shipping phases. Q uality of bahiagrass pas tures was determined to be 55.0% TDN and 8.7% CP (DM basis) from hand plucked samples collected at the beginning of the trial and analyzed by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). Samples were taken at 20 locations per pasture according to procedures determinated by Vendramini et al. (2006). P astures were not fertilized prior to or during the experimental period. Stargrass ( Cynodon nlemfuensis ) hay was offered to individually fed heifers in the feedlot in amounts to ensure ad libitum cons umption. Hay quality was calcula ted to be 53.5% TDN and 10.1% CP (DM basis) from hay samples collected weekly and analyzed by a commercial laborato ry (Dairy One Forage Laboratory ) A com plete commercial mineral and vitamin mix and water were offered ad libitum throughout both phases of the experiment (Table 4 3) Sampling D uring the pre shipping phase heifers shrunk BW (16 h of feed restriction) was recorded on d 30. In the post shipping phase, heifers shrunk BW was recorded on d 1 (immedia tely foll owing arrival at feeding facility) and at the end of the experiment (d 29) Full BW was recorded on d 15, 0, 4, 8, 15, 22, 28 in order to calculate DMI as a % of BW S hrunk BW values were utilized to determine ADG during the pre and post shipping phases.
65 During the post shipping phase, daily samples of offered and refused hay were collected, weighed, and analyzed for DM. Random representative samples of supplements ingredients were collected on a weekly basis for each phase of the trial for DM and nutrient determination. Samples were dried in a 60oC forced air oven for 96 h, ground through a 1mm Wiley laboratory mill screen (Model 4, A. H. Thomas, Philadelphia, PA) and analyzed for nutrient composition according to analytical procedures of a commercial feed laboratory ( Dairy One Forage Laboratory Ithaca, NY). Blood samples were collected from heifers on d 0, 1, 4, 8, 15, 22, and 28 and analyzed for plasma concentrations of ceruloplasmin, haptoglobin, and cortisol Blood A nalysis All b l ood samples were collected via jugular venipuncture into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, placed on ice immediately after sampling, and centrifuged (GPR Centrifuge, Model 349702; Beckman Instruments Inc., Fullerton, CA) at 2,000 x g for 30 min for plasma separation The plasma of each sample was removed by transfer pipet to it respective vial and frozen at 20C on the same day of collection. A microplate spectrophotometer (PowerWave 340; BioTek Instruments, Inc., Winooski, VT) was used to determine plasma haptoglobin concentrations in duplicate samples by measuring haptoglobin/hemoglobin complexing ( HpHbB ) by the estimation of differences in peroxidase activity as described previously (Makimura and Suzuki, 1982). For haptoglobin concentrations CV was 12.6%, and for concentrations >1.0 mg of HpHbB/100 mL, the intraassay CV was controlled to values 7.1% The interassay CV was controlled to values 8.9%
66 A spectrophotometer (ThermoSpectronic Genesys 20; Thermo Fisher Scientific Inc., Waltham, MA) was used to determine plasma ceruloplasmin concentration. P lasma ceruloplasmin oxidase activity was measured in duplicate samples using colorimetric proc edures described by Demetriou et al. (1974). The intraassay and interassay CV were 4.6 and 8.8% respectively Ceruloplasmin concentrations were expressed as mg/dL as described by King (1965). Concentrations of plasma cortisol wer e determined using a Coa t A Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid phase 125I radioimmunoassay (RIA) via Gama Counter ( Packard Cobra Auto Gamma 85005, GMI Inc., Ransey, MN ). All samples were analyzed in duplicate with procedures previously validated for bovine samples (Eicher et al., 2000). Reference plasma samples containing 50 ng/mL of cortisol were ana lyzed for the calculation of intra and interassay CV (5.5 and 6.4%, respectively). The minimum detectable concentration of cortisol was 5 ng/mL. Stati stical A nalysis Data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001). The statistical model used for plasma measurements and DMI from individually fed heifers was: Yijk = + TRTi + Dj + TRTDij + HEIFERk(i) + Eijk where Y = response variabl e = mean TRT = fixed effect of treatment D = fixed effect of d ay TRTD = effect due to interaction of treatment and d ay
