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Effect of Ractopamine-Hydrochloride on Muscle Fiber Morphometrics, Satellite Cell Population, and Shelf-Life Properties ...

Permanent Link: http://ufdc.ufl.edu/UFE0022824/00001

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Title: Effect of Ractopamine-Hydrochloride on Muscle Fiber Morphometrics, Satellite Cell Population, and Shelf-Life Properties of Beef Cattle
Physical Description: 1 online resource (152 p.)
Language: english
Creator: Gonzalez, John
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: agonist, beta, cattle, cell, color, cow, fiber, hypertrophy, muscle, myonuclei, ractopamine, satellite, shelf, steer
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of ractopamine-hydrochloride (RAC) to affect muscle fiber morphometrics, satellite cell population, and shelf-life properties of various muscles of the beef carcass was explored over three studies. In the first two studies, RAC was supplemented to cull cows during the final 28-35 days of feeding and differentially affected muscles of these animals. In the first study, RAC increased (P < 0.05) the cross-sectional area (CSA) and diameter of type I muscle fibers in the Longissimus dorsi without affecting type IIA fibers. In the second study, RAC supplementation increased the CSA of type I fibers of the Infraspinatus (P < 0.05) and Vastus lateralis (P < 0.15), but did not affect (P > 0.05) the Longissimus dorsi or Semimembranosus. Of these four muscles, RAC increased (P < 0.05) the CSA of type IIA fibers in only the Infraspinatus and Semimembranosus. In both studies, RAC supplementation altered (P < 0.05) the muscle fiber isoform distribution, but did not increase (P > 0.05) the number of satellite cells or fiber associated nuclei counted. This indicates that all growth observed in type I or type IIA fibers happened independent of satellite cell incorporation into the muscle fiber. In the third study, RAC supplemented to steers did not affect (P > 0.05) the CSA of either fiber isoforms nor the muscle weights or dimensions of muscles of the round and loin. The muscle fiber isoform distribution of all muscles, except the Semimembranosus, was changed (P < 0.05). Ractopamine supplementation did not affect (P > 0.05) objective measures of color or nitric oxide metmyoglobin reducing ability during a five day simulated retail display. Ractopamine supplementation increased (P < 0.05) the amount of surface discoloration on steaks from the Rectus femoris, Semimembranosus, and Vastus lateralis during the final days of display when evaluated by trained panelists. Results from all three studies indicate that RAC supplementation to both cull cows and steers has a limited ability to increase muscle fiber CSA, but can affect surface discoloration during retail display.
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 John Gonzalez.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Johnson, Dalton D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022824:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022824/00001

Material Information

Title: Effect of Ractopamine-Hydrochloride on Muscle Fiber Morphometrics, Satellite Cell Population, and Shelf-Life Properties of Beef Cattle
Physical Description: 1 online resource (152 p.)
Language: english
Creator: Gonzalez, John
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: agonist, beta, cattle, cell, color, cow, fiber, hypertrophy, muscle, myonuclei, ractopamine, satellite, shelf, steer
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of ractopamine-hydrochloride (RAC) to affect muscle fiber morphometrics, satellite cell population, and shelf-life properties of various muscles of the beef carcass was explored over three studies. In the first two studies, RAC was supplemented to cull cows during the final 28-35 days of feeding and differentially affected muscles of these animals. In the first study, RAC increased (P < 0.05) the cross-sectional area (CSA) and diameter of type I muscle fibers in the Longissimus dorsi without affecting type IIA fibers. In the second study, RAC supplementation increased the CSA of type I fibers of the Infraspinatus (P < 0.05) and Vastus lateralis (P < 0.15), but did not affect (P > 0.05) the Longissimus dorsi or Semimembranosus. Of these four muscles, RAC increased (P < 0.05) the CSA of type IIA fibers in only the Infraspinatus and Semimembranosus. In both studies, RAC supplementation altered (P < 0.05) the muscle fiber isoform distribution, but did not increase (P > 0.05) the number of satellite cells or fiber associated nuclei counted. This indicates that all growth observed in type I or type IIA fibers happened independent of satellite cell incorporation into the muscle fiber. In the third study, RAC supplemented to steers did not affect (P > 0.05) the CSA of either fiber isoforms nor the muscle weights or dimensions of muscles of the round and loin. The muscle fiber isoform distribution of all muscles, except the Semimembranosus, was changed (P < 0.05). Ractopamine supplementation did not affect (P > 0.05) objective measures of color or nitric oxide metmyoglobin reducing ability during a five day simulated retail display. Ractopamine supplementation increased (P < 0.05) the amount of surface discoloration on steaks from the Rectus femoris, Semimembranosus, and Vastus lateralis during the final days of display when evaluated by trained panelists. Results from all three studies indicate that RAC supplementation to both cull cows and steers has a limited ability to increase muscle fiber CSA, but can affect surface discoloration during retail display.
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 John Gonzalez.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Johnson, Dalton D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022824:00001


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1 EFFECT OF RACTOPAMINE-HYDROCHLORIDE ON MUSCLE FIBER MORPHOMETRICS, SATELLITE CELL POPULA TION, AND SHELF-LIFE PROPERTIES OF BEEF CATTLE By JOHN MICHAEL GONZALEZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 John Michael Gonzalez

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3 To my love, Sara, and my Texas and Florida fa milies. Without each of you I could not have finished this.

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4 ACKNOWLEDGMENTS I am glad that dissertations are not required reading for most people, because I know that I will forget to thank some people. If I fo rget to mention you, please know that I am not ungrateful, but that there have been a ton of people in my life that have helped me get to this point and it would be impossible to remember you all. To begin, I would like to first thank my supervisory committee. To the Chair of my committee, Dr. D. Dwain Johnson, I would like to thank you for giving me the opportunity to come to the University of Florida and work under you. I really appreciate the opportunity because I know that I was not the strongest meat scientist coming out of my Masters, but you allowed me the chance to further my education here. You have taught me valuable lessons in ho w to conduct research, teach and interact with students, and follow through on my re search ideas. I consider it an honor to have worked under you. Appreciation is also extended to Dr. Sa lly Williams and Dr. J.P. Emond. Thank you for taking the time to serve on my supervisory committee and for bringing new views to my research. I also appreciate the positive attitude s, thoughts, and input that you both have provided me here at UF and towards my future career. A huge debt of gratitude is given to Dr. Sa lly Johnson for exposing me to muscle biology and allowing me to come into her laboratory and turn it upside down. I in itially thought that I would never learn the technique s that I know now, but you taught me that I am capable of learning and accomplishing anything I want. I appreciate the amount of time and money you spent on this project, and know that I could not have done th is without you. Thank you for pushing me to publish, teaching me how to handle the criticisms (even when they were not right), and always being so encouraging. I know you will miss all my great sentences, but if you ever feel the need to read them, just give me a call.

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5 I would next like to thank all my friends and cowo rkers here at the Univer sity of Florida. I owe a huge thank you to all the lab members in Dr Sally Johnsons labora tory: Sarah Reed, Ju Li, and Dane Winner. Thank you for answering a ll my questions and teaching me the techniques necessary to complete my work. A world of thanks goes to Sarah Reed, because without you I would still be trying to figure out calculations, and your friendship has been very valuable to me. Thank you for being my best friend in the la b and always listening to me when I was complaining about something. I wish nothing but the best for you and your husband. Thank you to the Meats Laboratory Crew: Larry Eubanks, Tommy Estevez, Byron Davis, Ryan Dijkhuis, Frank Robbins, and all the student workers. Larry, without you I could have never had roasts instead of steaks (and the associat ed headaches that came with those roasts) for my last study. Seriously, thank you for all your he lp, encouragement, and for bei ng my Gainesville dad. I also learned a great deal of what I know now from you. Ryan, thank you for all your help and the expertise you provided when we were conducting my research. It was a real pleasure working with you and please know that my last study woul d not have gone as smoothly without you. We had some great times in the office, and on the road. I would like to also extend many thanks to past graduates of the Un iversity of Florida Department of Animal Sciences, including Dr. Nathan Krueger, Dr. Jeff Rhone, and Dr. Alex Stelzleni. Without your advice and encouragement, I could have never made it through school. Jeff, thanks for watching all those sporting ev ents with me and always giving me words of encouragement. Alex, thank you for all of your advice and help with my study. I really do appreciate all the faith you have in me as a fellow colleague. Thank you all for being my friends.

