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The Effects of Anti-Phospholipase A2 Antibody Supplementation on Feed Efficiency, Animal Performance, and the Acute Phas...

Permanent Link: http://ufdc.ufl.edu/UFE0045052/00001

Material Information

Title: The Effects of Anti-Phospholipase A2 Antibody Supplementation on Feed Efficiency, Animal Performance, and the Acute Phase Response of Background and Finishing Beef Cattle Diets
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Mercadante, Vitor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: a2 -- acute -- beef -- ceruloplasmin -- diets -- finishing -- haptoglobin -- phase -- phospholipase -- proteins -- response
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: a2cattleTo determine whether supplementation of anti-phospholipase A2 antibody (aPLA2)animal performance, and the acute phase response of transportation, and background and finishing beef cattle diets, two experiments were conducted.Experiment I: Individual performance and daily dry matter intake (DMI) was measured on 70 cross-bred weaned calves during a 70-d period using a GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada) at the University of Florida NFREC Feed Efficiency Facility (FEF). Calves were fed a growing diet, and were blocked by weight and sex, and then randomly assigned to pens to receive either no additional supplement (CON, n = 35) or receive a supplement at an inclusion rate of 0.6% of estimated daily DMI of aPLA2 antibody (aPLA2; n = 35). After the 70-d feed efficiency (FE) trial calves were loaded into a commercial livestock trailer and were driven for »1.600 km during 24 hr. Upon return to the FEF, calves were relocated to the same pens and groups, and received the same diets and treatments. Blood samples from each calf were collected on days 0, 1, 3, 5, 7, 14, 21 and 28 relative to transportation and were analyzed for determination of concentrations of plasma ceruloplasmin and haptoglobin. Initial BW (242.0 ± 3.7 kg; P = 0.92), BW d 70 (313.0 ± 4.1 kg; P = 0.79), and ADG (1.01 ± 0.02 kg; P = 0.95) were similar between treatments.  However, daily DMI was greater (P = 0.01) for CON (8.53 ± 0.15 kg) than aPLA2 (9.18 ± 0.15 kg).  In addition, RFI was greater (P = 0.002) for CON (0.389 ±0.110 kg/d) than aPLA2 calves (-0.272 ± 0.110 kg/d). After transportation there were no differences between treatments on BW loss (26.0 ± 0.6 kg; P = 0.86), BW d 28 (339.0 ± 4.1 kg; P = 0.72), ADG (1.28 ± 0.03 kg/d; P = 0.72), G:F (0.164 ± 0.004; P = 0.83), and concentrations of plasma haptoglobin (0.08 ± 0.02; P = 0.41).However, concentration of plasma ceruloplasmin was greater (P P = 0.79), ADG (1.25 ± 0.02 kg/d, P = 0.33),average daily DMI (7.1 ± 0.1 kg, P =0.43), and RFI (-1.25 ± 0.06 kg/d, P= 0.61). However, during the growing forage diet period daily DMI tended (P = 0.07) to be lower for aPLA2 0.2% and 0.4% treatments (6.51 ± 0.25 kg and 6.70 ± 0.25 kg, respectively) compared to CON (7.38 ± 0.25 kg) treatment, with MT (7.09 ± 0.25 kg) treatment being intermediate. Steers from aPLA2 0.2% and aPLA2 0.4% treatments (-0.12 ± 0.13 kg/d and -0.22 ± 0.13 kg, respectively) had lower (P P = 0.04) RFIthan CON (-0.37 ± 0.10 kg/d) steers. During the transition to grain-based dietphase WBC were greater (P = 0.04) foraPLA2 0.2% (13.61 ± 0.42 k/µL) than aPLA2 0.4% and MT treatments (12.16 ± 0.42and 12.37 ± 0.42 k/µL, respectively), with CON being intermediate (12.87 ± 0.42k/µL), and concentrations of lymphocytes also were greater (P = 0.01) for aPLA2 0.2% (7.66 ± 0.28k/µL) than aPLA2 0.4% and MT treatments (6.71 ± 0.28 and 6.70 ± 0.28 k/µL,respectively), with CON being intermediate (7.11 ± 0.28 k/µL). Concentrations of plasma ceruloplasmin was reduced (PP < 0.05) for CON (0.18 ± 0.05 mg/mL) steers compared to aPLA2 (0.26 ± 0.05 mg/mL) treatments. Mean daily DMI was negatively correlated with concentrations of plasma ceruloplasmin and haptoglobin. In conclusion, beef cattle supplemented with aPLA2 had improved FE when fed growing forage diets, but not grain-based diets, and had reduced concentrations of plasma ceruloplasmin after 24 hr transportation. However, CON steers had reduced concentrations of plasma ceruloplasmin and haptoglobin during the transition to grain-based diet compared to steers on aPLA2 and MT treatments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vitor Mercadante.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Lamb, Graham.
Local: Co-adviser: Dilorenzo, Nicolas.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045052:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045052/00001

Material Information

Title: The Effects of Anti-Phospholipase A2 Antibody Supplementation on Feed Efficiency, Animal Performance, and the Acute Phase Response of Background and Finishing Beef Cattle Diets
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Mercadante, Vitor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: a2 -- acute -- beef -- ceruloplasmin -- diets -- finishing -- haptoglobin -- phase -- phospholipase -- proteins -- response
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: a2cattleTo determine whether supplementation of anti-phospholipase A2 antibody (aPLA2)animal performance, and the acute phase response of transportation, and background and finishing beef cattle diets, two experiments were conducted.Experiment I: Individual performance and daily dry matter intake (DMI) was measured on 70 cross-bred weaned calves during a 70-d period using a GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada) at the University of Florida NFREC Feed Efficiency Facility (FEF). Calves were fed a growing diet, and were blocked by weight and sex, and then randomly assigned to pens to receive either no additional supplement (CON, n = 35) or receive a supplement at an inclusion rate of 0.6% of estimated daily DMI of aPLA2 antibody (aPLA2; n = 35). After the 70-d feed efficiency (FE) trial calves were loaded into a commercial livestock trailer and were driven for »1.600 km during 24 hr. Upon return to the FEF, calves were relocated to the same pens and groups, and received the same diets and treatments. Blood samples from each calf were collected on days 0, 1, 3, 5, 7, 14, 21 and 28 relative to transportation and were analyzed for determination of concentrations of plasma ceruloplasmin and haptoglobin. Initial BW (242.0 ± 3.7 kg; P = 0.92), BW d 70 (313.0 ± 4.1 kg; P = 0.79), and ADG (1.01 ± 0.02 kg; P = 0.95) were similar between treatments.  However, daily DMI was greater (P = 0.01) for CON (8.53 ± 0.15 kg) than aPLA2 (9.18 ± 0.15 kg).  In addition, RFI was greater (P = 0.002) for CON (0.389 ±0.110 kg/d) than aPLA2 calves (-0.272 ± 0.110 kg/d). After transportation there were no differences between treatments on BW loss (26.0 ± 0.6 kg; P = 0.86), BW d 28 (339.0 ± 4.1 kg; P = 0.72), ADG (1.28 ± 0.03 kg/d; P = 0.72), G:F (0.164 ± 0.004; P = 0.83), and concentrations of plasma haptoglobin (0.08 ± 0.02; P = 0.41).However, concentration of plasma ceruloplasmin was greater (P P = 0.79), ADG (1.25 ± 0.02 kg/d, P = 0.33),average daily DMI (7.1 ± 0.1 kg, P =0.43), and RFI (-1.25 ± 0.06 kg/d, P= 0.61). However, during the growing forage diet period daily DMI tended (P = 0.07) to be lower for aPLA2 0.2% and 0.4% treatments (6.51 ± 0.25 kg and 6.70 ± 0.25 kg, respectively) compared to CON (7.38 ± 0.25 kg) treatment, with MT (7.09 ± 0.25 kg) treatment being intermediate. Steers from aPLA2 0.2% and aPLA2 0.4% treatments (-0.12 ± 0.13 kg/d and -0.22 ± 0.13 kg, respectively) had lower (P P = 0.04) RFIthan CON (-0.37 ± 0.10 kg/d) steers. During the transition to grain-based dietphase WBC were greater (P = 0.04) foraPLA2 0.2% (13.61 ± 0.42 k/µL) than aPLA2 0.4% and MT treatments (12.16 ± 0.42and 12.37 ± 0.42 k/µL, respectively), with CON being intermediate (12.87 ± 0.42k/µL), and concentrations of lymphocytes also were greater (P = 0.01) for aPLA2 0.2% (7.66 ± 0.28k/µL) than aPLA2 0.4% and MT treatments (6.71 ± 0.28 and 6.70 ± 0.28 k/µL,respectively), with CON being intermediate (7.11 ± 0.28 k/µL). Concentrations of plasma ceruloplasmin was reduced (PP < 0.05) for CON (0.18 ± 0.05 mg/mL) steers compared to aPLA2 (0.26 ± 0.05 mg/mL) treatments. Mean daily DMI was negatively correlated with concentrations of plasma ceruloplasmin and haptoglobin. In conclusion, beef cattle supplemented with aPLA2 had improved FE when fed growing forage diets, but not grain-based diets, and had reduced concentrations of plasma ceruloplasmin after 24 hr transportation. However, CON steers had reduced concentrations of plasma ceruloplasmin and haptoglobin during the transition to grain-based diet compared to steers on aPLA2 and MT treatments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vitor Mercadante.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Lamb, Graham.
Local: Co-adviser: Dilorenzo, Nicolas.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045052:00001


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1 THE EFFECTS OF ANTI PHOSPHOLI P A SE A2 ANTIBODY SUPPLEMENTATION ON FEED EFFICIENCY, ANIMAL PERFORMANCE AND THE ACUTE PHASE RESPONSE OF BACKGROUND AND FINISHING BEEF CATTLE DIETS By VITOR RODRIGUES GOMES MERCADANTE 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 2012

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2 2012 Vitor Rodrigues Gomes Mercadante

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3 To my grandfather, Oripes

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4 ACKNO WLEDGMENTS I am deeply thankful to Dr. G. Cliff Lamb for allowing me the opportunity to come to UF a nd for believing i n my potent ial when he invited me to work i n his group during my Masters program. Throughout the years he has been much more than just a n advisor he is a great mentor, a role model and a good friend. I would also like to thank Dr. Nicolas DiLorenzo, he is an outstanding researcher who has been extremely helpful during my research trials and became a dear friend. In addition thank you to Dr. John D. Arthington for all support, friendship and many good laughs! I consider myself extremely lucky and blessed for having committee members that w ere deeply involved i n my formation allowing me to learn so much and grow as a professional and as a p erson. F or that I will always be grateful. My desire to further my studies came during my training in vet school, under the He was my first advisor, and introduced me to research and extension. It was through him that I met Dr. Lamb and came to the U.S. A on an internship program. Zequinha is a great man with strong values proud to say that I was one o f his students and that he is a personal friend. During the past two years I have made many good frien ds. My incredible lab mates Dr. Guilherme Marquezini, Tera Black, Kalyn Waters, Darren Henry and Francine Messias We worked as true team, and together we have made collecting sample s working cattle and even milking beef cows a lot more fun! Also, thank you to Philipe Moriel for his friendship and for helping me with lab work. To all my friends in the A nimal Sciences Department the amazing NFREC crew in Marianna and all others

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5 who are just too many to list here. I am eternally thankful for the support a nd good moments we shared. You all have made grad school a lot easier for me I cannot thank my family enough for the unconditional support throughout the years My father Guilherme, my mother ngela, and my brothers Rafa and Joo are always present and ve ry close to me even when thousands of miles away M y beloved grandparents Oripes and Gija who are the reason I am passionate about agriculture. Thanks to m y in laws Hans and Waleria for the friendship and kindness. And lastly, m y wife Paula, who is my be st friend and the love of my life! She is the fuel that keeps me going and my drive to succeed in life.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 2 LITERATURE REVIEW ................................ ................................ .......................... 20 Quantification of Feed Efficiency ................................ ................................ ............ 20 Fe ed Conversion Ratio ................................ ................................ ..................... 20 Residual Feed Intake ................................ ................................ ....................... 20 Immune System ................................ ................................ ................................ ...... 21 Acute Phase Response ................................ ................................ .................... 22 Cytokines ................................ ................................ ................................ .......... 23 Acute Phase Proteins ................................ ................................ ....................... 24 Pho spholipase A2 ................................ ................................ ............................ 25 Stress and Immunity ................................ ................................ ............................... 26 Transportation Stress ................................ ................................ ....................... 28 Concentrate Diet Levels and Stress ................................ ................................ 29 Immune Regulation of Growth ................................ ................................ ................ 30 Strategies to Improve Performance and Feed Efficiency ................................ ........ 32 Management Strategies ................................ ................................ ................... 32 Dietary Strategies ................................ ................................ ............................. 34 Antibiotics as Gro wth Promotants ................................ ................................ .... 35 Antibodies as Feed Additives ................................ ................................ ........... 36 Antibody Against sPLA2 ................................ ................................ ................... 37 Rationale ................................ ................................ ................................ ................. 38 3 EFFECTS OF INCLUSION OF ANTI PHOSPHOLIPASE A2 ANTIBODY TO BACKGROUNDING DIETS ON PERFORM ANCE, FEED EFFICIENCY AND THE A CUTE PHASE RESPONSE OF GROWING BEEF CALVES ....................... 39 Materials and Methods ................................ ................................ ............................ 41 Animals and Treatments ................................ ................................ ................... 41 Ultrasonic Ca rcass Traits ................................ ................................ ................. 42 Temperament Traits ................................ ................................ ......................... 42

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7 Blood Collection and Analyses ................................ ................................ ......... 43 Feed Sample Collection and Analyses ................................ ............................. 44 Statistical Analyses ................................ ................................ .......................... 44 Conclusion ................................ ................................ ................................ .............. 51 4 EFFECTS OF ANTI PHOSPHOLIPASE A2 ANTIBODY SUPPLEMENTATION ON DRY MATTER INTAKE, FEED EFFICIENCY, ACUTE PHASE RESPONSE AND BLOOD DIFFERENTIALS OF STEERS FED FORAGE AND GRAIN BASED DIETS ................................ ................................ ................................ ........ 62 Materials and Methods ................................ ................................ ............................ 64 Animals and Treatments ................................ ................................ ................... 64 Blood Collection and Analyses ................................ ................................ ......... 65 Feed Sample Collection and Analyses ................................ ............................. 67 Ultrasonic Carcass Traits ................................ ................................ ................. 67 Statistical Analyses ................................ ................................ .......................... 67 Results and Discussion ................................ ................................ ........................... 69 Animal Performance ................................ ................................ ......................... 69 Acute Phase Pr oteins and Blood Differentials ................................ .................. 72 Conclusion ................................ ................................ ................................ .............. 74 LIST OF REFERENCES ................................ ................................ ............................... 93 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 102