67 HEIFER = random effect of heifer within treatment E = residual error The statistical model used for ADG analysis for data obtained from individually fed heifers was: Yij = + TRTi + HEIFERj(i) + Eij where Y = response variable = mean TRT = fixed effect of treatment HEIFER = random effect of heifer within treatment E = residual error The statistical model used for ADG analysis for data obta ined from heifers on pasture during the pre shipping phase was : Yijk = + TRTi + HEIFERj(k ) + PENk(i ) + Eijk where Y = response variable = mean TRT = fixed effect of treatment HEIFER = random effect of heifer within pen PEN = random effect of pen within treatment E = residual error Results are reported as least square means. Means were separated using LSD. Significance was determined at P
68 significant interactions were reported. Pearson correlation coefficients among plasma measurements, ADG and DMI were generated using the CORR procedure of SAS (SAS, 2001). Results Measurements of Performance N o differences ( P = 0.99) were reported in ADG between treatments in the pre shipping phase (0.12 kg/ d for both CO and MG treatments; SEM = 0.06). In addition, no post shipping differences ( P SEM = 0.13) and G:F ratio (0.13 and 0.11 for CO and MG treatments, respectively; SEM = 0.01) among heifers maintained in the individual feeding facility were detected N o treatment effects ( P < 0.27) or treatment x day interactio n ( P < 0.77) were detected for total DMI (2.01 and 1.92% of BW for CO and MG; SEM = 0.06; Figure 41) and voluntary hay DMI (1.03 and 1.10 % of BW for CO and MG, respectively; SEM = 0.04) of heifers following transportation. A day effect w as observed (P < 0.01) Plasma Measurements and Coefficients of Correlation N o treatment ( P ay interactions ( P ceruloplasmin (31.5 vs. 29.1 mg/dL for CO and MG, respectively; SEM = 1.40; Figure 42) and plasma cortisol ( 3.97 and 3.62 ug/mL for CO and MG, respectively; SEM = 0.39; Figure 4 3) concentrations were detected A t reatment ( P < 0.01) effect was detect ed for plasma haptoglobin concentration ( 0.021 and 0.013 absorption at 450 nm x 100 for CO and MG, respectively; SEM = 0.002). Control fed heifers had greater haptoglobin concentrations on d 1, 3 and 7 (Treatment x day interaction, P < 0.002; Figure 4 4 ) compared to MG fed heifers. The effect of d ay was significant ( P < 0.01) for all analyses completed. Pearson correlation coefficient s among ceruloplasmin, haptoglobin, cortisol DMI and ADG are presented in Table 4 4. Significant positive ( P s were detect ed between ceruloplasmin x co rtisol, and haptoglobin
69 x DMI. The r elationship between ADG x DMI ten ded ( P = 0.09) to be positive. A negative ( P 0.05) correlation was observed between ceruloplasmin x ADG. Discus sion Performance The mean s between supplement treatments for ADG, DMI and G:F were nonsignificant, even if though differences were numerically less for MG than CO fed heifers. Th ese data are in agreement with Espinoza et al. (1995) and Kucuk et al. ( 2004) who suggeste d no effect of CSFA supplementation on the performance of growing and finishing steers. Differences in per formance responses in Exp. 1 compared to Exp. 2 may be a result of adaptation to the supplemental fat. H eifers in Exp. 2 were offered MG supplements during the 30d pre shipping phase. This allowed for a period of adaptation to MG, which may have allowed for a more consistent DMI in the post shipping phase of the study. In contrast, the steers in Exp. 1 were not provided a period of MG a cclimation prior to the start of the postshipping phase of the study. Further, in Exp. 2, heifers were provided their fat supplements in a limit fed concentrate offered sep arately from the ground hay whereas fat was mixed in the TMR in Exp. 1. Complete c onsumption of the daily supplement was observed for all heifers, suggesting that the heifers were adapted to their fat treatment. A ccording to Grummer et al. (1990 ) and Zinn (1988), a period of adaptation to fat supplementation is essential to maintain optimal feed intake; especially when CS of FA are the source of fat being supplemented. Inflammatory Reaction The s ignificant d ay effect for plasma co ncentrations of Cp and cortisol was detected in the study, wh e re independent of fat treatment; concentrations increased after shipping. According to The Merck Veterinary Manual (1997), normal values of Cp in cattle range from 16.8 to 34.2 mg/dL, and in this experiment C p range d from 17.8 to 45.8 mg/dL. The peaks at d 1