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6 Finally, I would like to thank both my Texas a nd Florida families. Because I come from a big Mexican family I will not name you all. Thank you for the encouragement, Joe, Becca, and Dad. Cheeto and Livie, thank you for reminding me that Im not ready for kids. Mom, I love you and please know that this accomplishment is as much yours as it is mine. Wright family, thank you for all the love and support, and for ma king me feel a part of your family. Mrs. Wright, please know that everything I accomplish in life, I owe to you. Bunny and Mary, thank you for being my Florida family. I know at times I drive you two crazy, but please know that I do love you both for allowing me into your family. Last, but certainly not least, all the love in my heart goes to Sara. I really wish I could express how I feel about you, but there is not enough room for it here. I want to thank you for not onl y helping me complete al l my studies and aiding me in writing my manuscripts, but for providing the most loving environment anyone could ever ask for. I was pretty lonely in Florida before I met you, but all that went away when I met you. You set up a wonderful home that I always knew I could go home to at the end of the day, and that made this experience a thousand times more enjoyable. I look forward to the life you, me, Bexar, and Astro have ahead of us, sandy bed and all. I know that you are going to be a wonderful vet and a great mother. To end, I just want to let everyone know that I am not exactly sure where my career will take me or how successful I will be. But all I really need in life to make me happy is to know that each and every one of you all is proud of me.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................12 ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 INTRODUCTION .................................................................................................................. 15 2 LITERATURE REVIEW .......................................................................................................19 Beta-Adrenergic Receptors ..................................................................................................... 19 Effect of Beta-Adrenergic Agonists on Muscle Fiber Isoform s ............................................. 23 Effect of Beta-Adrenergic Agonists on Muscle Fiber Morphom etrics .................................. 26 Beta-Adrenergic Agonists Mechanism of Musc le Growth and the Effect on Tenderness ..... 30 Effect of Beta-Adrenergic Agonists on Swine Growth Performance and Carcass Characteristics ............................................................................................................... ......38 Effect of Beta-Adrenergic Agonists on Cattle Gro wth Performance and Carcass Characteristics ............................................................................................................... ......50 Effect of -Adrenergic Agonists on Fr esh Meat Shelf-Life ................................................... 58 3 EFFECT OF RACTOPAMINE-HCl AND TRENBOLONE ACETATE ON LONGISSI MUS MUSCLE FIBER AREA DIAMETER, AND SATELLITE CELL NUMBERS IN CULL BEEF COWS ..................................................................................... 62 Introduction .................................................................................................................. ...........62 Materials and Methods ...........................................................................................................63 Animals and Diets ........................................................................................................... 63 Harvesting and Sample Collection .................................................................................. 64 Immunohistochemistry .................................................................................................... 65 Statistics .................................................................................................................... .......66 Results .....................................................................................................................................67 Discussion .................................................................................................................... ...........68 Conclusion .................................................................................................................... ..........73 4 DIFFERENTIAL RESPONSE OF CULL COW MUSCLES TO THE HYPERTROPHIC ACTIONS OF RACTOPAMINE-HCl .................................................... 80 Introduction .................................................................................................................. ...........80 Materials and Methods ...........................................................................................................81

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8 Animals and Diets ........................................................................................................... 81 Harvesting and Sample Collection .................................................................................. 81 Immunohistochemistry .................................................................................................... 82 RNA Extraction and Real-Time PCR Analysis ...............................................................83 Statistics .................................................................................................................... .......84 Results .....................................................................................................................................84 Discussion .................................................................................................................... ...........85 Conclusion .................................................................................................................... ..........89 5 EFFECT OF RACTOPAMINE-HCl ON THE F IBER TYPE DISTRIBUTION AND SHELF-LIFE OF SIX MUSCLES OF STEERS .................................................................... 95 Introduction .................................................................................................................. ...........95 Materials and Methods ...........................................................................................................96 Animals and Pre-Harvest Diets ....................................................................................... 96 Harvesting and Sample Collection .................................................................................. 96 Immunohistochemistry .................................................................................................... 97 Steak Cutting, Packaging, and Display ...........................................................................98 Nitric Oxide Metmyoglobin Reducing Analysis .............................................................98 Subjective and Objective Color Analysis ........................................................................ 99 Statistics .................................................................................................................... .....100 Results ...................................................................................................................................100 Discussion .................................................................................................................... .........101 Conclusion .................................................................................................................... ........106 6 EFFECT OF RACTOPAMINE-HCl ON LIVE AND CARCASS CHARACT ERISTICS WHEN FED TO STEE RS DURING THE FINAL 28 DAYS OF FEEDING ....................................................................................................................... ......112 Introduction .................................................................................................................. .........112 Materials and Methods .........................................................................................................113 Animals and Pre-Harvest Diets ..................................................................................... 113 Harvesting and Carcass Data Collection ....................................................................... 114 Whole Muscle Extraction and Measurement ................................................................ 114 Warner-Bratzler Shear Force Analysis .......................................................................... 115 Statistics .................................................................................................................... .....116 Results ...................................................................................................................................116 Discussion .................................................................................................................... .........118 Conclusion .................................................................................................................... ........123 7 OVERALL CONCLUSIONS AND I MPLICATIONS ........................................................ 128 APPENDIX A REPRESENTATIVE PHOTOMICROGRAPHS OF INFRASPINATUS MUSCLE IMMUNOSTAINED FOR DE TEC TION OF SATELLITE CELLS .................................. 133

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9 B REPRESENTATIVE PHOTOMICROGRAPH OF LONGISSIMUS DORSI MUSCLE IMMUNOSTAINED FOR DE TECTION OF FIBER ASSOCIATED NUCLEI ................ 134 LIST OF REFERENCES .............................................................................................................135 BIOGRAPHICAL SKETCH .......................................................................................................150

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10 LIST OF TABLES Table page 3-1 Longissimus muscle type I and type II fiber percentage and least square means of fiber cross -sectional area and diameter from cull cows fed four different feeding regimens ...................................................................................................................... .......74 3-2 Least squares means of LM fiber associat ed nuclei per fiber, a nd sate llite cells per hundred fibers of cull cows fed f our different feeding regimens ....................................... 75 4-1 Composition of basal diet ................................................................................................. .90 4-2 Sequence of bovine-specific PCR primers used for determ ination of the expression of mRNA for 1and 2-adrenergic receptors and myosin heavy chain isoforms ............. 91 4-3 Least squares means of muscle fiber m yosin heavy chain isofor m distribution, crosssectional area and diameter from four mu scles of cull-cows fe d three levels of ractopamine-HCl ............................................................................................................... .92 4-4 Myonuclei per fiber cross-section from cull-cows fed four different levels of ractopam ine-HCl ............................................................................................................... .93 4-5 Real time PCR Ct values for 2-adrenergic receptor, myosin heavy chain isoform expression from the Longissimus dorsi and Semimembranosus of cull-cows fed four levels of ractopamine-HCl .................................................................................................94 5-1 Least squares means of muscle fiber m yosin heavy chain isofor m distribution, crosssectional area and diameter from six muscles from steers fed with and without ractopamine-HCl .............................................................................................................. 108 5-2 Percent nitric oxide metmyoglobin reduced and ratios of oxyand m etmyoglobin accumulation from steaks originating from six muscles of steers fed with and without ractopamine-HCl displayed under simulated retail display conditions for 5 days .......... 109 5-3 Least squares means of HunterLab MiniScan X E L*, a*, and b* values from steaks originating from six muscles of steers fed with and without ractopamine-HCl displayed under simulated retail display conditions for 5 days ....................................... 110 5-4 Least squares means of visual panel sc ores for steaks from six muscles displayed under simulated retail display conditions for 5 days from cattle fed with and without ractopamine-HCl .............................................................................................................. 111 6-1 Composition of basal diet1 and carrier for top dress ........................................................ 124 6-2 Least squares means of live performance ch aracteristics of steers supplemented with and without ractopam ine-HCl1 ........................................................................................125

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11 6-3 Least squares means of offal weight s and carcass characteristics of steers supplem ented with and without ractopamine-HCl .......................................................... 126 6-4 Least squares means of muscle weig hts, dim ensions, color measurements, and Warner-Bratzler shear force values from six muscles of the round from cattle fed with and without ractopamine-HCl ..................................................................................127