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8 LIST OF TABLES Table page 3 1 Nutrient composition of background diets fed to beef calves during a 70 d feed efficien cy trial. ................................ ................................ ............................. 52 3 2 The effects of aPLA2 supplementation on animal performance and feed efficiency of growing beef cattle receiving backgrounding diets. ........................ 53 3 3 The effects of aPLA2 supplementation on animal performance, feed efficiency and concentrations of plasma acute phase proteins after 24 hr transportation. ................................ ................................ ................................ ..... 54 4 1 Nu trient composition of diets fed to steers during a transition from a forage based to grain based diet using a three steps adaptation period over 21 d. ...... 75 4 2 Overall animal performance, feed e fficiency and ultrasound carcass traits of steers transitioned from a forage based to grain based diets using a 21 d .............................. 76 4 3 Animal performance and feed efficiency of steers fed a forage based diet during Phase I. ................................ ................................ ................................ ... 77 4 4 Animal performance and feed efficiency of steers transitioned from a forage based to a grain based die Phase II. ................................ ................................ ................................ ............. 78 4 5 Animal performance and feed efficiency of steers fed a grain based diet during Phase III. ................................ ................................ ................................ 79 4 6 Blood differentials and concentrations of plasma acute phase proteins of steers transitioned from a forage based to grain ................................ ................................ 80

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9 LIST OF FIGURES Figure page 3 1 A verage daily DMI calculated on a biweekly basis during the 70 d feed efficiency trial.. ................................ ................................ ................................ .... 55 3 2 Mean chute score (on 5 point scale, with 1 being calm and 5 being aggressive) by day of beef calves duri ng a 70 d feed efficiency trial .................. 56 3 3 Mean exit velocity (seconds for a calf to travel 1.83 m from squeeze chute) by day of beef calves during a 70 d feed efficiency trial.. ................................ ... 57 3 4 Average daily DMI of beef calves during 15 d following 24 hr transportation.. ... 58 3 5 Concentration of plasma haptoglobin by day of beef calves following 24 hr transportation ................................ ................................ ................................ ...... 59 3 6 Concentration of plasma ceru loplasmin by day of beef calves following 24 hr transportation ................................ ................................ ................................ ...... 60 3 7 Correlation between mean concentration of plasma ceruloplasmin and average daily DMI of beef calves after 24 hr transporta tion, when combining treatments ................................ ................................ ................................ ........... 61 4 1 Experiment outline of steers transitioned from a forage based to a grain ................................ ............. 81 4 2 Decrease in daily DMI after diet change on d 1 (step 1), 8 (step 2), and 15 (step 3) of steers transitioned from a forage based to grain based diet over a ................................ ................. 82 4 3 Average DMI by day of steers transitioned from a forage based to a grain .................. 83 4 4 Average DMI by day of steers transitioned from a forage based to a grain .................. 84 4 5 Average DMI by day of steer s transitioned from a forage based to a grain .................. 85 4 6 Concentrations of plasma ceruloplasmin by day of steers transitioned fr om a forage based to a grain aptation period during Phase II. ................................ ................................ ................................ .. 86 4 7 Concentrations of plasma haptoglobin by day of steers transitioned from a forage based t o a grain during Phase II. ................................ ................................ ................................ .. 87

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10 4 8 Concentrations of plasma ceruloplasmin by day of steers transitioned from a forage based to a grain based d during Phase II. ................................ ................................ ................................ .. 88 4 9 Concentrations of plasma ceruloplasmin by day of steers transitioned from a forage based to a grain step during Phase II. ................................ ................................ ................................ .. 89 4 10 Concentrations of plasma haptoglobin by day of steers transitioned from a forage based to a grain n period during Phase II. ................................ ................................ ................................ .. 90 4 11 Correlation between plasma concentration of haptoglobin and average DMI by day of steers transitioned from a forage based to a grain based diet over tep ................................ ............ 91 4 12 Correlation between plasma concentration of ceruloplasmin and average DMI by day of steers transitioned from a forage based to a grain based diet ................................ ..... 92

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11 LIST OF ABBREVIATIONS AA Arachidonic acid ACTH Adrenocorticotropic hormone ADF Acid detergent fiber ADG Average daily gain aPLA2 Anti phospholipase A2 aPLA2 0.2% aPLA2 supplementation at 0.2% of diet DM treatment aPLA2 0.4% aPLA2 supplementation at 0.4% of diet DM treatment APP Acute phase proteins APR Acute phase response CON Control treatment CP Crude protein cPLA2 Cytosolic phospholipase A2 CV Coefficien t of variation DM Dry matter DMI Dry matter intake FCR Feed conversion ration FE Feed efficiency GH Growth hormone IGF I Insulin like growth factor I IL 1 Interleukin 1 IL 6 Interleukin 6 LED Light emitting diode LPS Lipopolysaccharides

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12 MT Monensin and tylosin supplementation treatment NDF Neutral detergent fiber PLA2 Phospholipase A2 PUFA Poly unsaturated fatty acids RFI Residual feed intake sPLA2 Secretory phospholipase A2 TDN Total digestible nutrient TLR Toll like receptors TMR Total mixed ration TN F Tumor necrosis factor alpha WBC White blood cells count

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13 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 THE EF FECTS OF ANTI PHOSPHOLIPA SE A2 ANTIBODY SUPPLEMENTATION ON FEED EFFICIENCY, ANIMAL PERFORMANCE, AND THE ACUTE PHASE RESPONSE OF BACKGROUND AND FINISHING BEEF CATTLE DIETS By Vitor Rodrigues Gomes Mercadante December 2012 Chair: Graham Cliff Lamb Co chair: Nicolas DiLorenzo Major: Animal Sciences To determine whether supplementation of anti phospholipase A2 antibody (aPLA2 ) animal performance, and the acute phase response of transportation, and background and finishing beef cattle diets, two experiments were conduc ted. Experiment I: I ndividual performance and daily dry matter intake ( DMI ) was measured on 70 cross bred weaned calves during a 70 d period using a GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada) at the University of Florida NFREC Feed Efficiency Facility (FEF) Calves were fed a growing diet and were blocked by weight and sex and then randomly assigned to pens to receive either n o additional supplement (CON n = 35) or receive a supplement at an inclusion rate of 0.6% of estimated daily DMI of aPLA2 antibody ( aPLA2; n = 35). After the 70 d feed efficiency ( FE ) trial calves were loaded into a commercial livestock trailer and were driven for 1.600 km during 24 hr. Upon return to the FEF, calves were relocated to the same pens and groups, and rec eived the same diets and treatments Blood samples from each calf were collected on days 0, 1, 3, 5, 7, 14, 21 and 28 relative to transportation and were analyzed for determination of concentrations of plasma ceruloplasmin and haptoglobin. Initial BW

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14 (24 2. 0 3.7 kg; P = 0.92), BW d 70 (313.0 4.1 kg ; P = 0. 7 9), and ADG (1.01 0.0 2 kg ; P = 0.9 5 ) were similar between treatments. However, daily DMI was greater ( P = 0.0 1) for C ON (8. 53 0.1 5 kg) than aPLA2 ( 9 1 8 0.1 5 kg). In addition, RFI was greater ( P = 0.0 02 ) for C ON (0. 389 0. 110 kg/d) than aPLA2 calves ( 0. 272 0. 110 kg/d). After transportation t here were no differences between treatments on BW loss ( 26.0 0.6 kg; P = 0.86), BW d 28 (339.0 4.1 kg; P = 0.72), ADG (1.28 0.03 kg/d; P = 0.72), G: F (0.164 0.004; P = 0.83), and concentrations of plasma haptoglobin (0.08 0.02; P = 0.41). However, concentration of plasma ceruloplasmin was greater ( P < 0.001) for CON calves (14.3 0.3) compared to aPLA2 calves (13.0 0.3). Experiment II: Individu al daily DMI was measured on 80 cross bred steers during a 141 d period at the FEF. On d 0, steers were blocked by BW and randomly assigned to receive a growing forage diet containing the following treatments: 1) no additive (CON; n = 20); 2) 30 mg of mone nsin and 8.8 mg of tylosin per kg of diet DM (MT; n = 20); 3) same as CON, but including aPLA2 at 0.4% of the diet DM (BB0.4%; n = 20); 4) same as CON, but including aPLA2 at 0.2% of the diet DM (B B0.2%; n = 20). On d 60 steers were transitioned into grain based diet ( 90 % concentrate) over a continuing to receive their supplement treatments and were maintained on the high grain diet until the end of the trial on d 141 On d 0, d 60, d 81 and d 141 individual shrunk BW was recorded. Blood samples were collected on d 60, 63, 65, 67, 70, 72, 74, 77, 79, 81 and 84, and for determination of concentration of plasma ceruloplasmin haptoglobin, and blood differentials. No treatment differences were detected on overall performance BW on d 141 (388.0 5.1 kg, P = 0.79), A DG (1.25 0.02 kg/d, P = 0.33), average daily DMI (7.1 0.1 kg, P = 0.43), and RFI ( 1.25 0.06 kg/d, P = 0.61).

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15 However, during the growing forage diet period daily DMI tended ( P = 0.07) to be lower for aPLA2 0.2% and 0.4% treatments (6.51 0.25 kg an d 6.70 0.25 kg, respectively) compared to CON (7.38 0.25 kg) treatment, with MT (7.09 0.25 kg) treatment being intermediate. Steers from aPLA2 0.2% and aPLA2 0.4% treatments ( 0.12 0.13 kg/d and 0.22 0.13 kg, respectively) had lower ( P < 0.05) R FI than CON (0.31 0.13 kg/d) steers with MT (0.05 0.13 kg/d) steers being intermediate. During the grain based diet period, the aPLA2 0.2% ( 0.12 0.1 0 kg/d), aPLA2 0.4% (0.3 6 0.1 0 kg/d), and MT (0. 10 0.1 0 ) steers had greater ( P = 0.04 ) RFI than CON ( 0.3 7 0.1 0 kg/d) steers. During the transition to grain based diet phase WBC were greater ( P = 0.04) for aPLA2 0.2% (13.61 0.42 k/L) than aPLA2 0.4% and MT treatments (12.16 0.42 and 12.37 0.42 k/L, respectively), with CON being intermediate (12.87 0.42 k/L), and concentrations of lymphocytes also were greater ( P = 0.01) for aPLA2 0.2% (7.66 0.28 k/L) than aPLA2 0.4% and MT treatments (6.71 0.28 and 6.70 0.28 k/L, respectively), with CON being intermediate (7.11 0.28 k/L). Conce ntrations of plasma ceruloplasmin was reduced ( P < 0.0001) for CON (22.2 0.8 mg/dL) steers compared to aPLA2 (24.4 0.8 mg/dL) treatments and c oncentrations of plasma haptoglobin was reduced ( P < 0.05) for CON (0.18 0.05 mg/mL) steers compared to aPL A2 (0.26 0.05 mg/mL) treatments. Mean daily DMI was negatively correlated with concentrations of plasma ceruloplasmin and haptoglobin. In conclusion, beef cattle supplemented with aPLA2 had improved FE when fed growing forage diets, but not grain based d iets, and had reduced concentrations of plasma ceruloplasmin after 24 hr transportation. However, CON steers had reduced concentrations of plasma ceruloplasmin and

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16 haptoglobin during the transition to grain based diet compared to steers on aPLA2 and MT tre atments.

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17 CHAPTER 1 INTRODUCTION In a world where the population continues to grow, global agriculture will need to increase its production by 70% in order to feed the projected 9.1 billion people by 2050. Of the production increase, 80% will need to come f rom increases in yields, and only 20% from expansion of land (FAO, 2009) To fulfill the demand for food, more specifically beef, efficiency needs to be a primary f ocus of the beef production chain. Efficiency can be defined as the ability to accomplish a task with a minimum expenditure of time and energy, and it is commonly presented as a ratio of outputs to inputs. Many different measures of efficiency can be appli ed to beef production systems and feed efficiency (FE) is one of those. Rising feed costs, global competition, and societal concerns about energy policy and the environment have created new economic challenges for the beef industry, since nearly two thi rds of the costs of producing beef is directly tied to the cost of feed inputs (Arthur and Herd, 2005) In order to achieve FE and more efficient body weight gain, feedlots in the United States have developed unique feeding and management strategies. The combina tion of extensive grain processing with greater consistency in quality of grain feeds compared to roughages, and the necessity to achieve greater productivity for increased demand in beef, lead feedlots to utilize diets with decreased concentrations of rou ghage (Galyean et al., 2011) Greater demands for grain in ethanol production and export markets associated with recent droughts have caused decreased grain production, resulting in increased global grain prices. Thus, feedlots and stocker cattle operations need to focus on optimizing grain utilization through processing fi nding alternative energy sources, selection of more feed efficiency animals and

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18 development of strategies to improve FE while maintaining high perfor mance (Galyean et al., 2011) In a ddition to the economic benefits of improving overall FE of beef production there is an important environmental benefit. Manure is an inevitable byproduct of livestock production and f eed intake and manure output have a strong positive relationship Reduction in greenhouse gas emission s from enteric fermentation in cattle is a high priority, plus enteric methane formation represen ts an energy loss to the animal; thus, improving FE is a novel way of reducing feed costs, methane production and nitrogen excretion without compromising growth rates and the economic viability of beef systems (Hegarty et al., 2007) The use of antibiotics has greatly impacted animal agriculture by improving FE and productivity of animal protein production systems. However, antimicrobial resistance is a grow ing public issue and the use of antibiotics as growth promotants seems to be subjected to future limitations and more restricted regulations (Galyean et al., 2011) The addition of antibiotics to cattle diets modifies t he rumen micro flora, improving rumen fermentation (Richardson et al., 1976) However, there is also a positive effect on animal health, with decreased acidosis and rumenitis (Nagaraja and Tit gemeyer, 2007) The activation of the immune system is costly in terms of animal growth and FE, with redirection of nutrients and consumption of body reserves of energy and protein (Johnson, 1997) Novel strategies using avian derived antibodies to modulate and reduce the activation of the immune system have been successfully used to imp rove performance and FE of livestock (Cook, 2011)

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19 Therefore, the development of alternative strategies and novel technologies to improve FE and sustain animal performance, ensuring the economic viability of beef production enterprises and the supply of th e global growing demand for beef, are of vital importance and need further investigation.