70 followed by a period of decreasing concentration of Cp confirm the presence of an acutephas e inflammatory reaction in heifers fed treatments by d 1. This response is in agreement to a previous study by Ar thington et al. (2008) that observed peak Cp during the first 72 h after transport followed by a gradual decrease in the concentration of this protein. In addition, Cullens (2005) observed no differences in plasma Cp concentrations in post partum lactating dairy cows supplemented with CSFA (MegalacR) compared to cows receiving no supplement fat although lactating heifers had lower concentration of plasma Cp when fed CSFA A treatment x d ay interaction was observed for Hp due to a g reater concentration i n CO compared to MG fed heifers on d 1, 3 and 7 after shipping. This response suggests that CO fed heifers experienced a greater pro inflammatory response to shipping compared with MG fed heifers. Because in this experiment Hp was detected in plasma of all heifers suggesting that each was experiencing an acutephase pro inflammatory reaction. Supplementation with MG resulted in decreased Hp concentrations compared wit h CO fed heifers. This decrease in Hp appears to be an anti inflammatory response to the stress caused by shipping, and this type of response might be expected when animals are supplemented with a n n3 and not an n 6 (i.e. MegalacR) FA source The inclusion of PUFA into diets has been shown to modulate immune cell function (Calder et al., 2002). Schmitz & Ecker (2008) stated that the main difference between n6 and n3 FA derived eicosanoids is that most of the mediators formed from ARA are pro inflammatory; whereas those formed from EPA and DHA are anti inflammatory. MegalacR is a CS of PUFA rich in LA (30% of total FA) which have been shown to stimulate cytokines (Fa r ran et al., 2008) and other physiological mediators such as PGE2 and LTB4 (Yaqoob & Calder, 1993).
71 Lessard et al., ( 2004) suggested that the regulation of cytokine synthesis can be influenced by differences in the ratio of n3 and n 6 PUFA in blood and other tissues. Lessard et a l. (2003) fed whole flaxseed (16% LA; 57% LNA), CS of palm oil (8% LA; 0.3% LNA) and micronized s oybeans (57% LA; 7 % LNA) to lactating dairy cows from calving to 108 d of lactation. Twenty one d after calving and 20 d after artificial insemination, serum concentrations of LNA and the n 3 to n6 ratio was greater for cows fed whole flaxseed than cows f ed microni zed soybeans or CS of palm oil. Blood serum concentrations of PGE2 were reduced in cows fed flaxseed for a minimum of 60 d, which is in agreement to the proposed anti inflammatory effects of n 3 supplementation (Lessard et al., 2003) Other research has shown conflicting results relative to the anti inflammato ry and inflammatory effects of n 3 and n6 FA. Do Amaral (2008) reported lowe r plasma Cp concentrations in lactating heifers fed CS of palm and fish oils after calving compared to those fed an n 6 FA supplement. G reater plasma Fb concentrations were found in multiparous lactating cows fed an n 6 FA supplement compared to cows fed an n 3 FA supplement Yaqoob & Calder (2007 ) stated that DHA and LNA can be converted to EPA in animal cells. Be cause EPA is able to compete with ARA for the same enzymes and receptors, EPA can inhibit the production of eicosanoids such as PG 2 and LT 4 series from ARA (Calder, 1999); thereby reducing inflammation. The concentration of LNA and other n3 FA in rumina nt tissu es is less compared with other FA, such as LA and stearic acid. Therefore, small tissue changes in n3 FAs concentration may cause significant physiological transformation in cellular activation and communication, mainly due to synthesis of eicosa noids. In the post shipping phase of Exp. 2, 50% of the diet was stargrass hay, and during the pre shipping phase, heifers were grazing bahiagrass pasture. In general, forages are recognized to
72 have greater n 3 FA concentration compared with grains which have greater n 6 FA content (Jurgens, 2002). The natural protection of the forage provided by cell wall components may be more adequate to prevent the release of fat and thus BH of PUFA, compared to the prot ection of other diet components such as grains an d oils (Petit, 2002). Perhaps the combination of both factors: greater concentration of protected n 3 FA provided by forage, and greater changes in PUFA profile (mainly LA) caused by the ruminal BH, would allow increased absorption of n3 FA in the small intestine by MG fed heifers, and consequently, would influence the magnitude of an inflammatory response. According to Farran et al. (2008), BH can be affected by many factors including: nature and amount of dietary lipid fed, type of protective treatment and the nature and amount of forages and concentrates included in the diet. The reason to protect fat is to provide a greater amount of U FA in the small intestine, although CSFA are not completely inert to the actions of rumen bacteria (Wu et al., 1981). Sukhija & Palmquist (1990) suggested that the FA profile of CS may affect properties of inertness. According to the authors, CS of SFA are less dissociated than