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12 LIST OF FIGURES Figure page 3-1 Representative photomicrogr aphs of LM fibers immunostain ed for type I and type II fibers from (CON) control diet fed co ws, (RAC) control di et + RAC fed cows, (TBA) control diet + implant fed cows and (RAC/TBA) control diet + RAC + implant fed cows. Scale bar equals 100 m. ...................................................................... 76 3-2 Number of Pax7 positive nuclei counted per field during a 72 h period postm ortem. Samples were taken at 0, 24, 48, and 72 hour s postmortem and subjected to the Pax7 staining protocol used in the pres ent study. Area of field equals 41.5 mm2. ..................... 77 3-3 Histograms of LM fiber cross-sectional areas of all type I fibers sam pled from (Control) control diet fed cows, (RAC) control diet + RAC fed cows, (TBA) control diet + implant fed cows, and (RAC/TBA) control diet + RAC + implant fed cows. ........ 78 3-4 Histograms of LM fiber cross-sectional ar eas of all type IIA fibers sam pled from (Control) control diet fed cows, (RAC) control diet + RAC fed cows, (TBA) control diet + implant fed cows, and (RAC/TBA) control diet + RAC + implant fed cows. ........ 79 A-1 Photomicrographs of immunohistochemical stains used to identify satellite cells. .........133 B-1 Photomicrograph of Longissimus dorsi m uscle immunostained for fiber associated nuclei detection. -Dsytrophin identified as green st ain and propidium iodide stain (red stain) identified nuclei. .............................................................................................134

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF RACTOPAMINE-HYDROCHLORIDE ON MUSCLE FIBER MORPHOMETRICS, SATELLITE CELL POPULA TION, AND SHELF-LIFE PROPERTIES OF BEEF CATTLE By John Michael Gonzalez December 2008 Chair: D. Dwain Johnson Major: Animal Sciences The ability of ractopamine-hydrochloride ( RAC ) to affect muscle fiber morphometrics, satellite cell population, and shelflife properties of various musc les of the beef carcass was explored over three studies. In the first two studies, RAC was supplemented to cull cows during the final 28-35 days of feeding and differentially a ffected muscles of these animals. In the first study, RAC increased ( P < 0.05) the crosssectional area ( CSA ) and diameter of type I muscle fibers in the Longissimus dorsi without affecting type IIA fibers In the second study, RAC supplementation increased the CS A of type I fibers of the Infraspinatus ( P < 0.05) and Vastus lateralis ( P < 0.15), but did not affect ( P > 0.05) the Longissimus dorsi or Semimembranosus Of these four muscles, RAC increased ( P < 0.05) the CSA of type IIA fibers in only the Infraspinatus and Semimembranosus In both studies, RAC supplementation altered ( P < 0.05) the muscle fiber isoform distri bution, but did not increase ( P > 0.05) the number of satellite cells or fiber associated nuclei counted. This indicates that all growth observed in type I or type IIA fibers happened independent of satellite cel l incorporation into the muscle fiber. In the third study, RAC supplemente d to steers did not affect (P > 0.05) the CSA of either fiber isoforms nor the muscle weights or dime nsions of muscles of the round and loin. The

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14 muscle fiber isoform distribution of all muscles, except the Semimembranosus was changed ( P < 0.05). Ractopamine supplementation did not affect (P > 0.05) objective meas ures of color or nitric oxide metmyoglobin reducing ability dur ing a five day simula ted retail display. Ractopamine supplementation increased ( P < 0.05) the amount of surf ace discoloration on steaks from the Rectus femoris, Semimembranosus and Vastus lateralis during the final days of display when evaluated by trained panelists. Results from all three studi es indicate that RAC supplementation to both cull cows and steers has a limited ability to increase muscle fiber CSA, but can affect surface discolor ation during retail display.

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15 CHAPTER 1 INTRODUCTION For over 25 years, researchers have exam ined the use of -adrenergic agonists ( BAA) to alter growth rate and body com position of livestock. Ricks et al. (1984a) and Beermann et al. (1985) were two of the first researchers to report the advantages associated with BAA supplementation to livestock. Structurally, BAAs are similar to norepinep hrine and epinephrine, which are natural catecholamines (Mills, 2002). The physiological activity of a BAA depends on its absorption, metabolism, and elimination rates. In addition, the distribution of the BAA to target tissues also affects a BAAs physiological activity (Smith, 1998). -adrenergic agonists elicit their actions by binding to one or more receptors identified as 1, 2, and 3 receptors. These receptors are located on the membranes of most mammalian cells. Their distribution varies between tissues and species, and can also vary within a given tissue between species (Mersmann, 1998). An agonist binds to a re ceptor at three points on the molecule: a -hydroxyl group, an aliphatic nitrogen, and an aromatic ri ng (Smith, 1998). Omissions or substitutions at these points results in agonist differences in receptor bindin g or activity (Ruffolo, 1991). Because these agonists alter growth by redir ecting nutrients from adipose tissue deposition to skeletal muscle accretion, BAAs are known as repartitioning agents (R icks et al., 1984a). Beermann et al. (1985) suggested using BAAs as a means to redu ce the cost of producing meat animals by reducing feed grain input costs. Smith (1998) identified up to 15 BAAs used in livestock production. Of those 15, clenbuterol, salbutamol, cimaterol, ractopamine, and L644,969 are commonly used in research st udies. Ractopamine-hydrochloride ( RAC ) and zilpaterolhydrochloride are the only two BAAs approved for use in the United States. Ractopamine was developed by Elanco and approved for use in pigs by the Federal Drug Administration in 1999. This product is currently sold unde r the trade name Paylean. Four years later, in 2003, Elanco

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16 developed and received approval for the use of RA C in beef cattle. This product is sold under the trade name Optaflexx and is approved for use in beef cattle during th e final 28-42 days of feeding prior to harvest. Historically, cows are culled in the fall for vari ous reasons that include infertility, old age, and poor reproductive performance. Income from culling practices can account for 15-20 percent of an operations animal proceeds (Feuz, 1999). Little consideration is given to feeding cull cows as a means to improve carcass quality. However, when market conditions are favorable, feeding cull cows is an attractive option. Matulis et al. (1987) reported that as time on feed is increased, cull cow carcass weight, fat thickness, marbling and quality grade are improved. Carcasses from fed cull cows demonstrate brighter, more youthful lean color and larger ri beye areas (Miller et al., 1987). The increased nutrients and energy intake causes a mark ed increase in fat deposition with little improvement in muscle accretion dur ing the final days of finishing (Brown and Johnson, 1991; Boleman et al., 1996; Cranwell et al., 1996a). Therefore, RAC supplementation may remedy this by directing nutrients away fr om fat deposition and toward muscle accretion. In swine, RAC supplementation profoundly incr eases lean deposition, while reducing the fat of the carcass (Yen et al., 1990; Dunshea et al., 1993). In young beef cattle, RAC modestly increases muscling as indicated by increases in ribeye area, but rarely affects fat deposition (Walker et al., 2006; Winterholler et al., 2007). Postnatal muscle growth is accomplished by muscle fiber hypertrophy. Hypertrophy involves an increase in fiber cross-sectional area via protein accretion. In order to maintain the incr eased protein synthesis demands of the growing fiber, nuclei are added to main tain an appropriate myonuclear domain (Kim and Sainz, 1992). Satellite cells proliferate, differentiate, and fuse with the muscle fibers to provide additional nuclei and DNA for the synthesis of protein (Aberle et al., 2001). In pigs, RAC causes muscle

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17 growth by stimulating increases in type II fiber cross-sec tional area without affecting type I fiber size (Aalhus et al., 1992). Beermann et al. (1987) and Kim et al. (1987) reported that DNA concentration per gram of protein was lower in beta-agonist supplemented lambs. Both groups concluded that muscle growth was due to a reduc tion in protein degradati on and not satellite cell activity. However, a study has ye t to measure the effect of RAC on actual satellite cell and fiber associated nuclei numbers. Therefore, the cellu lar mechanisms responsible for the increases in the cross-sectional area of the ribeye and other muscles of the beef carcass due to RAC supplementation are not well documented and require further investigation. A common effect of BAA supplemen tation to livestock is muscle fiber isoform shifts. In pigs, RAC supplementation shifts muscle fiber myosin heavy chain isoforms toward more glycolytic isoforms (Aalhus et al., 1992; Depreux et al., 2002). Vestergarrd et al. (1994) reported that cimaterol increases the percentage of glycolytic fibers at the expense of oxidative fibers. With the shift toward glycolytic fibe rs, NADH content may be reduced and affect shelflife. NADH is an integral part of the metmyogl obin reducing system that chemically reduces metmyoglobin to deoxymyoglobin (Mancini and Hunt, 2005). However, the effect of RAC supplementation on the myosin heavy chain isoform shift in beef ca ttle and the effects of this shift on shelf-life remain unexplored. While numerous studies examine the effects of RAC on gross carcass characteristics of beef cattle, few have focused on the cellular even ts behind these effects. The objective of this dissertation is to: (1) examine the effect of RAC supplementation on muscle fiber morphometrics of various muscles throughout th e beef carcass of young and old animals; (2) examine the effect of RAC supplementation on satelli te cell and fiber associated nuc lei populations; (3) examine the effect of RAC supplementation on muscle fiber myosin heavy chain isoform distribution of

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18 various muscles throughout the beef carcass of both young and old animals; (4) determine if RAC induced shifts in myosin heavy chain isofor m distribution affect th e shelf-life of steaks during retail display.