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20 CHAPTER 2 LITERATURE REVIEW Quantification of Feed Efficiency The relationship between feed intake and its utilization by the animal relies on the complexity of bi ological processes and interactions with the environment. Thus, selection of cattle based solely on feed intake is rarely used (Arthur and Herd, 2005) The complexity to determine the energetic efficiency of cattle has resulted in multiple measurements of FE to be developed, such as feed conversion ratio (FCR; Brody, 1945), residual feed intake (RFI; Koch et al., 1963) partial efficiency of growth ( Kellner, 1909 ), relative growth rate ( Fitzhugh Jr. and Taylor, 1971 ) and the Kleiber ratio ( Kleiber, 1947) (Nkrumah et al., 2004) Feed Conversion Ratio Feed conversion ratio is the most common measurement of FE, due to its ease of calculation, and it is also referred to as feed to gain. It is the ratio between the feed consumed to the amount of body weight gain over a specific period of time (Brody, 1945) In beef cattle FCR has been reported to be highly correlated with growth (Archer et al., 1999) and as a selection tool FCR has the potential to increase growth rate in growing animals. How ever, selection for FCR could also result in a larger mature size of the herd, with consequent increased feed intake (Nkrumah et al., 2007) Therefore, selection for FCR may result in unfavorable effects on overall production system efficiency (Archer et al., 1999; Nkrumah et al., 2004) Residual Feed Intake The concept of RFI was first used to adjust feed intake for body weight and weight gain, dividing feed intake in two portions, the expected feed intake for a given level of

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21 performance, and the res idual portion. The residual portion may be used to identify animals that deviate from their expected feed intake, with animals having negative RFI values being more efficient (Archer et al., 1999) However, differences in RFI have been associated with differences in carcass fatness, carcass leanness, and meat quality, and although the results in the literature are inconsistent, a reduction in carcass merit due to selection for lower RFI anima ls may not be desirable to the beef cattle industry (Herd et al., 2003; Nkrumah et al., 2004, 2007) Immune S ystem The immune system has evolved to protect the organism from pathogens and generates an variety of cells a nd molecules capable of specifically recognizing and eliminating foreign invaders and cancerous cells, all of which act together in a dynamic network (Kindt, 2007) There are two types of immunity that collaborate to protect the organism, innate immunity and adaptive immunity. The innate immunity is less specific and provides the first line of defense against invading pathogens (Lippolis, 2008) The innate immune system includes physi cal, chemical, and cellular barriers. The key cells that play a role in the innate immune response are neutrophils, macrophages, monocytes, natural killer cells, dendritic cells, and cells that release inflammatory mediators, such as basophils, mast cells and eosinophils (Kindt, 2007; Carroll and Forsberg, 2007) Adaptive immunity is highly specific and capable of recognizing and eliminating specific antigens (Kindt, 2007) The adaptive immunity is further subdivided into humoral and cell mediated. Humoral immunity is mediated by B lymphocytes, which produce antibodies specific to antigens and become memory cells. In cell mediated

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22 immunity, the T lymphocytes provide protection against intracellular pathogens and tumor cells (Galyean et al., 1999) Although the innate and adaptive immune systems are classified as distinct, they do not operate i ndependently, but rather in conjunction with one another and with help of molecules and nonimmune cells in a complex network at the initiation of the inflammatory response (Lippolis, 2008) The t oll like receptors (TLR) are a family of cell surface receptors that bind to various molecules specific to pathogens and act as some of the earliest surv eillance mechanisms against infections. The TLR interact with the pathogen associated molecular patterns which are different components of bacterial cell walls, such as lipop eptides and lipopolysaccharides (Parker et al., 2007; Lippolis, 2008) Upon stimulation the TLR acts to stimulate the cell to respond to infection and facilitates cellular responses via signaling pathways. The complex interactions amon g these cells are mediated by regulatory proteins called cytokines. Cytokines regulate and modulate a variety of cell functions and physiological processes, including the inflammatory response (Kindt, 2007; Parker et al., 2007; Carroll and Forsberg, 2007; Lippolis, 2008) Acute P hase R esponse The acute phase response (APR) is an important component of the innate imm une system, which is characterized by various reactions and physiological changes of the body in response to disturbances of its homeostasis, such as infections, disease, stress, and trauma (Carroll and Forsberg, 2007; Cooke and Bohnert, 2011) characteristic reactions during the APR includes fever, shift in liver metabolism and gene regulation, plasma mine ral alterations, and changes in behavior, such as lethargy, anorexia, hyperalgesia (J ohnson, 1997) and decreased social and sexual behavior

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23 (Cooke et al., 2009a) The APR is stimulated by the release of p r oinflammatory cytokines from macrophages and monocytes at t he site of inflammation or infection The initial release of proinflammatory cytokines is augmented by their p aracrine actions, which cause further release of these cytokines and eventually results i n a systemic release of cytokines (Carroll and Forsberg, 2007; Carroll et al., 2009b) Cytokines The term cy tokine was originally used to distinguish a group of immunoregulatory proteins from other cellular growth factors. Unlike hormones, cytokines are not produced by specialized cells, but rather by a number of diverse cells types, such as white blood cells, T helper cells, dendritic cells, monocytes and macrophages, but can also be secreted by other nonimmune cells in response to stimuli, such as injury, trauma, infection and stress. Likewise, there is not one specific cell type that is the sole target of mos t cytokines (John son, 1997; Kindt, 2007; Parker et al., 2007; Lippolis, 2008) The APR is stimulated by the release of the pro inflammatory cytokines tumor necrosis factor (TNF ), i nterleukin 1 (IL 1), and interleukin 6 ( IL 6 ) from macrophages and monocytes at the sit e of inflammation or infection (Johnson, 1997) These proinflammatory cytokines func tion as chemo attractants induc ing expression of adhesion molecules, which cause responding immune cells to localize to the site of infection, acting locally to amplify the cellular immune response, but they can also act systemically changing behavior, met abolism, and neuroendocrine secretions (Johnso n, 1997; Carroll and Forsberg, 2007; Lippolis, 2008)

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24 Acute Phase Proteins The initial release of proinflammatory cytokines is augmented by their paracrine actions resulting in their systemic release and stimulating production and secretion of the acute p hase proteins (APP) from the liver. Under normal circumstances, the APP have various biological functions, such as proteinase inhibitors, enzymatic functions, coagulation proteins, metal binding proteins, and transport proteins (Petersen et al., 2004; Carroll and Forsberg, 2007; Carroll, 2008) However, during an inflammatory response, the proinflammatory cytokines mediate the hepatocyte production of the APP leading to a dram atic change in APP concentration. The APP can be classified according to the magnitude of their increase (positive APP) or decrease (negative APP) in serum concentrations during the APR. Positive APP such as haptoglobin and ceruloplasmin are induced primar ily by IL 6 and are characterized by a later increase in serum concentrations reaching its peak 24 to 48 hr for haptoglobin, and 72 to 168 hr for ceruloplasmin, after the initiation of the APR and may remain elevated as long as two weeks (Arthington et al., 2003; Eckersall and Bell, 2010) Other positive APP such as serum amyloid A, fibrinogen, and C reactive protein are primarily induced by IL 1 and are characterized by an early increase in serum concentrations, occurring within four hours of the initiation of the APR and also have a rapid normalization of their serum levels, and ne gative APP such as albumin and transferrin, may have a 10 to 30% decrease in serum concentrations within 24 hr of the APR initiation (Petersen et al., 2004) Haptoglobin has numerous biological functions, b ut its primarily function is to bind free hemoglobin released from damaged erythrocytes, forming stable complexes in the blood and restricting the availability of free iron to invading bacteria, reducing their

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25 growth capacity, thus having a bacteriostatic effect (Petersen et al., 2004; Eckersall and Bell, 2010; Ceciliani et al., 2012) Ceruloplasmin is the major copper carrying protein in the blood and also participates in iron homeostasis. Copper deficiency has been correlated with immune suppression in cattle, with decreased concentrations of plasma ceruloplasmin (Arthington et al., 1996) In cattle, increased serum concentrations of haptoglobin and ceruloplasmin were found after experimentally induced inflammation (Carroll et al., 2009b, 2011; Cooke and Bohnert, 2011; Cooke et al., 2012b) trauma and castration (Petersen et al., 2004; Wa rnock et al., 2012; Ceciliani et al., 2012) inflammatory diseases and vaccination (Petersen et al., 2004; Ganheim et al., 2007; Eckersall and Bell, 2010) road transportation, weaning and commingling (Arthington et al., 2003, 2008; Fike and Spire, 2006; Qiu et al., 2007; Araujo et al., 2010) and when feeding backgrounding and finishing diets (Berry et al., 2004a; Ametaj et al., 2009; Zebeli et al., 2010) Phospholipase A2 Phospholipase A2 (PLA2) are a complex family of potent inflammator y enzymes that are upregulated upon extracellular stimuli. The PLA2 enzymes catalyze the hydrolysis of the sn 2 position of membrane glycerophospholipids, yielding free fatty acids and lysophospholipids (Kudo and Murakami, 2002) In mammals, secretory PLA2 type IIA (sPLA2) is a potent inflammatory marker that is upregulated by endotoxins and proinflammatory cytokines, and it is recognized as one of the first li ne of defenses against microbial infection, playing a central role as one of the first steps that triggers the inflammatory cascade. Inflammatory effector cells such as neutrophils and macrophages store sPLA2 in secretory granules and release it upon cellu lar activation. The sPLA2 also has an antimicrobial capacity by causing the disruption of bacteria,

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26 through hydrolysis of the bacterial membrane phospholipids. The sPLA2 cleaves the fatty acid located in the sn 2 position on the outer leaflet of the cell m embranes, releasing free fatty acids, such as arachidonic acid (AA). The release of sPLA2 upregulates cytosolic PLA2 (cPLA2), and cPLA2 cleaves AA from the sn 2 position on the inner leaflet of the cell membrane, releasing free AA in the cytosol. Arachidon ic acid acts, via cyclo oxygenase and lipoxygenase pathways, to serve as substrate to production of eicosanoids, such as prostaglandins, leukotrienes, and thromboxanes that are potent proinflammatory mediators (Kudo and Murakami, 2002; Cook, 2004; Nevalainen et al., 2008) In mice, sPLA2 secretion in the lumen of the gastrointestinal tract (GIT) has been reported to be stimulated by endotoxins, and its activity to have a great impact on the content and properties of the GIT phospholipid barrier, resulting in an increase in its permeability to bacterial me mbrane lipopolysaccharides (LPS ; Rozenfeld et al., 2001; Zayat et al., 2008) Stress and Immunity The concept of stress was always be in the same physiologic manner in an effort to maintain homeostasis. The sum of all physical, emotional, or are named stressors and elicit coordinated physiologic responses in order to maintain homeostasis, primarily by activation of the hypothalamic pituitary adrenal axis and the sympathetic nervous system (Pack and Palkovits, 2001) Upon exposure to stressors, corticotropin releasing hormone and vasopressin are secreted in the hypothalamus, stimulating adren ocorticotropic hormone (ACTH)

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27 secretion from the anterior pituitary gland, ACTH then stimulates production of steroids from the adrenal gland, cholesterol uptake and the release of glucocorticoids, such as cortisol, from the adrenal cortex (Friend, 1991) Glucocorticoids ar e essential for the maintenance and restoration of homeostasis by inducing metabolism of carbohydrates and proteins, regulating the growth and reproductive axes, stress responses, and the immune functions (Cooke and Boh nert, 2011) However, excessive concentrations of glucocorticoids in cattle have been linked to reduced rates of reproduction, suboptimal growth and performance, reduced milk production and suppression of immune function by reducing the production and rel ease of various cytokines, including the pro inflammatory cytokines IL 1, IL 6, and TNF (Friend, 1991; Johnson, 1997; Cooke and Bohnert, 2011) Glucocorticoids also enhance synthesis and secretion of catecholamines, which control several physiological processes. The most important catecholamines secreted by the adrenal medulla are adrenaline, noradrenaline, and dopamine. The main effects of catecholamines consist of increased heart rate, pupil and bronchiole dilatation, vasoconstriction in the skin and gut, vasod ilatation in muscles, and increases glucose production by the liver via gluconeogenesis, all of which compose the flight or fight response, also catecholamines control the stress response via regulation of ACTH release from the pituitary gland and stimulat ion of cortisol secretion from the adrenal cortex (Carroll and Forsberg, 2007) Throughout the beef production cycle, cattle experience several environmental, managerial, and nutr itional stressors that can negatively impact productivity. In livestock, stressors may be assigned to three categories: psychological, physical, and

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28 physiological. Psychological stress usually is associated with fear when experiencing commingling, restrain ing, and exposure to new environments. Thermal stress, hunger and thirst, pain, and disease represent physical stress, whereas physiological stress results from deviations in homeostasis, such as nutrient deficiencies and endocrine disorders (Friend, 1991; Grandin, 1997) The immune system response to stress is dependent on the type of stress encountere d. Acute stress usually is immunoenhancing, in a manner to prepare the body to potential infection and ultimately activation of the APR. However, chronic stress is immunosuppressive and long term exposure to glucocorticoids shifts the immune system from a preparatory into a suppressive mode (Carroll and Forsberg, 2007) Transportation Stress Transportation is one of the most common and intense acute physical stressors that cattle e ncounter throughout the beef production cycle. The event of transportation leads to physiological, nutritional, and immunological changes that affect subsequent health and performance of cattle, with decreased feed intake, weight loss, increased heart and respiration rate, increased blood concentrations of cortisol, increased concentrations of catecholamines, and activation of the APR with production and secretion of proinflammatory cytokines and APP (Loerch and Fluharty, 1999; Arthington et al., 2003; Fike and Spire, 2006; Duff and Galyean, 2007; Araujo et al., 2010) Following transportation, total white blood cell (WBC) counts and differentials are altered with an increase in neutrophils and monocytes and decrease in lymphocytes, with a consequent increase in neutrophil to lymphocyte ratio (Ishizaki and Kariya, 2010; Hulbert et al., 2011)