CS of UFA at any given ruminal pH. In agreement, Juchem (2007) observed BH rates greater than 85% for LA and 92% for LNA when fish oil were supplemented in CS or oil form Despite BH, continuous, long term PUFA supplementation has shown to modulate immune responses in cattle (Bil by et al., 2006; Silvestre, 2009), because certain amounts of PUFA ar e able to successfully escape microbial BH in the rumen, and be absorbed in the small intestine ( Palmquist & Jenkins, 1980). If BH occurs, the concentration of LA in the small intestine compared to that cons umed by the animal will be less, and like ly the concentrations of intermediate FA, such as trans C18:1 and CLA will be greater. A greater dietary supply of LA and LNA is known to increase milk
73 C18:1 concentrations (Dhiman et al., 1995) and CS of fish oil result in a greater concentration of trans fatty acids in the rumen (Baumgard et al., 2000). Lundy, III et al. (2004) observed a greater concentration of trans C18:1 in the omasum of dairy cows supplemented with CS or amides of soybean oil (56% LA) than cows supplemented with unprotected soybean oil. Thi s observation is in agreement with Harfoot & Hazlewood (1978) who suggested that greater concentrations of PUFA in ruminal contents may cause the accumulation of trans C18:1 by inhibiting the conversion of trans C18:1 to C18:0 (stearic acid), especially when PUFA are present as free acids. If this theory is applied to the current experiment, MG fed heifers would have had a greater absorption of trans fatty acids and CLA, which may affect FA composition, especially the n 3 to n6 ratio, of cellular membranes Monounsaturated fatty acids, such as oleic acid (C18:1) are non essential since they can be synthesized de novo In vitro studies have shown that oleic acid is able to suppress lymphocyte inflammation (Miles & Calder, 1998), additionally, Jeffery et al. (1996) observed that feeding diets containing high oleic sunflower oil and olive oil (80% C18:1) decreased the proliferation of spleen lymphocytes in rats compared with feeding low fat or safflower oil (75% LA) diets. Besler & Grimble (1995) demonstrated that feeding rats for eight wk with diets containing 5 or 10% butter (rich in oleic acid) or olive oil completely suppressed increases of tissue zinc content, liver protein synthesis, and serum Cp concentrations in response to subcutaneous Escherichia coli endotoxin, when compared with maize oil (47% LA) diet. These studies are in agreement with Yaqoob (2002) who stated that MUFA rich oils have physiological effects which are similar to fish oils. Pariza et al. (2000) demonstr ated that CLA supplementation t o LPS challenged rats appeared to stimulate an anti inflammatory response. A dditionally, Cook et al. (1993) observed that CLA derived eicosanoi ds could affect synthesis of PGs negatively;
74 therefore decreasing synthesis of TNF it is suggested that increased availability of MUFA, mainly trans C18:1, and CLA may modulate anti inflammatory immune responses in mammals The APP are often u sed as a marker of stress and which is commonly confused with an undes irable response (Silvestre, 2009). Howeve r, the acute phase reaction provides an early nonspecific defense mechanism against insult before specif ic immunity is achieved (Peterse n et al., 2004). In turn, Calder et al. (2002) suggested controversy exists in the literature concerning the effects of n6 and n3 FA on the immune response, and the discrepancies among experiments could be due to differences among species, source of PUFA added to diets, time of supplementation, and physiological status of the animals. The concentrations of cytokines in blood or in neutrophills were not evaluated in this study. However, several authors (Johnson, 1997; Spurlock, 1997; Calder, 1999) have reported that synthesis of APP, such as Hp, is directly stimulated by cytokines, mainly IL 6 and TNF is suggested that MG fed heifers experienced decreased blood Hp concentrations due to a reduced inflammatory reaction may caused in response to PUFA supplementation, even if LA is one of the FA present in more abundance in the MG product
75 Table 4 1. In gredient and nutrient composition of grainbased supplements fed heifers during the pre and post shipping phases of the study. Treatments 1 CO MG Ingredient (% of dietary DM) Soybean Hulls 74.3 72.6 Cracked Corn 10.3 Cottonseed Meal 14.1 21.5 Megalac R 5.9 Limestone 1.2 Component ( DM basis) NE g Mcal/kg 0. 8 0.85 TDN, % 63. 3 67.1 CP % 16.9 19.4 NDF % 54.9 55.0 EE % 3.0 7. 9 Ca, % 0.5 1.2 P % 0. 3 0.3 1 CO = grain based supplement with out MegalacR (Church & Dwight Co, Priceton, NJ); MG = g rain based supplement with MegalacR.