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19 CHAPTER 2 LITERATURE REVIEW Beta-Adrenergic Receptors Al most every cell type in the mammalian body possesses -adrenergic receptors ( BAR). In its plasma membrane BARs can vary in num ber and type. Receptors contain more than 400 amino acids in a continuous chain that form seven hydrophobic transmembrane domains, anchoring the receptor to the plasma membrane. The ligand binding site of the receptor is located within the seven transmembrane domain and this domain also interacts with the GS protein. When an agonist binds to the seventransmembrane G-coupled receptor, it activates intracellular adenylyl cyclase activity. The ac tivation of adenylyl cyclase increases the intracellular levels of cyclic AMP. Cyclic AMP binds to the regulatory subunit of protein kinase A, releasing the catalytic subun it, which can phosphorylate numerous intracellular proteins. The phosphorylation of these proteins can either increase the transcripti on of a gene, as with skeletal muscle accretion, or limit enzymes, such as the enzymes responsible for long-chain fatty acid synthesis (Liggett and Raymond, 1993; Me rsmann, 1998; Norman and Litwack, 1997). Scientists have identified the three BAR subtypes in mammals as 1, 2, and 3. Each BAR subtype exhibits a distinct RNA transcript size, protein size, and amino acid sequence. The three BAR subtypes maintain approximately 50% hom ology in amino acid sequ ence within a single species. Across species, indivi dual BAR subtypes display 75% to 90% homology. Differences in sequence allow for variation in ligand affinity and receptor activation. Therefore, one ligand may have different affinities and a receptor act ivation capability for different BAR subtypes within a species. Similarly, ligand affinity and receptor activation capabilities vary across species for a specific BAR subtype. This leads to extensive diversity in the responses generated by ligands. Generally, BAR subtypes exhibit ligand selectivity for norepinephrine as 1> 2> 3.

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20 For epinephrine, 1-receptor and 2-receptor bind similarly. The 2-receptor has a greater affinity for epinephrine than norepine pherine (Mersmann, 1998; Mills, 2002). Because BAR subtypes generate diverse respons es to different ligands, determining the distribution of these subtypes in mammalian tissu es holds importance. Different species can have different BAR subtype concentrations distributed throughout th eir tissues. In swine, the 1receptor represents the most abundant BAR. Th is subtype comprises 80 % and 60% of the BAR in adipose tissue and skeletal muscle, respectively. However, Sillence and Matthews (1994) suggest that the 2-receptor comprises the majority of the BARs in beef cattle skeletal muscle and adipocytes. Van Liefde et al. (1994) sugge st that cattle adipocyte BARs are composed of approximately 75% 2-receptors and 25% 1-receptors. Sissom et al. (2007) found that the expression of the 2-receptor in skeletal muscle was 10-fold greater than the other two subtypes, indicating that the 2-receptor is the most abundant receptor in bovine skeletal muscle. Mammalian tissue minimally expresses the 3-receptor. This receptor is found mostly in adipose tissue, and only comprises approximate ly 10% of the BAR (Mills, 2002). To avoid indefinite activation of BAR by ligands, the agonist can be removed or degraded, or the receptor may be inactivated by several m echanisms. These processes serve as safety mechanisms to prevent overstimulation of r eceptors during times of chronic exposure to agonists. The extent of desensitization of a receptor depends on the de gree and duration of the receptor/agonist interaction (Johnson, 2006). Thes e mechanisms of receptor inactivation include phosphorylation of the BAR by a kinase or comp lete removal of the BAR from the plasma membrane (Mersmann, 1998). Phosphorylation of the receptor by closely related G proteincoupled receptor kinases, such as PKA, serves as the principle mechanis m of short-term agonistpromoted desensitization. Due to phosphorylation, -arrestin binds and pa rtially uncouples the

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21 agonist-occupied form of the receptor from the Gs protein. Therefore, this limits receptor function by preventing continua tion of the signal by the Gs protein. In addition, -arrestins may bring other proteins into the re ceptor microenvironment. These pr oteins then act to metabolize cAMP and thus block the effects of receptor activa tion. Sequestration may also play a major role in short-term receptor regulation. This m echanism commonly occurs during periods of prolonged agonist exposure. Duri ng sequestration, receptors become internalized, which results in a loss of receptors from the cell surface. Sequestration takes longe r to reverse because dephosphorylation of the receptor must occur wh ile the receptor is in ternalized (Johnson, 2006). The body can also regulate BARs by the comple te removal of receptors from the cellular membrane. This process is termed downregulati on and can differ markedly from cell to cell and tissue to tissue. Downregulation activates the process of receptor degradation by ubiquitination of the BAR through an E3 ligase. In order to restore original leve ls of membrane BAR, cells and tissues require transcription and posttranslational conversion of BAR mRNA to protein. In addition to this process, downr egulation also can occur by modul ation of BAR gene expression through the cAMP pathway (Johnson, 2006). Several current studies report a variety of effects of -adrenergic agonists (BAA) on mRNA expression of the three BAR subtypes. The earliest studies reporting BAA induced downregulation of BARs involve rats (Sillence et al., 1991). Kim et al. (1992) supplemented 10 ppm of cimaterol for up to 28 days and found that BAR binding sites in the plantaris mu scle were reduced by 26.8, 42.2, 37.7, and 37.8% at 3, 7, 14, and 28 days, respectively. Re searchers concluded that the decrease in growth promoting effects seen in the study was due to alterations in the number of BAR binding sites. In a recent study involving Holstein steers, Walker et al. (2007) re ported that supple menting 200 mg of ractopamine-HCl ( RAC ) per day during the final 28 days of a 56 day feeding trial decreased

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22 mRNA expression of both 1and 2receptors in the Longissimus dorsi ( LD). Sissom et al. (2007) reported that expression of the 3-receptor was not affected by RAC alone, but decreased when 200 mg/d of RAC was combined with a Revalor-200 implant. G unawan et al. (2007) supplemented RAC to swine and reported that 2-receptor expression decreased significantly by week 2 of supplementation. Since RAC supplementation tends to shift muscle fiber type from a slow to fast isoform, the resear chers suggested that losses in -adrenergic receptor expression were due to the loss of slow fibers which have greater expression of -adrenergic receptors. In contrast to these findings, other results s uggest that RAC has the ability to increase BAR expression or have no effect on BA R expression. In beef cattle, Winterholler et al. (2007) and Sissom et al. (2007) reported that RAC suppl ementation did not affect the expression of 1and 3-receptors. The lack of effect on -adrenergic receptor expression of both receptor subtypes also was reported in pigs (Gunawan et al., 2007; Mills, 2002; Spurlock et al., 1994). In contrast, Winterholler et al. (2007) fed 200 mg/steer of RAC daily for the final 28 days of feeding and reported that the expression of 2receptors tended to increase. The authors suggested the increase in expression was due to weak binding of RAC to 2receptors, which triggered the synthesis of new 2receptor protein. The authors also suggested that th e synthesis of new protein could be due to muscle attempting to ov ercome the down regulation that occurs due to chronic administration of RAC. In agreement w ith this study, Sissom et al. (2007) examined the effect of 200 mghd-1d-1of RAC on heifers treated with various implant strategies, with similar results. Researchers found that RAC supplementation combined with a Revalor-IH/Finaplix-H implant strategy increas ed mRNA levels of 2-receptors, and increased mRNA levels of 3receptor. The authors hypothesized that the in creases in expression of BAR two and three may be due to manipulation of the muscle fiber isoforms elicited by RAC supplementation.