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29 Development of immunosuppression, due to chronic stress related to transpor tation combined with commingling and weaning stress, also may lead to increased morbidity and mortality of bovine respiratory disease in newly received feedlot steers. Negatively affecting feedlot performance and carcass merit, resulting in substantial eco nomic losses (Galyean et al., 1999; Duff and Galyean, 2007) C oncentrate Diet Level s a nd Stress Digestion of feedstuffs in the retilculorumen occurs in an anaerobic ecosystem, where microbes convert fermentable substrates into organic acids, which are then ab sorbed through the rumen wall. In beef cattle fed high concentrate diets, rumen pH usually ranges from 5.6 to 6.5, and fluctuates depending on intake of fermentable carbohydrates, an animals capacity to provide buffer through saliva production, and the rat es of absorption and utilization of the organic acids. Rumen pH is critical for the normal function of the rumen because of its profound effects on rumen microbial populations, fermentation products and physiological functions, such as motility and absorpt ive functions (Nagaraja et al., 1985) When feeding high grain diets, ruminal pH can fall below 5.6, having significant impact on ruminal function and animal health, leading to the development of acidosis and rumenitis (Nagaraja and Titgemeyer, 2007) Endotoxin or LPS is a cell wall component of gram negative bacteria. Death and disintegration of bacteria in the rumen are n ormal events and endotoxin is commonly present in rumen fluid. The rumen fluid concentration of endotoxin is greater in grain fed compared to forage fed cattle, due to greater quantities of gram negative bacteria and greater rates of bacterial death caused by lower rumen pH values (Nagaraja et al., 1978a; b; Andersen et al., 1994) The development of acidosis with accumulation of endotoxins in rumen fluid may lead to inflammation and degenerativ e processes of the

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30 rumen and intestinal mucosa, resulting in translocation of endotoxins into the bloodstream with activation of the APR and release of inflammatory mediators, such as AA metabolites and proinflammatory cytokines (Andersen et al., 1994; Gozho et al., 2007; Emmanuel et al., 2007, 2008; Khafipour et al., 2009; Ametaj et al., 2009; Dong et al., 2011) Beef cattle in feed yards undergo acidotic challenges when they are transitioned from forage based into grain based diets. The effects of the change in diet on feed intake, rumen pH and physiological responses, such as the activation of the APR, are dependent on the percentage increase in concentrate feeds and the time allowed between diet changes for the rumen to adapt (Berry et al., 2004a; Nagaraja and Titgemeyer, 2007; Ametaj et al., 2009) Immune Regulation of Growth The immune system has a priority in energy and nutrient utilization relative to ani mal growth. Upon activation of the immune system, the organism diverts nutrients away from growth and development, redirecting it into defense processes. The redirection of nutrients is followed by an immediate decrease in feed intake, wasting of skeletal muscle, increase lipolysis, and production of APP by the liver (Johnson, 1997; Cook, 2011) A mechanism to explain the anorectic and metabolic effects of immunological challenged livestock animals, with reduced performance and feed efficiency, was proposed and stated that pathogens and LPS stimulate leukocyte p roduction of proinflammatory cytokines, leading to anorexia and fever by acting directly on the central nervous system, and mediating systemic activation of cytokine receptors on nonimmune tissues such as, muscle and adipose, with increased lipolysis, musc le

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31 catabolism, hepatic production of APP, increased glucocorticoids, increased corticosteroids, reduced growth hormone (GH) and insulin like growth factor I (IGF I) secretion (Johnson, 1997; Spurlock, 1997; Gifford et al., 2012) Muscle protein d egradation is mediated by IL 1, IL 6, and TNF liver synthesis of APP. At least 60% of the amino acids used for hepatic synthesis of APP are derived from body protein reserves (Johnson, 1997) Although IL 1 and TNF may increase uptake of amino acids in vivo, only IL 6 has been shown to stimulate uptake of amino acids in vitro and is considered the primary mediator of this metabolic response to inflammation (Johnson, 1997; Spurlock, 1997) It appears that IL 1 and TNF secretion of IL 6 (Klasing et al., 1987; Johnson, 1997; Spurlock, 1997; Gifford et al., 2012) Nutrient intake is decreased during immune challenges, leading to a shift from accretion to mobilization of adipose tissue, with an in crease in plasma triglycerides in order to sustain the energy demand of the organism. Hepatic fatty acid synthesis is increased by TNF on adipose tissues acting in synergism with IL 1, stimulating lipolysis and resulting in hypertriglyceridemia and increased very low density lipoproteins, that transport fatty acids t o peripheral tissues for utilization (Johnson, 1997; Elsasser et al., 2008; Gifford et al., 2012) In cattle, decreased concentrations of GH and IGF 1 upon immunological challenge with injection of LPS, are correlated with greater plasma c oncentrations of IL 1 and TNF (Spurlock, 1997; Carroll, 2008; Elsasser et al., 2008) Peripheral

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32 inflammatory responses may stimulate production of proinflammatory cytokines in the brain via stimulation of vagal afferent nerves. The proinflammatory cytokines act in conjunction with each other to depress feed intake, alter social and sexual behavior and inducing fever (Johnson, 1997; Spurlock, 1997) However, IL 1 has been shown to be more potent in suppressing nutrient intake, altering glucose metabolism, and inducing behavior associated with illness, such as lethargy an d depression, and TNF potent at inducing lipolysis and muscle degradation ( Johnson, 1997) In addition, IL 6 is known as the primary cytokine responsible for the shift in hepatic protein synthesis towards the production of APP (Joh nson, 1997; Carroll, 2008) Strategies to Improve Performance and Feed Efficiency Feed is a major cost to beef production systems, and in order to achieve FE and maintain adequate body weight gain and performance, beef production systems have developed un ique feeding and management strategies to manipulate and improve the efficiency of rumen fermentation and overall animal health. These strategies aim to minimize the activation of the immune system and to maximize performance and feed efficiency (Loerch and Fluharty, 1999; Duff and Galyean, 2007; Cook, 2011) Management S trategies Upon arrive to a feedlot, beef calves have been exposed to multiple stressors that lead to physiological, metabolic and behavior responses consequently resulting in r educed nutrient intake, decreased performance and increased morbidity and mortality of diseases, such as bovine respiratory disease (Loerch and Fluharty, 1999; Duff and Galyean, 2007) Management practices of beef calves prior to entry to the feedlot involve a complete preconditioning program to ensure that animals have been weaned greater than 30 days prior to shipping. The preconditioning program usually involves

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33 vaccination program, castration of bull calves, and acclimation to feed and water bunks, thereby reducing t he stress response associated with these events once arriving at the feedlot (Duff and Galyean, 2007) In addition, strategies to improve health and performance after feedlot arrival are extremely important to ensure proper nutrient intake and immunological support, allowing ac hievement of high performance rates after arrival (Peterson et al., 1989; Loerch and Fluharty, 1999; Duff and Galyean, 2007) Early weaning, at 70 days of age, follow ed by concentrate supplementation has been shown to reduce the APR associated with transportation and entry to the feedlot, compared to calves weaned directly before transport and feedlot entry. The early weaned steers also had improved feedlot performance with greater average daily gain (ADG) and greater FCR than control steers (Arthington et al., 2008) Similarly in an alternative study, early weaned beef c alves had decreased concentrations of pro inflammatory cytokines followed by LPS challenge compared to normal weaned calves, and early weaned calves had higher concentrations of interferon innate immune system of early weaned calves may be more competent at recognizing and eliminating the endotoxin (Carroll et al., 2009a) Temperament has been shown to also impact animal performance. Animals with greater temperament scores, more aggressive or excitable, had decreased ADG and dry matter intake (DMI ; Grandin, 1997) Strategies to acclimate anim als to handling facilities and personnel, as well as selection for calm temperament, have been successfully used to reduce temperament scores, decrease the APR associated with handling, and consequently increase animal performance (Voisinet e t al., 1997; Cooke et al., 2009a,b; 2012a)

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34 Dietary S trategies Diets with higher nutrient density for newly received feedlot ca lves have been used in order to compensate for the lower feed intake of those animals. Diets must be formulated to supply sufficient nutrients so that the animal does not use its own body reserves (Berry et al., 2004b) A series of studies using diets with high crude protein and energy contents for newly arrived feedlot cattle were reviewed, and indicated that calves receiving diets with greater concentrate and protein density, during the first weeks after feedlot entry, had increased overall performance and improved health (Loerch and Fluharty, 1999; Berry et al., 2004a; b; Duff and Galyean, 2007) Supplementation of rumen protected polyunsaturated fatty acids (PUFA) has been shown to modulate the immune response in cattle, with reduced conc entration of APP after transportation and feedlot entry. However, PUFA supplementation had a negative impact on ADG and DMI (Araujo et al., 2010) Concentrations of most minerals needs to be increased in receiving diets to compensate for the lower DMI of newly received calves. A num ber of minerals, specifically selenium, copper, zinc, and chromium, have been shown to be important for the support and maintenance of the immune system in cattle (Duff and Galyean, 2007) Mineral deficiencies can affect immunity by reducing antibody production and responses, c ell mediated immunity, and NK cell activity (Carroll and Forsberg, 2007) However, it remains unclear whether mineral supplementation beyond physiological requirements enhances imm unity of livestock, and the results appears to be dependent on source, organic or inorganic, and on interactions with vitamins (Galyean et al., 1999; Carroll a nd Forsberg, 2007; Duff and Galyean, 2007)

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35 Antibiotics as G rowth P romotants The use of antibiotics in animal feeds as antimicrobial therapy for disease and as reveal ed that antibiotic supplementation had no effect on animal performance in germ free animals (Cook, 2004, 2011) Monensin, a carboxylic polyether ionophore antibiotic, naturally produced by strains of Streptomyces cinnamonensis (Haney, M E and Hoehn, 1967) selectively inhibits gram positive bacteria, modifying the rumen flora and metab olism, improving digestive efficiency of cattle. Feeding monensin increases the rumen concentration of propionic acid and decreases concentrations of acetate and butyrate acids (Richardson et al., 1976) it also red uces the production of methane, thus enhancing animal performance from the improved retention of carbon and energy in rumen fermentation, increased digestibility of dry matter, starches and amino acids (Bergen and Bates, 1984; Beckett et al., 1998) Monensin treatment has also been successfully used to decrease the ri sk of acidosis and sub acute acidosis in cattle consuming high concentrate diets, due to reduction in lactic acid concentration in the rumen (Nagaraja et al., 1985; Burrin and Britton, 1986; Nagaraja and Titgemeyer, 2007) Recently in a meta analysis on the effects of feeding monensin for growing and finishing beef cattle, authors concluded that monensin improved FE, r educed DMI, improved ADG, and a linear effect of monensin dose was found for FE (higher dose improving FE), DMI (higher dose reducing DMI), and for ADG (higher dose reducing ADG; Duffield et al., 2012) Feeding high grain diets may result in the development of liver abscesses in cattle. Generally, the incidence of liver abscesses averages from 12 to 32% in most f eedlots

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36 (Nagaraja and Chengappa, 1998) Liver absces ses are a major economic liability to the beef industry, as the number one cause for liver condemnation in the United States, and even more important by its great impacts on animal performance with reduced DMI, weight gain, decreased FE, and decreased carc ass dressing percentage (Meyer et al., 2009) Tylosin is an antibiotic effective primarily against Gram positive bacteria, and its inclusion on high grain diets for finishing cattle has been shown to reduce the incidence of liver abscesses, as well as improve ADG, FE and carcass dressi ng percentage (Galyean et al., 1992; Stock et al., 1995; Nagaraja and Che ngappa, 1998; Meyer et al., 2009) A ntibodies as F eed A dditives Public health concerns that the use of antibiotics in animal agriculture may generate microbial resistance resulted in the development of alternative strategies to manipulate the gastrointe stinal micro flora and enhance animal performance (Salyers, 2002; Cook, 2004) The use of avian antibodies to modify ruminal bacteria populations and manipulate rumen fermentation to improve animal performance and FE, is a novel technology and an alternative to the use of antibiotics for li vestock diets (Cook, 2004, 2011; Cook and Trott, 2010) Feeding avian egg antibodies is a possible alternative because immunoglobulin Y (IgY), the main source of avian immunoglobulin, is resistant to heat, acid digestion, and proteolysis (Shimizu et al., 1988) Binding activity of purified IgY was reduced only when heated at 70 C for 15 minutes. Egg antibodies retained over 85% of their antirotavirus neutralizing activity at a pH of 2 for one hour, even though the antibody binding capacity was reduc ed by 50% (Cook and Trott, 2010) In addition, the hen offers many advantages for the production of polyclonal antibodies,

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37 with low cost mass production and a capacity to develop effective antib odies for specific targets (Cook, 2011) Avian derived polyclonal antibodies targeting specific rumen bacteria populations have been used to prevent deleterious effects associated with these bacteria, improving animal performance. Polyclonal antibody prepar ations against Streptococcus bovis and Fusobacterium necrophorum were successfully used as feed additives to beef steers receiving high grain finishing diets, to reduce rumen acidosis and liver abscess that are related to higher counts of those bacteria (Nagaraja and Titgemeyer, 2007) Steers receiving the polyclonal antibody preparation against Streptococcus bovis also had increased FCR, but had decreased dressing percentage (DiLorenzo et al., 2006, 2008) An alternative study compared a polyclonal antibody preparation against numerous rumen microorganisms with supplementation of monensin and found a tendency in the reduction of liver abscess f or the group receiving the antibody, and similar feedlot performance between monensin and antibody supplementation (Pacheco et al., 2012) Antibody A gainst s PL A2 The sPLA2 plays an important role in endotoxin induced prostaglandin synthesis in intestinal epithelial cells, mediating the disruption of the intestinal barrier, a potent proinflammatory mediator leading to the activation of the immune system with production of proinflammatory cytokines and the APP (Cook, 2004, 2011) An avian antibody specific against intestinal sPLA2, anti phospholipase A2 (aPLA2) was developed to regulate the intestinal inflammat ory response and the negative effects of the activation of the APR on animal growth and FE (Cook, 2011) In a series of studies with poultry, swine, and fish the inclusion of aPLA2 improved FE and growth, without increased incidence of infectious diseases (Yang et al., 2003; Cook, 2004, 2011;

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38 Schwartz et al., 2006; Barry and Yang, 2008) However, to our knowledge no studies on the use of aPLA2 as a feed additive for cattle diets have been reported. Rationale The activation of the immune system with production and secretion of proinflammatory cytokines and acute phase proteins is costly in terms of energy and anima l performance, with decreased ADG, DMI, and FE, establishment of fever and immediate redirection of nutrients, muscle waste, and lipolysis. Feed is a major input in beef production systems, and with feedstuff prices increasing FE is a primary concern to ma intain sustainability of beef production in an economic manner. Alternative strategies to the use of antibiotics, due to increased public concern and environmental agencies with development of bacterial resistance, need to be addressed. The use of specific egg antibodies targeting proinflammatory enzymes, such as the aPLA2, blocking the APR at the intestinal level are a novel approach to improve animal performance and FE. Thus, the objective of this research was to elucidate the effects of aPLA2 supplementa tion on performance and feed efficiency of beef cattle receiving background and finishing diets, and the effects of aPLA2 supplementation on the acute phase response due to transportation and feeding high concentrate diets.