76 Table 4 2. Fatty acid profile of supplemental fat source used in the formulation of experimental MG supplement (% of fatty acids) .1 Fat Supplement 2 MG C12:0 0.1 C14:0 0.9 C16:0 36.3 C16:1 0.2 C18:0 3.9 cis C18:1 26.7 trans C18:1 01 C18:2 28.5 C18:3 3.0 Others 3 0.3 1 C12:0 = Lauric acid; C14:0 = Myristic acid; C16:0 = Palmitic Acid; C16:1 = Palmitoleic acid; C18:0 = Ste a ric Acid; cis C18:1 = Oleic acid; trans C18:1 = Vacceni c acid; C18:2 = Linoleic Linolenic acid. The fatty acid profile of the fat supplement was determined according to the manufacturer. 2 MG = MegalacR (Church & Dwight Co, Priceton, NJ). 3 Others = Not de tected.
77 Table 4 3. Nutrient composition of mineral and vitamin mix supplement.1 Amount Macro elements (%) Calcium (Ca) 14.0 Phosphorus (P) 9.0 Sodium chloride (NaCl) 64.0 Potassium (K) 0.2 Magnesium (Mg) 0.3 Sulfur (S) 0.3 Micro elements (ppm) Cobalt (Co) 50 Copper (Cu) 1,500 Iodine (I) 210 M a n g a nese (Mn) 500 Selenium (Se) 40 Zinc (Zn) 3,000 Fluorine (F) 800 Iron (Fe) 800 Vitamins (IU/kg) Vitamin A 360,000 1 The nutrient composition of the mineral and vit amin mix supplement was supplie d by the manufacturer. (Cattle Select; Lakeland Animal Nutrition; Lakeland, FL).
78 Table 4 4. Correlations between plasma measurements, DMI and ADG of transport stressed heifers during post shipping phase of the study.1 Item Haptoglobin Ceruloplasmin Cortisol DMI Ceruloplasmin 0.32 0.13 Cortisol 0.10 0.40 0.65 0.05 DMI 0.51 0.03 0.03 0.01 0.90 0.88 ADG 0.14 0.40 0.18 0.35 0.51 0.05 0.39 0.09 1 Upper row = correlation coefficients Lower row = P values.