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23 Effect of Beta-Adrenergic Agon ists on Muscle Fiber Isoforms Adult skeletal muscle traditionally consis ts of four myosin heavy chain isoforms identified as types I, IIA, IIX, and IIB. Although these isoform designations are based on inherent ATPase activity, muscle fibers may al so be called by other nomenclature based on the assay used to identify them. Each isoform exhi bits subtle differences in amino acid sequences, which creates differences in prot ein functionality. Type I and IIA fibers are commonly referred to as red muscle fibers, while type IIX and IIB fibers are referred to as white fibers. The differences between red and white fibers lie in their content of the oxygen binding protein myoglobin. Red fibers contain more myoglobin due to their use of oxidative metabolism. White fibers operate on glycolytic metabolism and contai n less myoglobin than red fibers. Because red and white fibers differ in the type of metabolism they utilize, they contain different organelles and energy sources in their cellu lar matrix. Since red fibers operate on oxidative metabolism, they have a greater number of larger mitochondria, a greater lip id content, and lower glycogen content, compared to white fibers. The speed of contraction once stimulated also differs between fiber types. Type I fibers contra ct slower than type II fibers, with type IIB fibers contracting the fastest. Each fiber type also fa tigues at different rates, with type I being the least susceptible to fatigue, followed by IIA, and then IIX/IIB. In mo st mammalian species, type I and II fibers are randomly intermingled within muscle bundles, bu t the primary fiber type that comprises the muscle determines its functiona l ability (Aberle et al., 2001). At birth, bovine muscle contains approximately 40-50% type IIB fibe rs, 35-45% type IIA fibers, and 10-20% type I fibers. The percenta ge of each fiber type isoform varies slightly between breeds and can be affected by age. Afte r birth, the main fiber type shifts occur during the first two months of lif e. From birth until two months of ag e, type IIB fibers increase at the expense of type IIA fibers. After two months of age, the rate at which type IIA fibers convert to

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24 type IIB fibers decreases drama tically (Wegner et al., 2000). Juri e et al. (1999) report that the conversion of fast oxidative fibers to fast glycolytic fibers can o ccur at up to 12 months of age, with the fiber type frequency remaining consta nt after 12 months. Wegner et al. (2000) found that the percentage of the type I fiber isoform remains constant or changes slightly from birth until two years of age. In rats, muscles contract slowly at birth and increase in contraction speed during the first few weeks of life, indicating a slow to fast fi ber type transition (Kelly and Rubinstein, 1980). The postnatal shifts of muscle fiber isoform re ported above can be attr ibuted to the altered functional demands of the muscles observed. The mu scle fibers of beef cattle fed in confinement had a lower oxidative metabolic potential when co mpared to muscle fibers from beef animals raised in an extensive production system (Vestergaard et al., 2000). In addition to postnatal activity, other factors such as nutrition can play a role in fiber type shifting. During early postnatal development, under-nutri tion delays the shift of type I to type II fibers (Ward and Stickland, 1993; White et al., 2000 ). When comparing feedlot st eers to non-supplemented range steers, Beerwinkle et al. (1979) found that range steer muscle c ontained a larger proportion of type I fibers. These results were most likely due to the differing nutritional status of the two groups of cattle in the study, w ith the range cattle having a lower plane of nutrition. Nutritional supplementation in th e diet can also affect fibe r type isoform shifts. As mentioned earlier, numerous studies report that supplementation with RAC causes fiber type shifts in skeletal muscle. This is importa nt from a muscle growth and BAA efficiency standpoint. According to Aal hus et al. (1992), the response to BAA supplementation is dependant on the initial fiber type Therefore, different muscles, breeds, and species would have different responses based on the initial fibe r type composition. Numerous studies have

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25 demonstrated different combinations of fiber type shifts. With BAA supplementation, various rat muscles ( Soleus, Diaphragm and Extensor digitorum longus ) showed increases in the percentage of type IIA (Bricout et al., 2004; Rajab et al., 2000), type II X (Bricout et al., 2004; Criswell et al., 1996; Maltin et al., 1986; Polla et al., 2001), or type IIB (Maltin et al., 1986; Polla et al., 2001) fibers at the e xpense of type I fibers. Ractopamine-HCl supplementation to pigs increa sed the percentage of type IIB fibers at the expense of type IIA fibers in a variety of muscles. However, the percentage of type I fibers remained the same when evaluated by histologi cal techniques (Aalhus et al., 1992) and ELISA (Depreux et al., 2002). To furt her investigate the effect of BAA on muscle fiber isoform distribution, Gunawan et al. (2007) examined the effect of RAC supplementation on mRNA expression of the four myosin heavy chain isoforms in pigs. In the study, pigs were fed RAC for eight different time periods and harvested. Following harvesting, mRNA expression was quantified using real time PCR analysis. In ag reement with other pig data, this study indicated that type I fiber expression was unaffected by RAC administration. Type IIA expression decreased by 96 hours after administration and conti nued to fall for one week. By the end of the 4 week trial, type IIA mRNA expression returned to pre-supplementation levels. It appears that type IIB expression increased thr oughout the trial at the expense of type IIX expression, which decreased by week 2 and continued to fall until th e end of the trial. Therefore, these findings support other data demonstrating that RAC causes in creases in type IIB fibers at the expense of type IIA and IIX fibers. Studies analyzing the effect of RAC and other BAA on rumina nt muscle fiber distribution are not as extensive as in othe r species. Kim et al. (1987) reported that lambs fed 10 ppm of cimaterol for 8 weeks showed no difference in the proportion of type I and type II fibers in the

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26 Semitendinosus or the Longissimus dorsi In another study, Beermann et al. (1987) fed 10 ppm of cimaterol to lambs for seven and 12 weeks, finding mixed results based on muscle. In the Longissimus dorsi and Semimembranosus supplementation did not affect the percentage of type I fibers. However, in the Semitendinosus the percentage of type I fibers was slightly reduced. Examining beef cattle, Vestergaar d et al. (1994), uncovered results si milar to swine data. In the study, four pairs of monozygotic tw ins were fed either 0 or 0.06 mg of cimaterol per kilogram of live weight per day for 90 days. Da ta indicated that the percentage of IIB fibers increased due to a decrease in the percentage of type IIA fibers with supplementation. The percentage of type IIB fibers also increased due to a decrease in type I fibers. This contrasts swine data, in which BAA supplementation does not affect the percentage of type I fibers. Effect of Beta-Adrenergic Agonists on Muscle Fiber Morphometrics Muscle is comprised primarily of fibers whic h are gathered into groups of 20 to 40 muscle fibers known as primary muscle bundles. Vari able numbers of prim ary muscle bundles are grouped to form secondary muscle bundles. Variable numbers of secondary muscle fiber bundles are then grouped, yielding the whole muscle. Because the building blocks of muscle consist of muscle fibers, muscle mass is mainly determined by the number and size of fibers in any given muscle. In muscle, growth is achieved through both hyperplasia and hypertrophy. Muscle growth accomplished through hyperplasia consists of an increase in muscle fiber number. Muscle growth through hyperplasia occu rs primarily during postnatal development and to some extent for the first few months followi ng birth. Hypertrophic growth is accomplished by enlargement of individual muscle fibers. Commonly, hypertrophy of muscle fibers occurs through increases in the cross-sectional area ( CSA ) or diameter of the fibers of that muscle. Therefore, since the number of musc le fibers present in a muscle is set at birth, postnatal muscle

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27 growth is accomplished through muscle fiber hypertrophy (Aberle et al ., 2001; te Pas et al., 2004). The nutritional status of an an imal can affect myosin heavy chain distribution, as well as the CSA and diameter of muscle fibers. The magn itude of the nutritional e ffect on fiber size is dependent on the developmental st age of the animal and the muscle type observed (White et al., 2000). Studies have shown that the type I fibers in older, under-nourished animals are minimally reduced in CSA while the type II fibers are greatly decreased in CSA (Everitt et al., 2002; Goldspink and Ward, 1979; Stickland et al., 197 5). When comparing feedlot steers to nonsupplemented range steers, Beerwinkle et al. ( 1979) found that the range steer muscle had smaller fiber diameters and a larger proportion of type I fibers. These results were most likely due to the difference in nutritional status between the two groups of cattl e, with the range cattle having a lower plane of nutrition. More importantly, the decrease in fiber CSA and diameter resulting from poor nutrition can be reversed in mature animals (Goldspink and Ward, 1979). While improved nutritional status increases muscle fiber CSA and diameter, numerous studies report that supplementa tion with BAAs also increase these parameters. Many studies involving the effects of BAA supplementation on muscle fiber hypertrophy focus on the supplementation of clenbuterol to rats. Ho wever, the fiber type isoform affected by supplementation is not consistent across studies. In rats, RAC supplementation generally causes increases in type II fiber CSA. Criswell et al. (1996) found that clenbuterol supplementation does not affect the CSA of type I fibers, but increases the CSA of t ype IIA fibers. In agreement, Bricout et al. (2004) reported th at type IIA fiber CSA doubled in non-injured muscle, while type I fibers were not affected. However in the sa me study, injured muscle treated with clenbuterol showed an increase in the CSA of both type I and IIA fibers. Th is may indicate that the injury