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39 CHAPTER 3 EFFECTS OF INCLUSION OF ANTI PHOSPHOLIPASE A2 ANTIBODY TO BACKGROUNDING DIET S ON PERFORMANCE, FEED EF FICIENCY AND THE A CUTE PHASE RESPONSE OF GROWING BEEF CALVES Rising feed costs, global competition, and societal concerns about energy policy and the environment have created new economic challenges for the beef industry, since nearly two thirds of the costs of producing beef is directly tied to the cost of feed inputs (Arthur and Herd, 2005) Thus, beef cattle operations need to focus on optimizing feed utilization through fi nding a lternative energy sources, selection for feed efficient animals and development of strategies to improve feed efficiency (FE) while maintaining high performance rates (Galyean et al., 2011) The relationship between fee d intake and its utilization by the animal relies on the complexity of biological processes and interactions with the environment. The immune system has a priority in energy and nutrients utilization relative to animal growth. Upon activation of the immune system and the acute phase response (APR), the organism diverts nutrients away from growth and development, redirecting it into defense processes followed by immediate decrease in feed intake, wasting of skeletal muscle, increase lipolysis and production of acute phase proteins (APP) by the liver, plasma mineral alterations, fever and changes in behavior (Johnson, 1997; Cook, 2011; Cooke and Bohnert, 2011) The APR is stimulated by the release from macrophages and monocytes of the pro inflammatory cytokines tumor necrosis factor (TNF ), i nterleukin 1 (IL 1), and interleukin 6 ( IL 6 ) at the site of i nflammation or infection (Johnson, 1997) Transportation is one of the most common and intense acute physical stressors that cattle encounter throughout the production cycle and leads to physiological,

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40 nutritional and immunological changes that can affect subsequent health and performance (Loerch and Fluharty, 1999; Arthington et al., 2003; Fike and Spire, 2006; Duff and Galyean, 2007; Araujo et al., 2010) A theory e xplaining the mechanism by which immunological challenged livestock have poorer performance and FE, hypothesizes that pathogens and lipopolysaccharides (LPS) translocate into the bloodstream through activation of secretory phospholipase A2 (sPLA2) in the i ntestinal lumen, and once in the bloodstream LPS stimulates leukocyte production of proinflammatory cytokines with further activation of the APR, leading to anorectic and physiological responses that have direct effects on animal performance (Cook, 2011) T he sPLA2 is a potent inflammatory marker that is upregulated by LPS and proinflammatory cytokines. Upon activation, sPLA2 up regulates cytosolic phospholipase A2 (cPLA2) that will cleave arachidonic acid (AA) from the sn 2 position of the cell membrane. Fr ee AA on the cytosol will then serve as substrate for production of eicosanoids such as prostaglandins and leukotrienes, that are potent inflammatory mediators (Kudo and Murakami, 2002; Cook, 2011) Feed efficiency and growth was improved in a series of studies with poult ry, swine and fish using an egg derived antibody against sPLA2 (aPLA2) as a feed additive (Yang et al., 2003; Cook, 2004, 2011; Schwartz et al., 2006; Barry and Yang, 2008) However, the use of aPLA2 as a feed additive for cattle diets has not been reported. Therefore, the potential exists that cattle that consume aPLA2 may have decreased inflammatory response, and consequently enhanced performance and improved FE. The objective of this study was to determine whether supplem entation of

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41 aPLA2 would alter the overall performance of growing beef calves receiving a background diet, and the APR associated with transportation. Materials and Methods Animals and T reatments Phase I. Seventy cross bred weaned beef calves (steers, n = 53; and heifers, n = 17) were allocated in group pens (108 m2/pen; 2 pens with 11 animals each, and 4 pens of 12 animals each) at the University of Florida North Florida Research and Extension Center, Feed Efficiency Facility (FEF), in Marianna, FL. Calves were submitted to a 70 d feed efficiency trial and individual daily DMI was measured using the GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada) Each pen in the FEF was equipped with two GrowSafe feed bunks. Calves were blocked by weight and sex a nd then randomly assigned to pens to receive treatments. A 21 d period of adaptation to facilities and diets preceded the 70 d FE trial. Body weight (BW) was recorded at 14 d intervals over the 70 d period. Calves were assigned to receive a growing TMR die t (0.6 Mcal NE g /kg DM, 13% CP) containing 31% corn gluten feed pellets, 30.5% soy hulls, 3.0% molasses, 34% b ahiagrass hay, 0.5% mineral supplement, 0.5% salt and 0.5% limestone (Table 3 1). Calves received either an additional supplement of estimated dail y DMI containing an anti phospholipase A2 antibody (BIG BEEF, Aova Technologies, Madison, WI; aPLA2, n = 35) at an inclusion rate of 0.6% or no additional supplement (CON, n = 35). The antibody supplement was formulated using a premix with soy hulls contai ning the aPLA2 at a concentration of 30%, and the antibody premix was included in the TMR diet at a 2% level.

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42 Phase II. Immediately after completion of the 70 d feed efficiency trial, on d 0 calves were loaded into a commercial livestock trailer and driven within the state of FL for 1,600 km during a 24 h r period, before returning and being unloaded at the FEF, on d 1. Due to a limit in the maximum weight of the livestock trailer, four calves (two of each treatment, the heaviest and lightest calves) were not loaded into the trailer and were subsequently excluded from the statistical analysis for Phase II. Upon return to the FEF, calves were relocated to the same pens and groups, and received the same diets and treatments of Phase I. Body weight was determi ned on d 0 before shipping and upon arrival on d 1, and a shrunk BW (16 h r following removal from feed) at the final of Phase II, on d 28. Ultrasonic C arcass T raits Phase II. On d 28 of Phase II, ultrasound was used to determine fat thickness (BF) using a n Aloka real time ultrasound scanner (3.5 MHz linear array transducer, Aloka 500V, Corimetrics Medical Systems, Inc., Wallingford, CT) and image capturing software. Scanning was performed on the right side of each animal, between the 12 th and 13 th ribs. Tw o measurements were taken per animal at each scanning session. Final BF values were calculated as the average between the two measurements recorded. Temperament T raits Phase I. Temperament traits evaluated were chute score (CS) and exit velocity (EV). The subjective measurement of the behavioral response to restraint within the squeeze chute was assigned on a 1 to 5 scale (1 = calm, docile and quiet; 2 = restless; 3 = nervous; 4 = excited and flighty; 5 = aggressive) by the same trained evaluator at all mea surement sessions. Exit velocity was the speed (m/s) at which each animal exited

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43 the squeeze chute and passed by light emitting diode (LED) optical sensors placed at a distance of 1.83m. Both traits were measured on 14 d intervals during the 70 d feed effi ciency trial. Blood C ollection a nd A nalyses Phase II. Blood samples from each calf were collected from the jugular vein into 10 mL evacuated glass vials containing 143 IU of Na heparin (Vacutainer, Becton Dickinson Inc., Franklin Lakes, NJ) on days 0, 1, 3, 5, 7, 14, 21 and 28 relative to transportation. Blood samples were immediately placed on ice, and centrifuged at 1,500 x g at 4C for 15 min. The plasma was transferred by pipette into polypropylene vials (12mm x 75mm; Fisherbrand, Thermo Fisher Scient ific Inc., Waltham, MA) and stored at 20C until further analysis. A spectrophotometer (ThermoSpectronic Genesys 20; Thermo Fisher Scientific Inc., Waltham, MA) was used to determine concentrations of plasma ceruloplasmin. The plasma ceruloplasmin oxidase activity was measured in duplicate samples using colorimetric procedures as described in the literature (Demetriou et al., 1974) Concentrations of ceruloplasmin were expressed as mg/dL (King, 1965) The intra and interassay coefficient of variation (CV) for ceruloplasmin were 2.1 and 4.8%, respectively. A micropla te spectrophotometer (Power Wave 340; BioTek Instrument s, Inc., Winooski, VT) was used to determine concentrations of plasma haptoglobin in duplicate samples by measuring haptoglobin/hemoglobin complexing by the estimation of differences in peroxidase activity as described previously (Makimura and Suzuki, 1982) an d results are expressed as arbitrary units from the absorption reading at 450 nm of wavelength x 100. The intra and interassay CV for haptoglobin were 5.9 and 4.9%, respectively.

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44 Feed S ample Collection and Analyses Representative samples of each treatmen t TMR diet were taken before feeding and at 28 d intervals throughout Phases I and II. All samples were bagged and frozen immediately after collection until drying. All feed samples analyzed for nutritive values were dried at 55 C for 48 h in a forced air oven. At conclusion of the drying period all samples were ground in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA) using a 1.0 mm screen. After grinding, samples were composited for analysis on an equal weight basis. The composited samples were analyzed for DM, CP, TDN, ADF, NDF, Ca and P in duplicate by a commercial laboratory using NIR procedures (Dairy One Forage Laboratory, Ithaca, NY). Statistical A nalyses Phase I. Linear regression of BW against day on trial was used to establish ADG, using the SLOPE function of EXCEL (Microsoft, Redmond, WA). Gain to feed ratio was computed as the ratio of ADG to daily DMI. Residual feed intake was calculated as the actual DMI minus expected DMI, with expected DMI derived from the regression of actual DMI on ADG and mid test metabolic body weight using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC ). The repeated measures statement of the MIXED procedure of SAS was used to analyze differences in CS and EV. The statistical model included treatment, d ay, treatment by day interaction and sex, with day as the repeated variable (d 0, 14, 28, 42. 56 and 70), animal as subject and block as random effect. The MIXED procedure of SAS was used to identify differences in initial BW, final BW, ADG, daily DMI, G: F and RFI. The statistical model included treatment, pen within

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45 treatment and sex. Statistical differences were reported at P < 0.05 and tendencies were identified when P < 0.10, with means being reported as LS means SEM. Phase II. Individual calf ADG wa s determined by the difference between the final shrunk BW and the initial shrunk BW upon arrival on d 1, divided by the number of days on trial. Body weight loss (BWL) after transportation was calculated as the difference between BW on d 0 and BW on d 1, the percentage of BWL (%BWL) was calculated as the ratio between BW d 0 and BWL multiplied by 100, and G:F was calculated as the ratio of ADG to daily DMI. The MIXED procedure of SAS was used to identify differences in BW d0, BW d 1, BWL, %BWL, daily DMI, G:F and BF. The statistical model included treatment, pen within treatment and sex. The repeated measures statement of the MIXED procedure of SAS was used to analyze differences in concentrations of haptoglobin and ceruloplasmin. The statistical model inc luded treatment, day, sex and treatment by day interaction, with day as the repeated (d 0, 1, 3, 5, 7, 14, 21 and 28), animal as the subject and block as random effect. For differences in daily DMI after transportation, the repeated measures statement of t he MIXED procedure of SAS was used. The statistical model included treatment, day, sex and treatment by day interaction, with day as repeated (d 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14), animal as the subject and block as random effect. The GLM procedure of SAS was used to assess the correlation between concentrations of ceruloplasmin and haptoglobin with DMI on d 0, 1, 3, 5, 7 and 14 following transportation. Statistical differences were reported at P < 0.05 and tendencies were identified when P < 0.10, with means being reported as LS means SEM.

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46 Results and Discussion Phase I A nimal performance and FE d ata for Phase I are summarized i n Table 3 2 There were no differences between treatments in initial BW, final BW, and ADG during Phase I. Mea n daily DMI during the 70 d FE trial was significantly greater ( P < 0.01) for CON (9.18 0.15 kg) compared to aPLA2 (8.53 0.15 kg). Daily DMI was greater ( P < 0.01) for CON calves during wk 1, 2, 7, and 8, and tended ( P < 0. 10 ) to be greater during wk 9 and 10 of the 70 d trial, compared to calves in the aPLA2 treatment (Figure 3 1). Supplementation of aPLA2 reduced daily DMI, while maintaining similar ADG and final BW to CON calves, improving FE. Residual feed intake was significantly lower ( P < 0.01) f or aPLA2 calves ( 0.272 0.110 kg/d) than for CON calves (0.389 0.110). There was also a tendency ( P = 0.09) for the aPLA2 treatment to have greater G:F than the CON treatment (0.117 0.003 and 0.110 0.003, for aPLA2 and CON, respectively). Calves re ceiving aPLA2 supplementation had a 7% reduction in average daily DMI compared to CON calves (Table 3 2). Similarly, supplementation of aPLA2 improved FE and growth of broilers by 3.8 and 5.3%, respectively (Cook, 2004, 2011) Several other reports of improvements on FE and growth of swine and fish supplemente d with aPLA2 have been reported, without increasing morbidity and mortality due to infectious diseases (Yang et al., 2003; Schwartz et al., 2006; Barry and Yang, 2008) Germ free and antibiotic fed chickens have decreased thickness and weight of the intestine that is largely attributed to accumulation of immune cells and enzymes in ord er to build a defense barrier against microorganisms on the intestinal lumen (Lev and Forbes, 1959; Drew et al., 2003; Cook, 2004) Lipopolysaccharides are commonly present in rumen fluid, and translocation of LPS into the bloodstream can occur,

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47 especially under immu nological stress and ruminally acidotic conditions (Andersen et al., 1994; Gozho et al., 2007; Emmanuel et al., 2007, 2008; Khafipour et al., 2009; Ametaj et al., 2009; Dong et al., 2011) The tran slocation of LPS is mediated by sPLA2, with disruption of the intestinal phospholipid barrier, release of free AA in the cytosol and increased production of prostaglandins, with further activation of the APR and production of proinflammatory cytokines (Johnson, 1997; Rozenfeld et al., 2001; Zayat et al., 2008; Cook, 2011) Regulation of the intestinal immune stimulation could reduce intestinal thickness improving nutrient absorption, reduce the energy costs associated with t he maintenance of the intestinal immune barrier, and reduce the detrimental effects of the activation of APR with production of proinflammatory cytokines over animal performance, explaining the decreased DMI and FE improvement observed on calves treated wi th aPLA2. It has been shown that cattle with more excitable temperament (greater CS and EV) have increased secretion and circulating concentrations of adrenocorticotropic hormone (ACTH) and cortisol, via activation of the hypothalamic pituitary adrenal axi s (Curley et al., 2008) Glucocorticoids, such as cortisol, are essential to the maintenance of homeostasis and act on the regulation of carbohydrate and protein metabolisms, and on the re gulation of growth and reproductive axes. However, excessive levels of cortisol have been linked with suboptimal growth and performance of cattle (Friend, 1991; Johnson, 1997) and cattle with calmer temperament during handling have increased ADG compared with cattle that are agitated during routine handling (Voisinet et al., 1997) A day effect ( P < 0.01) was detected for CS and EV, with an increase in EV (Figure 3 3), and decreased CS (Figure 3 2) during the 70 d FE trial for both treatments.