79 Figure 4 1. Least squares mean s for DMI of pen fed heifers during the post shipping phase of the study. Heifers were fed grain based supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. A d ay effect was detect ed (P < 0.001). 1.25 1.50 1.75 2.00 2.25 2.50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 DMI (%BW)Days after shipping CO MG
80 Figure 4 2. Post shipping concentrations of plasma ceruloplasmin of heifers fed grainbased suppl ements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. Heifers were loaded onto a trailer on d 0 and arrived in the feedlot on d 1. A day effect was observed (P < 0.001). 15.0 20.0 25.0 30.0 35.0 40.0 0 1 3 7 14 21 28 Ceruloplasmin (mg/dL)Days after shipping CO MG
81 Figure 4 3. Post shipping concentrations of plasma cortisol of heifers fed grain based supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. Heifers were loaded onto a trailer on d 0 and arrived in the feedlot on d 1. A day effect was observed (P < 0.001). 2.0 2.7 3.4 4.1 4.8 5.5 0 1 3 7 14 21 28 Cortisol ( g/mL)Days after shipping CO MG
82 Figure 4 4. Plasma concentrations of haptoglobin of heifers fed grainbased supplements containing MegalacR (MG) or no supplemental fat (CO) from 30 d before to 27 d after shipping. Heifers were loaded onto a trailer on d 0 and arrived in the feedlot on d 1. The asterisks at d 1, 3 and 7 indicate that CO fed heifers had greater plasma concentration of haptoglobin compared to MG fed heifers (Treatment x d interaction; P < 0.001). A d e ffect was observed ( P < 0.001). 0.00 0.01 0.02 0.03 0.04 0.05 0 1 3 7 14 21 28 Haptoglobin (abs @ 450 nm x 100)Days after shipping CO MG
83 CHAPTER 5 GENERAL CONCLUSION The data from these experiments suggest MG supplementation to growing cattle during the feedlot receiving period negatively affect s ADG, DMI and G:F negatively mainly if they have not been ex posed to it previously. One potential explanation for this response is palatability. A poor acceptability of CS of PUFA by cattle may impact DMI, particularly over a short term, feedlot receiving period (approximately 30 d). A period of adaptation to MG m ay be a useful prior to transport and feedlot entry. Supplementation of MG appears to impact the inflammatory reaction of transport stressed cattle. However, it appears likely that supplementation of MG, prior to the immune challenge, is required to illic it this response. This supposition is derived from the current studies, where heifers supplemented with MG for 30 d prior t o shipping experienced a decreased acutephase reaction compared to CO fed heifers. In contrast, the same results were not observed in transport ed steers that started MG supplementation only after shipping stress and received into the feedlot The reason why MG fed heifers experienced a reduced inflammatory response is unknown. However, one explanation might be related to the concentr ation of MUFA and LA in the diet. In addition, the ruminal BH rate may influence the amount and type of FA absorbed in the small intestine. Further research is required to understand the effects of the supplemental PUFA sources, as well as the timing of P UFA supplementation on measures of performance and inflammation of transport stressed beef calves.
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96 BIOGRAPHICAL SKETCH Davi Brito de Araujo was born in Mogi Mirim, a small town located in the Citrus Belt of the S tate o f So Paulo/Brazil in 1981. He is the oldest son of Eduardo Netto de Araujo and A rlete Aparecida Brito de Araujo; and brother of Marlia Brito de Araujo. He is also the oldest grandson of Eivany Julianetti de Brito, who owns a cattle ranch in Aquidauana / MS, and an orange grove and feedlot ranch in Mogi Mirim /SP, where he spent all his childhood and when hi s background in cattle began. D avi started the School of Veterinary Medicine at the So Paulo State University (UNESP) Botucatu / SP, in 2000. The UNESP Botucatu Vet School has been ranked among the Top 2 programs in the country during the last 15 years. During the five years of the vet school program, Davi participated at the Student Enterprise of Beef and Dairy Production (CONAPEC Jr.) with his first advisor Dr. Jos Luiz Moraes Vasconcelos, who offered the opportunity for Davi to study as an intern in the USA during his last two s emeste rs of his Vet School program. In January 2005, Davi started an internship at UC Davis VMTRC ( Tulare, CA) advised by Dr Jos Eduardo Portela Santos, where he had the chance to work with nutrition, reproduction, and health of dairy cattle. On July 2005, he moved to Ona, Florida, where he started another internship, advised by Dr. John Arthington at the Range Cattle REC. Davi received his DVM degree in December of 2005, and came back to the RCREC in March of 2006 to work on another internship; as a result he started a MS degree in the summer of 2007. In 2008, Davi started a MAB which is combined with his first MS in the Department of Food Resource and Economics advised by Dr. Allen Wysocki.
97 At Ona, Davi have w orked with Dr. Arthington on evaluation of strategies to improve performance and health of immune challenged beef cattle Fatty acids and trace minerals supplementation to calves during the receiving period of feedlot are the highlights of his research. During his four years in the US, Davi has already published with other authors, three manuscripts in refere ed journals and 16 abstracts.