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28 status of muscle affects its response to BAA. Herrera et al. (2001) also reported that clenbuterol increased the CSA of type I and type II fibe rs by 30 and 13 percent, respectively, in the unweighted hindlimbs of 30 month old rats. Th e 30 month old rats and 12 month old rats responded to clenbuterol treatment differently. In 12 month old rats, type I fibers increased in CSA due to clenbuterol supplementation while ty pe II fibers were unresponsive. This may indicate that an animals age affects the responsivene ss to BAA administration. Polla et al. (2001) also noted an age effect on BAA responsiveness. Twenty-one day old rats were supplemented with clenbuterol for 28 da ys. The CSA of type I, IIA, and IIB fibers from four distinct muscles were analyzed. In the D iaphragm and S uperficial tibialis anterior type IIB CSA increased. Type I and IIA fibers of the Soleus decreased in CSA. Researchers concluded that growth stimulation by clenbut erol administration is age dependent and only detectable in young adult rats. In addition, the researchers hypothesized that decreases in the CSA of both type I and IIA fibers was due to both hypotrophy and shift in fiber types between the two isoform types. Rat research also has demons trated that long term admi nistration of BAA limits its effectiveness in increasing fiber CSA. Using ma le rats, Maltin et al. (1986) found that the growth promoting effects of clenbuterol were se lective for muscle fibe r isoform and decreased over time. Rats were fed clenbuterol for 4 or 21 days and the CSA of the muscle fibers from the Soleus and Extensor digitorum were analyzed. The Extensor digitorum responded to supplementation in a time dependent manner. Afte r four days of treatment, the cross-sectional area of slow-oxidative and fastoxidative-glycolytic fibers increased significantly. However, after 21 days of supplementation, the CSA of neith er fiber isoform responded, indicating that the effects of supplementation last for a limited time period. In agreement, Zerman et al. (1988)

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29 found that chronic administration of clenbuterol increased the CSA of type I fibers of the S oleus for up to 2 weeks, but had no effect thereafter. While numerous rat studies examine the e ffects of BAA on muscle fiber CSA and diameter, few studies focus on othe r species. In an early study examining the effect of BAA on lambs, Beermann et al. (1987) reported that cimate rol increased type I an d type II muscle fiber CSA in the Semitendinosus by 30.4 and 29.3 percent, respectively. However, Kim et al. (1987) found that only type II fiber CSA of the Semitendinosus and Longissimus dorsi increased by 50 percent when using the same dosage level. The in ability of cimaterol to affect the type I fiber CSA in the study may have been due to a shorter supplementation period employed by the researchers. This may indicate that supplemen tation duration affects which fiber types respond to supplementation. In the study by Beermann et al. (1987), researchers noted that BAAs are variably effective across muscles. Type I and II fiber CSA of the Longissimus dorsi increased by 13 and 15 percent, respectively. However, grea ter increases were observed in the Semitendinosus Maltin et al. (1990) also noticed this trend when supplementing clenbute rol to Friesian bull calves. Mean general fiber area incr eased by 50 percent in the Semimembranosus while the mean fiber area increased by 13 percent in the Triceps Currently, research has not determined why muscles react differently to BAA supplementation. Some scientists hypothesize that the disparity between muscles is due to the fiber type composition and thus the receptor density and affinity differences between fiber types. According to Martin et al. ( 1989) type I fibers contain more receptors than type IIB fibers, but receptors on type IIB fibers have a higher affinity for agonists. Therefore, since the Semitendinosus has more type IIB fibers than the Longissimus

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30 dorsi (Kirchofer et al., 2002), it di splays a higher affinity for -agonists and exhibits an increased response. Two studies examining swine and cattle support this hypothesis. Swine were supplemented with RAC, and the red fiber CSA of the Semimembranosus and Psoas major were not significantly affected. However, the CSA of both intermediate and white fibers increased by 24.3 and 16.8 percent, respectively (Aalhus et al., 1992). In agreement, Maltin et al. (1990) found that clenbuterol fed cattle ex hibited increased CSA of fast tw itch glycolytic and fast twitch oxidative glycolytic fibers, with no effect on slow oxidative fiber CSA. Ve stergaard et al. (1994) supplemented cimaterol to bulls and uncovered opposi ng results. The CSA of type I fibers of the Longissimus dorsi were significantly increa sed, while type IIA fibers were not significantly affected, but were reduced in CSA. In the same study, Semitendinosus type I and type IIB fibers were found to increase in CSA. Due to the l ack of a large number of studies examining BAA supplementation to food producing animals, more rese arch is needed to evaluate the effect of BAA on muscle fiber morphometrics. Beta-Adrenergic Agonists Mechanism of Musc le Grow th and the Effect on Tenderness As mentioned earlier, muscle growth occu rs through both hypertrophy and hyperplasia. If one were to put both into an equation explai ning the mechanism of growth, it would read as: growth equals the number of muscle cells plus protein accretion minus protein degradation. Therefore, hypertrophy through myof ibrillar protein accretion re presents the balance between myofibrillar protein synthesis a nd myofibrillar protein degrada tion (Forsberg et al., 1989). Initially, scientists studying BAAs found that BAAs divert nutrients away from adipose tissue towards muscle accretion. However, they did not know which part of the muscle growth equation was responsible for the increased lean accretion. Ricks et al. (1984a) and Beermann et al. (1985) first proposed that BAA supplementati on in livestock caused muscle growth through

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31 decreasing protein catabolism and/or increasing protein synthesis. Swine studies contributed the majority of data supporting acceleration of protein synthesis as the mechanism of muscle growth (Dunshea et al., 1993; Dunshea et al., 1998; W illiams et al., 1994). Mersmann (1998) supports these findings, stating that di fferent BAAs increase blood flow to certain regions of the body, thus increasing muscle accretion by the enhanced delivery of substrates and energy needed for protein synthesis. However, other investigators examining rats and sheep found no effects on muscle protein synthesis in BAA treated subjects and concluded that muscle grow th was due to altered protein degradation (Bohorov et al., 1987; Reed s et al., 1986). Most of the early work that investigated the mechanism behind increased muscle hypertr ophy focused on protein degradation rates. Beermann et al. (1987) and Kim et al. (1987) re ported that DNA concentration per gram of protein was lower in clenbuterol supplemented lambs. Based on these results, both groups concluded that muscle growth was due to a reduction in protein de gradation and occurred independent of satellite cell activit y. Similar observations were reported for rats (Maltin et al., 1986), lambs (Bohorov et al., 1987), and poultry (Gwartney et al ., 1992) fed clenbuterol. Forsberg et al. (1989) conducted a premie r study examining the effects of BAAs on calcium-dependent proteases which regulate muscle protein degradation. In their study, the researchers supplemented rabbits with cimaterol fo r 35 days before harvest. Results indicated that cimaterol did not affect cathe psin B, cathepsin D, or neutral serine. However, cimaterol did reduce and m-calpain by 58 and 57%, respectively. In addition, cimaterol reduced calpastatin activity by 52%. Wang and Beermann (1988) similarly found up to a 70% reduction in -calpain activity in lambs supplemented cima terol. Therefore, the researchers concluded that hypertrophy

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32 induced by cimaterol supplementation may invol ve an alteration of the calcium-dependent proteolysis system. In disagreement with the previously mentioned data, other studies report increases in calpastatin activity due to BAA s upplementation. Higgins et al. (1988) reported that clenbuterol increased calpastatin activity in sheep. Two other studies involving the supplementation of L644,969 to lambs also found increases in calpastatin activity. The first study reported that at 0 days postmortem, calpastatin activity in the LD of treated lambs was 74% higher, while at four days postmortem, calpastatin activity was 430% higher than controls. Increased calpastatin activity resulted in lower extractable calpain I and the appearance of degraded myofibrillar proteins detected on SDS-PAGE gels for the trea ted animals (Kretchmar et al., 1990). In the second study, BAA supplementation increased cal pastatin activity by 62.8% and 227.2% on day 0 and 7 postmortem, respectively. In disagreement with Kretchmar et al. (1990), (Koohmaraie et al., 1991) reported BAA supplementation increased and m-calpain activity at 7 days postmortem. However, despite increased ca lpain activity, SDS-PAGE gel electrophoresis revealed that BAA administration slowed the appear ance of myofibrillar proteins associated with postmortem proteolysis. These findings led the researchers to conclude that reduced muscle protein degradation plays a role in muscle protein accretion due to BAA supplementation. Bardsley et al. (1992) conducte d a series of experiments i nvolving the supplementation of BAA to various species (cattle, poultry, rats, and lambs) and found that BAAs affected the calcium activated proteolytic system differently in each species. Trials indicated that cattle were more responsive to BAA treatment than the other three species. Cimaterol included at low levels in the diet of beef cattle for 9 or 16 weeks increased Longissimus dorsi calpastatin expression by 99 and 76%, respectively. The next most res ponsive species was sheep, which displayed a