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48 There was no treatment effect on EV, however calves receiving aPLA2 had reduced ( P < 0.05) CS (Table 3 2), indicating an improvement in temperament and perhaps alleviating the negatives effects on animal perfor mance associated with the activation of the hypothalamic pituitary adrenal axis and greater concentrations of cortisol; however concentrations of cortisol were not measured in this experiment. Phase II. Animal performance and FE data for Phase II are summa rized in Table 3 3. There were no differences in BW before (d 0) and after (d 1) 24 hr transportation, and no differences in final BW at d 28. No difference in mean BWL after 24 hr transportation (26.2 0.6 kg and 25.9 0.6 kg for CON and aPLA2 calves, r espectively) was detected. Weight loss after transportation of beef calves range from 6 to 10%, and the amount of weight loss was directly dependent on body condition prior to transportation, pre transit diets and duration of transport (Fike and Spire, 2006) The percentage o f BW loss after 24 hr transportation did not differ ( P = 0.73) between treatments, calves from both treatments had a 8.1 0.1% reduction in BW during transportation. There were no differences in ADG, average DMI, and G:F between treatments during the 28 d following 24 hr transportation (Table 3 3). Beef calves have decreased DMI after transportation, especially within the first two weeks following transport, and the decrease in DMI was even more dramatically when there was a combination of stressors such as transport, commingling, and introduction to new diets that have greater contents of concentrate (Loerch and Fluharty, 1999; Arthington et al., 2003; Duff and Galy ean, 2007) There was no difference ( P = 0.26) in the decrease in DMI as a result of transportation from d 0 to d 1 (2.43 0.15 kg) (Table 3 3 ). However, the

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49 decrease in average daily DMI over the two weeks following transportation was not different. Cal ves in the aPLA2 and CON treatments had similar decrease in average daily DMI (10 and 13% decrease, respectively) within the first two weeks following transportation compared with average daily DMI on the two weeks that preceded transport. In this study ca lves were maintained on the same diets and group pens during pre and post transport periods, eliminating the stress associated with commingling and dietary changes, which may have attributed similar decreases in DMI following transportation. There were a treatment ( P = 0.02) and a treatment day ( P < 0.0001) effect on daily DMI during the first 15 d following transportation (Figure 3 4). Calves consuming the aPLA2 supplement had reduced average DMI compared to CON calves (8.45 0.21 kg and 8.82 0.20 k g, for aPLA2 and CON, respectively) during the first 15 d following transportation. Calves in the CON group had greater daily DMI than aPLA2 calves on d 1, 6 and 8 and lower daily DMI on d 7. The reduced DMI during the first 15 d following transportation o f aPLA2 calves is likely a result of the lower DMI during Phase I, and reinforce the hypothesis that calves receiving the aPLA2 have reduced energy requirements for maintenance of the intestinal local immune response allowing more available energy and nutr ients for the animal to cope with stress and physiological processes, resulting in reduced DMI and increased FE. The APR is stimulated by the release from macrophages and monocytes of the proinflammatory cytokines TNF IL 1, and IL 6 at the site of infl ammation but they may also act systemically changing behavior, metabolism, neuroendocrine secretions, inducing skeletal muscle waste and lipolysis (Johnson, 1997; Carroll and Forsberg,

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50 2007; Lippolis, 2008) An ultrasonography measurement of BF at d 28 was performed to determine fat thickness betwee n the 12 th and 13 th ribs; however, no difference ( P = 0.26) in BF was detected between treatments (Table 3 3). Concentrations of haptoglobin and ceruloplasmin peaked on d 3 (day effect, P < 0.0001) after transportation indicating that calves experienced a n APR (Arthington et al., 2008) Activation of the APR is a normal immunological reaction of the organism to stress, and is characterized by increased concen trations of proinflammatory cytokines and APP (Johnson, 1997) Although CON calves h ad numerically greater concentrations of haptoglobin at the peak on d 3, supplementation of aPLA2 did not affect ( P = 0.41) concentrations of plasma haptoglobin after 24 hr transportation and no treatment day interaction existed ( P = 0.21; Figure 3 5). I n addition, no treatment day interaction was detected ( P = 0.98) for concentrations of plasma ceruloplasmin. However, a treatment effect ( P < 0.001) existed for concentrations of plasma ceruloplasmin after transportation. Calves receiving aPLA2 supplemen tation had reduced concentrations of plasma ceruloplasmin throughout the 28 d that followed transportation compared to CON calves (Figure 3 6 ), indicating that aPLA2 supplementation successfully reduced the APR to 24 hr transportation. Independent of treat ments, mean concentrations of plasma ceruloplasmin was negatively correlated ( P < 0.001) with average daily DMI during the first two weeks after transportation (Figure 3 7), but no correlation ( P = 0.77) between average daily DMI and concentrations of plas ma haptoglobin was detected. Greater concentrations of ceruloplasmin after transportation have been negatively correlated with ADG, and

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51 positively correlated with concentrations of cortisol (Arthington et al., 2003; Cooke et al., 2009a; Araujo et al., 2010) Conclusion B eef calves receiving a growing diet supplemented with aPLA2 had reduced average daily DMI, while maintaining similar ADG than CON calves, resulting in lower RFI and improved FE during the 70 d trial. Supplementation of aPLA2 improved temperament by reducin g CS of calves, and had reduced concentrations of plasma ceruloplasmin after 24 hr transportation, indicating a reduction in the magnitude of the APR and its negative effects on animal performance.

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52 Table 3 1. Nutrient composition of background diets fed to beef calves during a 70 d feed efficiency trial. Treatments 1 Composition aPLA2 CON DM, % 92.0 92.8 CP, % DM 12.0 13.1 NE g 2 Mcal/kg DM 0.6 0.6 NDF, % DM 63.5 60.1 ADF, % DM 36.1 35.4 TDN, % DM 56.0 57.0 Calcium, % DM 0.6 0.6 Phosp horus, % DM 0.4 0.4 1 aPLA2 = inclusion of aPLA2 supplement at 0.6% of the diet DM; CON = no additive. 2 Net energy for gain.

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53 Table 3 2. The effects of aPLA2 supplementation on animal performance and feed efficiency of growing beef cattle receiving backgrounding diets. Treatments 1 Item aPLA2 C ON SEM P value Initial BW 2 kg 242.0 241.6 3.7 0.92 Final BW 2 kg 312.6 314.0 4.1 0.79 ADG 3 kg 0.99 1.00 0.02 0.95 Daily DMI 4 kg 8.53 9.18 0.15 0.01 G:F 0.117 0.110 0.003 0.09 RFI 5 kg/d 0.272 0.389 0.110 0.002 CS 6 2.21 2.32 0.05 0.03 EV 7 m/s 0.76 0.71 0.05 0.25 1 aPLA2 = inclusion of aPLA2 supplement at 0.6% of the diet DM; CON = no additive. 2 Live B W. 3 Calculated using linear regression of BW against day on trial. 4 Average daily DMI during the 70 d feed efficiency trial. 5 Residual feed intake. 6 Based on a 1 to 5 scale, with 1 being docile and 5 being aggressive. 7 M easure of seconds taken for an imal to travel 1.83 m from squeeze chute

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54 Table 3 3. The effects of aPLA2 supplementation on animal performance, feed efficiency and concentrations of plasma acute phase proteins after 24 hr transportation. Treatments 1 Item aPLA2 C ON SEM P value BW d 0 2 kg 317.2 315.6 3.9 0.73 BW d 1 3 kg 291.3 289.4 3.7 0.65 BW loss 4 kg 25.9 26.2 0.6 0. 86 % BW loss 5 8.2 8.3 0. 2 0. 73 BW d 28 6 kg 339.9 337.9 4.1 0.72 ADG, kg 1.27 1.29 0.03 0.72 Daily DMI 7 kg 8.73 8.95 0.15 0.45 DECDMI 8 kg 2.60 2.25 0.15 0.26 G:F 0.165 0.163 0.004 0.83 BF 9 mm 1.91 2.08 0.01 0.26 Haptoglobin 10 mg/mL 0.08 0.09 0.02 0.41 Ceruloplasmin 11 mg/dL 13.0 14.3 0.3 <0.001 1 aPLA2 = inclusion of aPLA2 supplement at 0.6% of the diet DM; CON = no additive. 2 Shrunk BW (16 hrs separation from feed) immediately before 24 hr transportation. 3 BW immediately after 24 hr transportation. 4 Difference in BW between d 0 and 1. 5 Perc entage BW loss after 24 hr transportation relative to BW on d 0. 6 Shrunk BW (16 hr separation from feed). 7 Average daily DMI during the 28 d following transportation. 8 Decrease in DMI on d 1 of Phase II relative to d 70 of Phase I. 9 Fat thickness at d 28 (measured between the 12 th and 13 th ribs by ultrasonography). 10 Least square mean of concentration of plasma haptoglobin during Phase II. 11 Least square mean of concentration of plasma ceruloplasmin during Phase II.

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55 Figure 3 1. Average daily D MI calculated on a biweekly basis during the 70 d feed efficiency trial. Means differ (P < 0.01). ** Means tend to differ (P = 0.06).

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56 Figure 3 2. Mean chute score (on 5 point scale, with 1 being calm and 5 being aggressive) by day of beef calv es during a 70 d feed efficiency trial. a,b,c Overall day means differ (P < 0.01).

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57 Figure 3 3. Mean exit velocity (seconds for a calf to travel 1.83 m from squeeze chute) by day of beef calves during a 70 d feed efficiency trial. a,b,c Overall day me ans differ (P < 0.01).

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58 Figure 3 4. Average daily DMI of beef calves during 15 d following 24 hr transportation. Means differ within day (P < 0.001). ** Means tend to differ within day (P = 0.06).

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59 Figure 3 5. Concentration of plasma haptoglobin by day of beef calves following 24 hr transportation (treatment effect, P = 0.41; treatment by day effect, P = 0.21).

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60 Figure 3 6. Concentration of plasma ceruloplasmin by day of beef calves following 24 hr transportation (treatment effect, P < 0.001).

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61 Figure 3 7. Correlation between mean concentration of plasma ceruloplasmin and average daily DMI of beef calves after 24 hr transportation, when combining treatments (P < 0.001, R2 = 0.035).

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62 CHAPTER 4 EFFECTS OF ANTI PHOSPHOLIPASE A2 ANTIBODY SUPPLE MENTA TION ON DRY MATTER INTAKE, FEED EFFICIENCY, ACUTE PHASE RESPONSE AND BLOOD DIFFERENTIALS OF STEERS FED FORAGE AND GRAIN BASED DIETS In order to achieve feed efficiency (FE) and more efficient body weight gain, feedlots in the United States have develo ped unique feeding and management strategies. The extensive grain processing and higher consistency in quality of grains feeds in comparison to roughages, aimed at achieving higher productivity, resulted in feedlots to using diets consisting of greater con centrates. However, greater demands for grain in ethanol production and export markets have a large impact on future global grain markets, resulting in increased grain prices. Thus, feedlots and stocker cattle operations will need to focus on optimizing gr ain utilization, fi nding alternative energy sources, selecting animals with greater FE and development of strategies to improve FE while maintaining high performance rates (Galyean et al., 2011) Beef cattle in feed yar ds undergo acidotic challenges when they are transitioned from forage based into grain based diets, resulting in decreased ruminal pH and physiological responses that are directly impacted by the percentage increase in concentrate and the time allowed for ruminal adaptation to occur between diet changes (Berry et al., 2004a; Nagaraja and Titgemeyer, 2007; Ametaj et al., 2009) Lipopolysaccharide (LPS) is a cell wall component of gram negative bacteria that is commonly present in ruminal fluid. Grain fed cattle have greater concentrations of LPS in rumen fluid compared to forage fed cattle, due to greater quantities of gram negative bacteria and greater rates of bacterial death caused by lower ruminal pH values (Nagaraja et al., 1978a; b; Andersen et al., 1994)

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63 The combination between the development of ruminal and intestinal mucosa inflammation with increased concentrations of LPS in the rumen fluid, results in translocation of LPS into the bloodstream with activation of the immune system through the acute phase response (APR) and release of inflammatory mediators (Andersen et al., 1994; Gozho et al., 2007; Emmanuel et al., 2007, 2008; Khafipour et al., 2009; Ametaj et al., 2009; Dong et al., 2011) The secretory phospholipase A2 (sPLA2) enzyme is a potent proinflammatory mediator and plays an important role in LPS induced prostaglandin synthesis in in testinal epithelial cells, mediating the disruption of the intestinal barrier (Cook, 2011) The APR is stimulated by the release from macrophages and monocytes of the pro inflammatory cytokines TNF IL 1 and IL 6 at the site of i nflammation, resulting in redirection of nutrients into defense processes, development of fever, alterations in behavior, decreased feed intake, wasting of skeletal muscle, increased lipolysis and hepatic production of acute phase proteins (APP) (Johnson, 1997; Cook, 2011; Cooke and Bohnert, 2011) Feeding monensin and tylosin selectively inhibits ruminal gram positive bacteria, with consequent improved digestive efficiency of cattle consuming high grain diets, due to increased ruminal concentration of propionic acid, impr oved FE, ADG, reduced DMI and lower incidence of acidosis and liver abscesses in cattle (Richardson et al., 1976; Nagaraja et al., 1985; Burrin and Britton, 1986; Galyean et al., 1992; Nagaraja and Titgemeyer, 2007; Meyer et al., 2009) However, antimicrobial resistance is a growing public issue and the use of antibi otics as growth promotants for beef cattle seems to be subjected to future limitations and more restricted regulations (Galyean et al., 2011)

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64 Recently, an egg derived antibody against intestinal sPLA2, anti phospholipa se A2 (aPLA2), was developed to regulate the intestinal inflammatory response and the negative effects of the activation of the APR on animal growth and FE, and it has been successfully used as a feed additive for poultry, swine, and fish, with improved FE and growth (Yang et al., 2003; Cook, 2004, 2011; Schwartz et al., 2006; Barry and Yang, 2008) Therefore, t he potential exist that steers consuming aPLA2 may have decreased inflammatory response due to a transition into high grain diets, and consequently enhanced performance and improved FE. The objectives of this study was to determine whether supplementation of aPLA2 would alter animal performance, voluntary DMI, FE, plasma concentration of APP and blood differentials (BD) of steers transitioned from a high forage into a high grain diet. Materials and Methods Animals and T reatments Eighty cross bred steers wer e allocated in group pens (108 m2/pen; 8 pens with 10 animals each) at the University of Florida North Florida Research and Extension Center, Feed Efficiency Facility (FEF), in Marianna, FL. Individual daily DMI was measured using the GrowSafe system (Grow Safe Systems Ltd., Alberta, Canada). Each pen in the FEF was equipped with two GrowSafe feed bunks. Steers were blocked by weight and then randomly assigned to pens to receive treatments. A 14 d period of adaptation to facilities and diets preceded the ini tiation of the 141 d trial. Shrunk BW (16 hr following removal from feed) was measured on d 0, prior to initiation of diet change on d 60, immediately following diet change on d 81 and at co nclusion of the study on d 141.

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65 Following the adaptation period, t he experiment was divided in three phases (Figure 4 1 ). In Phase I steers were fed a growing diet (0.80 Mcal NEG/kg of DM and 13% CP) comprised of 69% concentrate, 31% bermudagrass hay and a vitamins and minerals supplement containing the following treatme nts: 1) no additive (CON; n = 20); 2) inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM (MT; n = 20); 3) inclusion of a aPLA2 supplement at 0.2% of the diet DM (aPLA2 0.2%; n = 20); 4) inclusion of aPLA2 supplement at 0.4% of the diet DM (aPLA2 0.4%; n = 20). Phase II, on d 60 all steers were transitioned into a grain with three steps from 69% concentrate to 75% (step 1), 85% (step 2) and 90% concentrate (step 3). Phase III, on d 81 all steers re ceived a 90% concentrate diet (74% cracked corn; 1.32 Mcal NEg/kg of DM, 11.4% CP) for 60 d until the conclusion of the experiment on d 141. A soybean meal based premix containing 0.2 0.4% of aPLA2, or no antibody, was mixed to the treatment diets at 1% o f the diet DM. In addition, two pelleted supplements were included at a 5% of the diet DM to deliver either no antibiotics (CTL and all aPLA2 containing diets), or 30 mg of monensin plus 8.8 mg of tylosin per kg of DM (MT diet). Steers were maintained in t he same group pens throughout the 141 d trial. The nutrient content of all diets are presented on Table 4 1 Blood C ollection and A nalyses Phase II Blood samples from each steer were collected from the jugular vein into 10 mL evacuated glass vials contai ning 143 IU of Na heparin (Vacutainer, Becton Dickinson Inc., Franklin Lakes, NJ) on days 0, 3, 5, 7, 10, 12, 14, 17, 19 and 21 relative on ice, and centrifuged at 1, 500 g, at 4C for 15 min. The plasma was transferred by

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66 pipette into polypropylene vials (12mm x 75mm; Fisherbrand, Thermo Fisher Scientific Inc., Waltham, MA) and stored at 20C until further analysis. A spectrophotometer (ThermoSpectronic Genesys 20; Thermo Fisher Scientific Inc., Waltham, MA) was used to determine plasma ceruloplasmin concentration. The plasma ceruloplasmin oxidase activity was measured in duplicate samples using colorimetric procedures as described in the literature (Demetriou et al., 1974) Concentrations of ceruloplasmin were expressed as mg/dL (King, 1965) The intra and interassay coefficient of variation (CV) for ceruloplasmin were 6.0 and 10.4%, respectively. A microplate spectroph otometer (PowerWave 340; BioTek Instruments, Inc., Winooski, VT) was used to determine concentrations of plasma haptoglobin in duplicate samples by measuring haptog lobin/hemoglobin complexing by the estimation of differences in peroxidase activity as described previously (Makimura and Suzuki, 1982) and results are expressed as arbitrary units from the absorption reading at 450 nm 100. The intra and interassay CV for ha ptoglobin were 7.0 and 11.3%, respectively. Blood differentials, total red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), total white blood cell count (WBC), neutrophils, lymphocytes and neutrophil:lymphocyte ratio (N:L), monocytes, eosinophi ls and total platelets count were assessed using a hematology cell counter (IDEXX ProCyte DX Hematology Analyzer, Westbrook, ME), and individual whole blood samples. Blood samples were collected from the jugular vein into 2.0 mL polypropylene evacuated tub es containing 3.6 mg of K2 EDTA (BD Hemogard, Becton Dickinson Inc., Franklin Lakes, NJ), and immediately stored on ice until analysis, all samples were analyzed within 8 hr of collection.

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67 Feed S ample Collection and Analyses Representative samples of each treatment TMR diet were taken before feeding, at 28 d intervals throughout the 141 d trial. All samples were bagged and frozen immediately after collection until drying. All feed samples analyzed for nutritive values were dried at 55C for 48 hr in a force d air oven. At conclusion of the drying period all samples were ground in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA) using a 1.0 mm screen. After grinding, samples were composited for analysis on an equal weight basis. The composited sa mples were analyzed for DM, CP, TDN, ADF, NDF, Ca, and P in duplicate by a commercial laboratory using NIR procedures (Dairy One Forage Laboratory, Ithaca, NY). Ultrasonic Carcass Traits On d 0 and d 141, ultrasound was used to determine fat thickness (BF) and Longissimus dorsi muscle area (REA) using an Aloka real time ultrasound scanner (3.5 MHz linear array transducer, Aloka 500V, Corimetrics Medical Systems, Inc., Wallingford, CT) and image capturing software. Scanning was performed on the right side of each animal, between the 12 th and 13 th ribs. There were two measurements taken per animal at each scanning session. Final BF and REA values were calculated as the average between the two measurements recorded. Statistical A nalyses Individual steer ADG wa s determined by the difference between the shrunk BW at the end and the beginning of each phase, divided by the number of days on trial. Gain to feed ratio (G:F) was computed as the ratio of ADG to daily DMI. Residual feed intake (RFI) was calculated as th e actual DMI minus expected DMI, with expected DMI derived

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68 from the regression of actual DMI on ADG and mid test metabolic body weight using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC, 2011). The MIXED procedure of SAS was used to identify differen ces in BW, ADG, average daily DMI, G:F, RFI, BF and REA. The statistical model included treatment, pen within treatment. The repeated measures statement of the MIXED procedure of SAS was used to analyze differences in concentrations of haptoglobin and ceru loplasmin, treatment, day, and treatment day interaction, with day as the repeated (d 0, 3, 5, 7, 10, 12, 14, 17, 19 and 21), animal as the subject and block as random effect. The decrease in DMI (DECDMI) immediately after each change in diet during phase II was analyzed using the repeated measures statement of the MIXED procedure of SAS. The statistical model included treatment, day and treatment by day interaction, wi th day as the repeated variable (d 1, 8 and 15), animal as the subject and block as random. The CONTRAST function of the GLM procedure of SAS was used to conduct orthogonal contrasts between the aPLA2 0.2% and aPLA2 0.4% (0.2/0.4), both aPLA2 treatment c ombined against MT (aPLA2/MT), and both aPLA2 plus MT treatments against CON treatment (TRT/CON) on daily DMI, and concentrations of ceruloplasmin and haptoglobin, and BD during Phase II. The statistical model included treatment, day and the treatment da y interaction. The GLM procedure of SAS was used to assess the correlation between concentrations of haptoglobin and ceruloplasmin with DMI during phase II on d 0, 3, 5, 7, 10, 12, 14, 17, 19 and 21. Statistical differences were reported at P < 0.05 and te ndencies were identified when P < 0.10, with means being reported as LS means SEM.

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69 Results and Discussion Animal P erformance High concentrate diets have been linked to the activation of the APR in cattle (Emmanuel et al., 2008; Ametaj et al., 2009) which may negatively affect animal growth and FE as a result of de creased DMI, wasting of skeletal muscle and lipolysis (Joh nson, 1997; Cook, 2011) However, in the current experiment there were no statistical differences among treatments in measurements of performance and FE (Table 4 2 ). No treatment differences were detected in BW on d 0 (212.0 4.0 kg, P = 0.94), BW on d 1 41 (388.0 5.1 kg, P = 0.79), ADG (1.25 0.02 kg/d, P = 0.33), BF on d 0 (0.96 0.04 cm, P = 0.89) and on d 141 (1.43 0.11 cm, P = 0.73), REA on d 0 (25.3 3.4 cm2, P = 0.49) and on d 141 (46.4 2.6 cm2, P = 0.51), G:F (0.18 0.01, P = 0.98), averag e daily DMI (7.1 0.1 kg, P = 0.43), and RFI ( 1.25 0.06 kg/d, P = 0.61). Phase I. Animal performance and FE data during Phase I are summarized in Table 4 3 There were no differences on BW at d 60 (259.0 4.4 kg; P = 0.84) and G:F (0.11 0.01; P = 0.16) among treatments. However, there was a tendency (P = 0.08) for the MT treatment to have greater ADG compared to the aPLA2 0.2%, whereas CON and aPLA2 0.4% treatments were intermediate. Feeding monensin and tylosin improves DM digestive efficiency of cattle, increasing ruminal concentrations of propionic acid and decreasing concentrations of acetate and butyrate (Richardson et al., 1976; Beckett et al., 1998) increasing glucose supply to the animal, thus improving FE, reducing DMI, and improving ADG of growing and finishing cattle (Duffield et al., 2012) Similar to the results from Phase I of Chapter 3 (Table 3 2), aPLA2 supplementation tended to reduce daily DMI, and significantly reduced RFI compared to

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70 CON steers, improving FE of steers fed forage based diets. Daily DMI tended (P = 0.07) to be lower for both aPLA2 0.2% and 0.4% treatments compared to CON treatment, with MT treatment be ing intermediate. Steers from both aPLA2 treatments had lower RFI than CON steers (P < 0.05). Steers in the aPLA2 0.4% treatment had the lowest RFI, and MT steers had intermediate RFI values compared to other treatments. Regulation of the intestinal inflam matory response by blocking intestinal sPLA2, reduces the nutrient and energy costs of the immune intestinal barrier, therefore reducing the maintenance requirements and DMI of the animal, with an overall improvement in FE (Cook, 2011) Phase II. Performan ce and FE data during Phase II are summarized in Table 4 4 There were no differences among treatments on BW on d 81 (297.0 5.0 kg, P = 0.46), ADG (1.82 0.16 kg/d, P = 0.17) and G:F (0.26 0.02, P = 0.47) during Phase II. However, similar to Phase I a verage daily DMI was significant lower (P = 0.02) for aPLA2 0.4% steers compared to MT and CON steers, daily DMI for steers on aPLA2 0.2% was intermediate. Daily DMI was impacted by diet changes during Phase II. Supplementation of aPLA2 and monensin and t ylosin reduced DMI of steers during the transition to grain based diets. Using contrast analysis to assess differences in daily DMI during Phase II, there were no differences (P = 0.18) in daily DMI between the aPLA2 treatments (Figure 4 3 ). However, when comparing both aPLA2 treatments combined against CON treatment (Figure 4 4 ), and both aPLA2 plus the MT treatments combined against CON (Figure 4 5 ), CON steers had significantly greater (P < 0.0001) daily DMI during Phase II.

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71 Dry matter intake was decrea sed (day effect, P < 0.0001) on the day after diet change (d 1, 8 and 15) across all treatments. The mean DECDMI on the day after change in diets were significantly lesser (P = 0.03) for the MT compared with CON steers, and both aPLA2 treatments were inter mediate (Table 4 4 ). A treatment day effect (P < 0.001) was also detected for DECDMI (Figure 4 2 ). On d 1, CON steers had the greatest DECDMI compared to aPLA2 0.2% and MT steers. There was a tendency (P = 0.06) for aPLA2 treatments to have lesser DECDM I then CON, with MT steers having intermediate DECDMI on d 8. However, on d 15 DECDMI was significantly greater for both aPLA2 treatments than MT, and CON steers had reduced DECDMI compared to aPLA2 0.2% steers. The lower DMI of MT steers is likely a refle ction of the inhibition of gram positive bacteria in the rumen (Richardson et al., 1976) during the transition to grain based diets, increasing ruminal propionate concentration, and improving ruminal function, decre asing the negative impacts of the ruminal adaptation to higher concentrate contents of the diets on DMI and animal performance (Nagaraja et al., 19 85) Phase III. Animal performance data during Pha se III are summarized in Table 4 5 Supplementation of monensin and tylosin have been successfully used to decrease the risk of acidosis and sub acute acidosis in cattle consuming high grain diets, due to reduction in lactic acid concentration in the rumen, reducing the negative impact of acidosis on ruminal function ( Nagaraja et al., 1985; Burrin and Britton, 1986; Nagaraja and Titgemeyer, 2007) Monensin also increases ruminal concentration of propionic acid, increasing hepatic production of glucose (Richardson et al., 1976; Bergen and Bates, 1984) thus improving animal performance and FE of cattle (Duffield et al.,

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72 2012) However, in the current experiment there were no differences in BW at d 141, ADG, average daily DMI and G:F among treatments. Residual feed intake was significantly greater (P = 0.04) for aPLA2 0.4% treatment compared to CON. Steers in the aPLA2 0.4% treatment had the greatest RFI (0.36 0.10 kg/d) and CON steers had the lowest RFI ( 0.37 0.10 kg/d), with aPLA2 0.2% and MT steers remaining intermediate ( 0.12 0.10 and 0.10 0.10 kg/d, respectively). Feeding high grain diets reduces ruminal pH, and facilitated the development of acidosis and rumenitis (Nagaraja and Titgemeyer, 2007) Egg antibodies were shown to retain over 85% of their anti rotavirus neutralizing activity at a pH 2 for one hour, even though the antibody binding capacity was reduced by 50% (Cook and Trott, 2010) However, the effects of long exposures to lower pH, such as the rumen when feeding high grain diets, on egg antibody function have not been reported. Perhaps the failure of aPLA2 supplementation to improve FE during Phase III is a result of the denaturation of the antibody due to exposure of lower pH in the rumen, directly affecting the quantity of active antibody passing through the rumen into the intestine and its capacity to block the intestinal inflamma tory response, thereby alleviating the negatives effects of the activation of the immune system over animal performance Acute Phase Proteins and B lood D ifferentials Blood differentials and AP P data are summarized in Table 4 6 There were no significant di fferences among treatments on mean RBC (8.96 0.30 M/L, P = 0.23), HCT (34.58 0.82 %, P = 0.27), HGB (11.58 0.46 g/dL, P = 0.46), neutrophils (3.56 0.23 k/L, P = 0.21), N:L (0.53 0.03, P = 0.84), monocytes (2.42 0.71 k/L, P = 0.82) eosinophil s (0.22 0.04 k/L, P = 0.64) and platelets (406.9 34.2, P = 0.31).