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33 maximum of 39% increase in calpastatin activity Because all species do not respond similarly to cimaterol supplementation, Bardsley et al. (1992) suggested that one must consider the type of agonist, dosage, and duration of supplementatio n to achieve maximum effect on the calcium proteolytic system. Wheeler and Koohmaraie (1992) conducted a study supplementing L644,969 to cattle fed a high concentrate diet for 6 weeks prior to harves t. A 27% decrease in the fractional degradation rate of skeletal muscle myofibrillar protein was reporte d. At harvest, BAA administration had no effect on and m-calpain activity, but on days 0 and 7 postmortem calpastatin activity increased by 60 and 348% percent, respectively. The researchers concluded that BAA induced hypertrophy resulted from reduced proteolytic activ ity because of increased calpastatin activity. In agreement with this study, G eesink et al. (1993) and Garsse n et al. (1995) reported that clenbuterol administered to v eal calves significantly increased calpastat in activity by 37 and 67%, respectively. Geesink et al. (1993) al so reported clenbute rol tended to decrease -calpain by 34% and electrophoresis analysis indicated that clenbuterol administration reduced postmortem proteolysis by slowing the appearance of the 30 kDa m yofibrillar degradation band. Therefore, these findings indica te that BAAs stimulate muscle hypertrophy by altering calcium dependent protease activity, more specifically, calpastatin. Because BAAs stimulate muscle hypertrophy by decreasing muscle protein degradation through increased calpastatin activit y, a toughening effect on meat te nderness occurs. In skeletal muscle, calpastatin acts as the endogenous inhibitor of and m-calpain. In living muscle, and m-calpain degrade muscle protein to aid musc le protein turnover. These proteases require calcium for activation, and once activated postmortem, these proteases are responsible for tenderization (Aberle et al., 2001). In discussing the importance of the calpain proteolytic

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34 system, Koohmaraie and Geesink (2006) identifie d the BAA effect on calpastatin as the reason that meat from BAA treated animals becomes tougher. The first studies to report decreases in te nderness due to BAA supplementation involved lambs. In these studies, lambs were supplemented 4 ppm of the BAA L644,969 for 6 weeks prior to harvest. In the first study, treated lambs had 111 and 108% higher Warner-Bratzler shear force ( WBS) values on day 3 and 6 postmortem, respectivel y, when compared to controls (Kretchmar et al., 1990). The second study reported that BAA administration increased WBS values for treated lambs by approximately 10, 48, and 70% on days 1, 7, and 14, respectively. Over the entire 14-day storage period, WBS values fell by 52.6% for control animals and by 18.3% for treated animals. Myofibril fragmentation i ndex (MFI), a measure highly correlated with tenderness, also indicated that BAA-fed animal s produced less tender meat. Over the 14-day aging period, their MFI increased by only 3.3%, while control animals MFI increased by 42.3% (Koohmaraie et al., 1991). Studies involving beef cattle repo rt results similar to sheep data with respect to tenderness. Steaks from beef cattle fed the BAA L644,969 for 6 weeks prior to harvest had WBS values that were greater than the controls on day 7 and 14 pos tmortem. In addition, WBS values for treated animals did not change during the entire 14 da y aging period, indicating a lack of postmortem tenderization due to treatment. Myofibril fragme ntation index for the study also indicated that control animals had more postmortem proteolysi s than the treated animals throughout the 14 day aging period, which contributed to the greater WBS values repor ted (Wheeler and Koohmaraie, 1992). Geesink et al. (1993) examined the effect of the BAA clenbuterol on th e tenderness of four different muscles ( Longissimus, Semimembranosus, Triceps brachii, and Psoas major ) of veal

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35 calves and found that multiple muscles are affect ed by supplementation. However, the results indicate that not all muscles are affected e qually. Steaks from some treated muscles were tougher on day 1 or 7, while other muscles were unaffected. In a related study involving veal calves, Garssen et al. (1995) examined th e effects of clenbuterol and salbutamol on Longissimus lumborum tenderness. Warner Bratzler shear for ce values for treated animals were higher on days 2, 5, and 10 postmortem. When compared to the control group, the salbutamol group produced the largest increase in WBS, which fu rther indicates that various BAAs affect tenderness differently. In 2007, the FDA approved a new BAA for use in beef cattle. Interv et sells this BAA, Zilpaterol-HCl, whic h acts through the 2-receptor and seems to be more potent than RAC. Because this compound is new, limited literatu re exists documenting its effect on beef tenderness. However, it appears that this co mpound greatly affects tenderness. When fed to steers, zilpaterol increased shear values by 16 and 6% compared to control and RAC supplemented steers, respectively (Avendano-Reyes et al., 2006). More im portantly, zilpaterol increased shear values of supplemented steaks to an average of 5.11 kg, wh ich is nearly tough, as defined by Boleman et al. (1997) and Miller et al. (2001). Since Optaflexx (ractopamine-HCl) gained F DA approval for beef cattle in 2003, research on the effect of RAC on beef tende rness has progressed slowly. The beef industry is justifiably concerned about this issue, give n the literature involving other BAAs. For many years, Elanco, the manufacturer of the commercial form of RA C provided the only data on the effect of RAC on beef tenderness. These researchers used bot h objective and subjective measures to evaluate four concentration levels of RAC (0,100, 200, and 300 mghd-1d-1) on strip loin steak tenderness. Both methods indicated that RAC fed at 100 and 200 mghd-1d-1did not affect tenderness.

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36 Warner-Bratzler shear force values of steaks from the 300 mghd-1d-1group were 0.4 kg greater than control steaks. However, researchers concluded that this finding was not a major issue because these values were below 3.95 kg, which is considered acceptably tender to consumers. In addition, researchers stated that a 0.45 to 0.91 lb change in shear force is required for consumer recognition of a change. Trained sens ory panelists found that Ractopamine fed at all levels did not affect juiciness, fl avor, or the incidence of off-fla vors. However, tenderness scores mirrored the findings of the WBS data. Compared to controls, initial and sustained tenderness scores were lower for the 300 mghd-1d-1steaks. Once again, researcher s stated that changes in tenderness detected by the panelists would not be detected by consumers and should not be a concern (Schroeder et al., 2004a). Since these results were published, numerous university studies have concurred with the Elanco data. The first independent study that evaluated the effect of RAC on beef tenderness found that RAC fed at 300 mghd-1d-1 during the final 33 days of f eeding significantly decreased tenderness by approximately 0.6 kg. The researcher s attributed the decrease in tenderness to BAAs ability to reduced protein degradati on and proteolytic activ ity, decreased collagen solubility, and changed fiber type distribution within the muscle. In agreement with Elanco, these researchers concluded that the RAC i nduced decrease in tenderness was a non-issue because the steaks were well within the accepta ble range of shear force tenderness values (AvendanoReyes et al., 2006). Possibly due to these finding s, most university conducted studies have fed RAC at a rate of 200 mghd-1d-1. Of these studies, two have evaluated the effect of RAC on tenderness. In agreement with Schroe der et al. (2004a), RAC supplementation at this level did not affect tenderness. Re searchers attributed this lack of effect to a lack of growth in

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37 the LD muscle. They hypothesized that the abse nce of growth did not signal an alteration in protein degradation and postmortem prot eolytic activity (Q uinn et al., 2008). However, RAC supplemented at 200 mghd-1d-1 for the final 28 days of feeding to British, Continental crossbred, and Brahman crossbred st eers decreased longissimus muscle tenderness (Gruber et al., 2008). Daily RAC supplementatio n increased WBS and slice shear force by 0.38 and 1.4 kg, respectively. Ractopamine supplem entation increased the WBS of Brahman crossbred steers more than the Continental cros sbred and British steers. To counteract the toughening effect of RAC s upplementation, steaks were aged for 3, 7, 14, and 21 days postmortem. Aging steaks did reduce WBS va lues, but did not completely reduce the RAC toughening effect. Ractopamine supplementation also affected trained sensory panelist scores. Trained sensory panelists found that RAC supplemented steaks we re less tender and juicy, and tended to have reduced beef flavor. Re searchers noted that because RAC is a 1-agonist and this class of agonist causes muscle growth primaril y through an increase in protein synthesis, the decrease in tenderness detected by objective and subjective evaluations was not due to increased calpastatin activity. The resear chers hinted that RAC possibly increased the CSA and diameter of muscle fibers, decreasing tenderness, but th at this hypothesis required further examination (Gruber et al., 2008). Dijkhuis et al. (2008) examined the effect of three levels of RAC (0, 100, 200, and 300 mghd-1d-1) on the tenderness of selected muscles from cull cows. Results established that RAC does not affect the tenderness of musc les uniformly. The tenderness of the Infraspinatus, Rectus femoris, Semimembranosus, and Triceps brachii lateral head were affected by RAC supplementation. The tenderness of other muscles, such as the Adductor Gracilis Longissimus thoracis Teres major, Triceps brachii long head and Vastus lateralis were unaffected by RAC.