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73 Transition from forage to grain based diets has been shown to activate the APR of cattle (Berry et al., 2004a; Ametaj et al., 2009) through the development of acidosis and rumenitis with increased concentrations of LPS in the rumen fluid and translocation of LPS to the bloodstream (Nagaraja and Titgemeyer, 2007; Emmanuel et al., 2007, 2008) The APR activates leukocytes such as macrophages, increasing the production of pro inflammatory cytokines and resulting in a systemic inflammatory response, with development of leukocytosis, which is marked by an increase in lymphocy tes (Johnson, 1997) Mean WBC and lymphocytes among treatments were within the norma l interval for cattle (Merck Veterinary Manual, 2010) However, WBC were significantly greater (P = 0.04) for aPLA2 0.2% (13.61 0.42 k/L) than aPLA2 0.4% and MT tr eatments (12.16 0.42 and 12.37 0.42 k/L, respectively), with CON being intermediate (12.87 0.42 k/L). Concentrations of lymphocytes also were significantly greater (P = 0.01) for aPLA2 0.2% (7.66 0.28 k/L) than aPLA2 0.4% and MT treatments (6.71 0.28 and 6.70 0.28 k/L, respectively), with CON being intermediate (7.11 0.28 k/L). No differences in mean concentrations of plasma ceruloplasmin and haptoglobin between treatments during Phase II were detected (Table 4 6 ). There was a day effect (P < 0.01) for concentrations of both haptoglobin and ceruloplasmin in Phase II indicating that steers experienced an APR during the transition to grain based diets (Arthington et al., 2003; Emmanuel et al., 2007; Ametaj et al., 2009) Concentrations of plasma ceruloplasmin and haptoglobin peaked after each diet change (Figure 4 6 and Figure 4 7 respectively), and there was a treatment day effect (P = 0.01) on concentrations of plasma ceruloplasmin during Phase II (Figure 4 6 ). On d 10, steers in the aPLA2 0.4% treatment had greater (P < 0.05) concentrations of ceruloplasmin th an

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74 steers in MT and CON treatments. On d 17, steers in the aPLA2 0.2% treatment had greater (P < 0.05) concentrations of ceruloplasmin than CON and MT treatments. On d 21, aPLA2 0.2% steers had greater (P = 0.02) concentrations of ceruloplasmin than CON st eers, with MT and aPLA2 0.4% steers being intermediate. No treatment day effect was detected for concentrations of plasma haptoglobin (Figure 4 7 ). Concentrations of plasma ceruloplasmin was reduced (P < 0.0001) for CON steers compare d to aPLA2 treatmen ts (Figure 4 8 ), and compared to aPLA2 p lus the MT treatments (Figure 4 9 ). Concentrations of plasma haptoglobin was reduced (P < 0.05) for CON steers compar ed to aPLA2 treatments (Figure 4 10 ). When combining all treatments, mean concentrations of plasma ceruloplasmin (Figure 4 1 1 ) and haptoglobin (Figure 4 12 ) were negatively correlated (P < 0.001) with mean daily DMI during Phase II, in agreement with previously reports (Arthington et al., 2003; Cooke et al., 2009a; Araujo et al., 2010) and our findings from Phase II on Chapter 3. Conclusion Supplementation of aPLA2 improved FE of steers fed a growing diet containing 31% forage (DM basis) but not when feeding grain based diets. Steers from both aPLA2 treatments and MT steers had reduced DMI during the 21 d transition period from forage based to grain based diets. Steers on aPLA2 0.4% and MT treatments had decreased WBC and concentration of lymphocytes during the transition period compared to aPLA2 0.2% steers, with CON steers being intermediate. However, CON steers had reduced concentrations of plasma ceruloplasmin and haptoglobin during the transition to grain based diet compared to steers on aPLA2 and MT treatments. Mean daily DMI was negatively correlated with concentrations of plasma ceruloplasmin and haptoglobin.

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75 T able 4 1 Nutrient composition of diets fed to steers during a transition from a forage based to grain based diet using a three steps adaptation period over 21 d. Treatments 1 aPLA2 0.2% aPLA2 0.4% MT CON Composition, DM HF 2 Stp1 3 Stp2 4 HG 5 HF Stp1 St p2 HG HF Stp1 Stp2 HG HF Stp1 Stp2 HG DM, % 92.4 92.9 93.9 93.7 92.6 93.2 93.3 94.0 93.3 93.4 93.5 93.7 92.2 93.1 93.4 93.3 CP, % 13.9 10.4 13.2 13.5 13.1 11.5 13.2 13.5 13.9 10.6 13.2 15.0 15.9 11.4 13.5 13.8 NE g 6 Mcal/kg 0.8 0.9 1.1 1.2 0 .8 1.0 1.1 1.2 0.8 1.0 1.1 1.1 0.8 1.0 1.1 1.2 ADF, % 32.0 22.7 13.6 9.2 32.4 22.3 18.5 9.7 35.3 20.9 13.7 17.6 29.3 20.3 17.5 11.3 NDF, % 58.1 43.9 28.1 21.6 59.2 10.3 31.5 16.9 57.8 39.2 29.7 31.5 54.3 40.7 33.6 22.2 TDN, % 63. 0 68.0 73.0 75.0 63.0 69.0 71.0 77.0 63.0 69.0 72.0 71.0 65.0 69.0 71.0 75.0 Ca, % 0.9 0.7 0.8 0.6 0.8 0.8 1.0 0.9 0.9 0.7 1.0 1.0 1.0 0.8 0.9 0.8 P, % 0.5 0.4 0.4 0.4 0.5 0.4 0.4 0.4 0.5 0.4 0.4 0. 5 0.6 0.4 0.4 0.4 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no add itive. 2 Growing diet ( 31% bahiagrass hay and 69% concentrate) fed during Phase I. 3 Step 1 diet (75% concentrate) fed during Phase II. 4 Step 2 diet (85% concentrate) fed during Phase II. 5 High grain diet (90% concentrate) fed during step 3 on Phase II and Phase III. 6 N et energy for gain.

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76 Table 4 2 Overall animal performance, feed efficiency and ultrasound carcass traits of steers transitioned from a forage based to grain based diets using a 21 d d trial. Treatments 1 Item aPLA2 0.2% aPLA2 0.4% MT CON SEM P value BW 2 d 0, kg 211 .0 210 .0 216 .0 210 .0 4.0 0.94 BW 2 d 141, kg 380 .0 384 .0 392 .0 393 .0 5.1 0.79 ADG 3 kg 1.20 1.23 1.25 1.30 0.02 0.33 G:F 0.18 0.17 0.17 0.18 0.01 0.98 Daily DMI 4 kg 6.8 7.1 7.2 7.2 0.1 0.43 RFI 5 kg/d 0.14 0.01 0 04 0.09 0.06 0.61 BF 6 d 0, cm 0.95 0.91 1.01 0.97 0.04 0.89 BF 6 d 141, cm 1.47 1.38 1.42 1.44 0.11 0.73 REA 7 d 0, cm 2 25.3 23.7 26.4 25.6 3.4 0.49 REA 7 d 141, cm 2 45.4 45.9 46.7 47.5 2.6 0.51 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no additive. 2 Shrunk BW (16 hr separation from feed). 3 ADG calculated as the difference between BW on d 0 and d 141, divided by number of days on trial. 4 Average daily DMI during the 141 d trial. 5 Residual feed intake. 6 Fat thickness measured between the 12 th and 13 th ribs by ultrasonography. 6 Longissimus dorsi area measured between the 12 th and 13 th ribs by ult rasonography.

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77 Table 4 3 Animal performance and feed efficiency of steers fed a forage based diet during Phase I. Treatments 1 Item aPLA2 0.2% aPLA2 0.4% MT CON SEM P value BW 2 d 0, kg 211.0 210.0 216.0 210.0 4 .0 0.94 BW 2 d 60, kg 253.0 258.0 260.0 264.0 4.4 0.84 ADG 3 kg 0.69 0.79 0.83 0.81 0.04 0.08 Daily DMI 4 kg 6.51 6.70 7.09 7.38 0.25 0.07 G:F 0.11 0.12 0.12 0.11 0.01 0.15 RFI 5 kg/d 0.12 a 0.22 a 0.05 ab 0.31 b 0.13 0.03 a,b Significant differences of least squared means within a row ( P < 0.05). 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no additive. 2 Shrunk BW (16 hrs separation from feed). 3 ADG calculated as the difference between BW on d 0 and on d 60, divided by number of days on trial. 4 Aver age daily DMI during the 60 d of Phase I. 5 Residual feed intake.

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78 Table 4 4 Animal performance and feed efficiency of steers transitioned from a forage based to a grain Phase II. Treatments 1 Item aPLA2 0.2% aPLA2 0.4% MT CON SEM P value BW 2 d 60, kg 253.0 258.0 260.0 264.0 4.4 0.84 BW 2 d 81, kg 298.0 285.0 297.0 308.0 5.0 0.46 ADG 3 kg 2.14 1.29 1.76 2.10 0.16 0.17 Daily DMI 4 kg 7.15 ab 6.52 a 7.21 b 7.64 b 0.13 0.02 G:F 0.30 0.20 0.25 0.28 0.02 0.47 DECDMI 5 kg 2.37 ab 2.61 ab 1.59 a 3.18 b 0.40 0.03 a,b,c Significant differences of least squared means within a row ( P < 0.05). 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no additive. 2 Shrunk BW (16 hrs separation from feed). 3 ADG calculated as the difference between BW on d 60 and on d 81, divided by number of days on trial. 4 Average daily DMI during the 21 d of Phase II. 5 Average decrease in DMI immediately after change in diet.

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79 Table 4 5 Animal performance and feed efficiency of steers fed a grain based diet during Phase III. Treatments 1 Item aPLA2 0.2% aPLA2 0.4% MT CON SEM P value BW 2 d 81, kg 298.0 285.0 297.0 308.0 5.0 0.46 BW 2 d 141, kg 380.0 384.0 392.0 393.0 5.1 0.79 ADG 3 kg 1.37 1.65 1.58 1.42 0.06 0.23 Daily DMI 4 kg 7.14 7.61 7.41 6.98 0.11 0.13 G:F 0.19 0.22 0.21 0.20 0.01 0.78 RFI 5 kg/d 0.12 ab 0.36 a 0.10 ab 0.37 b 0.10 0.04 a,b Significant differences of least squared means within a row ( P < 0.05). 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusi on of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no additive. 2 Shrunk BW (16 hrs separation from feed). 3 ADG calculated as the difference between BW on d 81 and on d 141, divided by number of days on trial. 4 Average daily DMI durin g the 60 d of Phase III. 5 Residual feed intake.

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80 Table 4 6 Blood differentials and concentrations of plasma acute phase proteins of steers transitioned from a forage based to grain Treatments 1 Item 2 aPLA2 0.2% aPLA2 0.4% MT CON SEM P value RBC, M/L 9.01 8.79 9.25 8.78 0.30 0.23 HCT, % 34.34 34.00 35.76 34.22 0.82 0.27 HGB, g/dL 11.45 11.54 11.89 11.42 0.43 0.46 WBC, k /L 13.61 a 12.16 b 12. 37 b 12.87 ab 0.42 0.04 Neutrophils, k/L 3.79 3.27 3.53 3.63 0.23 0.21 Lymphocytes, K/L 7.66 a 6.71 b 6.70 b 7.11 ab 0.28 0.01 N:L 0.52 0.51 0.53 0.54 0.03 0.84 Monocytes, k/L 1.99 1.94 2.8 1 2.94 0.71 0.82 Eosinophils, k/L 0.25 0.18 0.20 0.25 0.04 0.64 Platelets, k/L 407.19 387.36 448.41 384.64 34.20 0.31 Ceruloplasmin, mg/dL 24.43 24.28 23.39 22.20 0.83 0.21 Haptoglobin, mg/mL 0.28 0.23 0.19 0.18 0.05 0.42 a,b Significant differences of least squared means within a row ( P < 0.05). 1 aPLA2 0.2% = inclusion of a aPLA2 supplement at 0.2% of the diet DM; aPLA2 0.4% = inclusion of a aPLA2 supplement at 0.4% of the diet DM; MT = inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM; CON = no additive. 2 RBC = red blood cell count; HCT = hematocrit; HGB = hemoglobin; WBC = white blood cell count; N:L = neutrophil to lymphocyte ratio.

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81 Figure 4 1 Experiment outline of steers transitioned from a forage based to a grain

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82 Figure 4 2 Decrease in daily DMI after diet change on d 1 (step 1), 8 (step 2), and 15 (step 3) of steers transitioned from a forage based to grain based diet over a differ, P < 0.001; yz least square means tend to differ, P = 0.06).

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83 Figure 4 3 Average DMI by day of steers transitioned from a forage based to a grain based diet o between aPLA2 0.2% and aPLA2 0.4% ( P = 0.18).

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84 Figure 4 4 Average DMI by day of steers transitioned from a forage based to a grain ing Phase II. Contrast between both aPLA2 treatments combined and CON ( P < 0.0001).

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85 Figure 4 5 Average DMI by day of steers transitioned from a forage based to a grain betw een both aPLA2 and MT treatments combined and CON (P < 0.0001).

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86 Figure 4 6 Concentrations of plasma ceruloplasmin by day of steers transitioned from a forage based to a grain during Phase II. Mea ns differ within day, P = 0.01.

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87 Figure 4 7 Concentrations of plasma haptoglobin by day of steers transitioned from a forage based to a grain during Phase II.

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88 Figure 4 8 Concentrations of plasm a ceruloplasmin by day of steers transitioned from a forage based to a grain during Phase II. Contrast between both aPLA2 treatments combined against CON (P < 0.0001).

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89 Figure 4 9 Concentrations of pla sma ceruloplasmin by day of steers transitioned from a forage based to a grain during Phase II. Contrast between both aPLA2 and MT treatments combined against CON (P < 0.0001).

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90 Figure 4 10 Concentrati ons of plasma haptoglobin by day of steers transitioned from a forage based to a grain during Phase II. Contrast between both aPLA2 treatments combined against CON (P < 0.05).

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91 Figure 4 1 1 Correlation between plasma concentration of haptoglobin and average DMI by day of steers transitioned from a forage based to a grain based diet treatments (P < 0.001, R2 = 0.02).

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92 Figure 4 1 2 Correlation between plasma concentration of ceruloplasmin and average DMI by day of steers transitioned from a forage based to a grain based diet treatments (P < 0.0001, R2 = 0.03).

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102 BIOGRAPHICAL SKETCH Vitor Rodrigues Gomes Mercadante was born in Piracicaba, So Paulo, Brazil in 1986 He was raised in Campo Grande in the state of Mato Grosso do Sul, where he had his first contact with beef cattle production. In 2005, Vitor entered the Veterinary Medicine School at the So Paulo State University in Botucatu, where he joined CONAPEC Jr., a student enterprise consulting on agricultural issues, as the director for beef projects. After graduation, Vitor moved to Gainesville FL, US to pursue his the immune system on performance and feed efficiency of beef cattle.