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38 However, the muscles affected by RAC did not respond similarly to supplementation. Ractopamine fed at each level signifi cantly increased shear force in the Infraspinatus Ractopamine fed at 100 mghd-1d-1increased tenderness in the Semimembranosus In the Rectus femoris, RAC fed at 200 mghd-1d-1resulted in steaks that were less tender than RAC steaks fed at the 100 mghd-1d-1dosage level. The same trend was seen in the Triceps brachii lateral head except steaks from the 300 mghd-1d-1 level were more tender than 100 mghd-1d-1steaks. Researchers hypothesized that the increases in tenderness with RAC supplementation may be due to increased protein turnover in the 200 and 300 mghd-1d-1 treatment groups, which diluted collagen cross-linking. Effect of Beta-Adrenergic Agonists on Sw ine Growth Performance and Carcass Characteristics The main advantage of feeding RAC, and most BAAs, is that they increase lean deposition while restricting fat deposition in various species. These mechanis ms translate into carcass and meat quality effects, as well as animal pr oduction improvements (average daily gain [ ADG ], average daily feed intake [ ADFI ], and feed:gain ratio [ F/G ]). Studies involving RAC supplementation in swine can be broken into two subgroups: effects on growth performance and effects on carcass and fresh meat characteristics. The three primary growth perfor mance characteristics improved by RAC supplementation include ADG, F/G, and ADFI. Watkins et al. (1990) found that when finishing pigs were supplemented with 0, 2.5, 5, 10, 20 or 30 ppm of RAC, ADG and F/G were improved linearly with increasing RAC level. In this study, supplementing RAC over a range of 5 to 15 ppm improved ADG and F/G over controls by 8.3 and 9.9%, respectively. Orthogonal polynomials for this study revealed that the dosage producing the maximum response in ADG was between 14 ppm and 16 ppm. Crenshaw et al. (1987) also studied these levels of RAC

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39 supplementation and found that as RAC was increas ed, ADFI showed a liner decrease. Sities et al. (1991) ran a similar study examining the effe cts of 5, 10 or 20 ppm of RAC supplementation to finishing swine and found a similar linear increase in ADG with increase in RAC dosage level. Prince et al. (1987) analyzed the same range of RAC supplementation as Watkins et al. (1990) and only found a tendency for RAC to improve ADG and F/G at the 5, 10, 20 and 30 ppm levels. The diminished effect may be due to th e inclusion of a five da y withdrawal period or insufficient number of animals. Crenshaw et al. (1987) found that RAC did not improve ADG or F/G. In agreement with this study, Hancock et al. (1987) found th at ADG was unaffected by these levels of RAC supplementation, however a quadratic response was de tected for F/G. Studies have demonstrated that RAC supplem entation to pigs during the finishing phase has the ability to positively affect ADG and F/G ra tios. The response is rapid, occurring in as little as 6 days, with a constant supplementation level of 5 ppm. These effects are maintained by feeding 5 ppm and 20 ppm of RAC for 34 days (Armstrong et al., 2004). The enhanced growth response of pigs to prolonged RAC supplementation initially rises rapidly, reaches a plateau, and then declines during the rest of the feeding pe riod (Dunshea et al., 1993; Williams et al., 1994). This trend indicates that prolonged RAC supplem entation can cause a fatigue in receptor and growth responses. Sainz et al (1993) noticed that RAC suppl ementation also improved weekly and ADG during the first three weeks of supplementation and not thereafter. See et al. (2004) observed the effects of feedi ng RAC at increasing (step-up) decreasing (step-down), and constant levels on growth performance. Re searchers concluded that the step-down method desensitized -adrenergic receptors and caused a re duction in the overall response to RAC supplementation. The step-up RAC supplementati on protocol enhanced growth performance

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40 (ADG and F/G) above the step-dow n or constant supplementation methods. Therefore, fatigue to RAC supplementation can affect response to supplementation, but the phase of production (weight and age) can affect magnitude of response. Older and heavier pigs experi ence a more pronounced stimulat ion in growth performance than young pigs (Sainz et al., 1993). This indicated that phase of growth when RAC is added to the diet affects how an animal responds to suppl ementation. In contrast to this study, Crome et al. (1996) compared the effect of RAC supplementation in two different weight groups of finishing pigs (light, 68 to 107 kg; heavy, 85 to 125 kg). Average daily gain was improved with no difference between groups. Average daily feed intake was improved by RAC supplementation and the heavier group experienced a greater decrease. Th e heavier group responded to RAC supplementati on with a greater decrease in F/G than the lighter group. Watkins et al. (1990) stated that the respons e of growth and carcass variables to RAC supplementation may depend on supplementation level as well as other factors, such as genetics and the dietary crude protein conten t of the diet. Genetic studies ha ve investigated the effect of crossbreeding and RAC supplementation on growth performance. In a study by Uttaro et al. (1993), results indicated that when 20 ppm of RAC was supplemented during the finishing phase of crossbred Canadian swine, ADG was impr oved by 150g/d and F/G was decreased by 0.52 kg. Yen et al. (1991) conducted a st udy where contemporary crossbred swine and crossbred Meishan swine were supplemented with 20 ppm of RAC with improved ADG and F/G, with no effect on ADFI. In contrast to these studi es, Gu et al. (1991) determined that when RAC was fed to five different genotypes of crossbred pigs, with differe nt lean growth potential s, there was no effect on ADG, ADFI, or F/G. Numerous studies wh ich supplemented RAC to contemporary pigs

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41 revealed no effect on ADG (Hancock et al., 1987, Prince et al., 1983; Ott et al., 1989). Contradictory results may be due to the geneti c capacity of various breeds to deposit lean. Pigs more genetically pred isposed to increased lean tissue deposition respond more favorably to RAC supplementation. Bark et al. (1992) compared low and high lean tissue genotypes and found that both genotypes main tained the same ADFI, however ADG was increased by 41% and F/G was decreased by 32% fo r the high lean tissue genotype, compared to the low lean tissue genotype. Stoller et al. (2003) reported that ADG of high lean pigs was greater than ADG of purebred Durocs and purebred Berkshire, when all lines were supplemented with 20 ppm of RAC. Yen et al. (1990) compar ed genetically obese and genetically lean pigs supplemented with 20 ppm of RAC. Average dail y feed intake and F/G ratio of both genetic lines were improved by supplementation, but ADG was unaffected by treatment. Lean pigs supplemented with RAC had higher F/G ratios than obese pigs supplemented with RAC. By contrast, Mimbs et al. (2005) found that during the first 7 days of supplementation, lean pigs fed RAC gained less than fat pigs fed RAC. Over the course of the entire study, RAC did not affect ADG between the two genotypes. In agreement, Mitchell et al. (1990) supplemented RAC to the diets of high lean deposition line of pigs fed 12 or 24% crude protein, and found no significant effect on ADG or F/G. These researchers determined that RAC improves growth by improving carcass characteristics, but has no effect on live perfor mance. However, this result may have occurred due to the low number of animals in each group. The extent of the improvement seen with s upplementation is influenced by many factors, including the factors previously discussed. The nutrient content of the diet, including energy and crude protein fed during supplementation also af fects the magnitude of the RAC response. Williams et al. (1994) compared four levels of dietary energy conten t and found that ADG and

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42 F/G were improved by RAC supplementation with a linear increase in ADG with increased energy content. Dunshea et al. (1993) comp ared six levels of protein (8.5, 11.2, 14.0, 16.7, 19.5 and 22%) and found that ADG increased with di etary protein content in RAC supplemented animals. Similarly, F/G improved (decreased) with increased dietary protein content. Adeola et al. (1990) reported that pigs fe d different dietary protein levels respond differently to RAC supplementation. Pigs fed a 17% protein diet de monstrated increased ADG and F/G, while pigs fed a 13 % protein diet demons trated decreased ADG and F/G. By contrast, Mitchell et al. ( 1991) reported that RAC did not affect ADG or F/ G at dietary crude protein levels of 12, 18, and 18 % restricted. The authors noted that th e lack of RAC effect on ADG was due to performance during the latter stages of the study, possibly because of receptor fatigue. Dunshea et al. (1998) found that RAC increased ADG and decreased F/G in both gilts and boars, but this was achieved inde pendently of dietary energy intake. As demonstrated by the above studies, RAC supplemen tation has an effect on growth performance and this response is determined by the level of supplementation, genetics, and diet. Not only do these factors affect growth performance, but also carcass and fresh m eat characteristics. Ractopamine supplementation mainly affects pi g carcasses via increased deposition of lean tissue and decreased depos ition of adipose tissue. This eff ect is positively correlated with improved dressing percentage. Dressing percentage increases linearly with increasing levels of RAC (Hancock et al., 1987; Stites et al., 1990 ; Watkins et al., 1990). Supplementation of RAC at 2.5, 5, 10, 20, and 30 ppm levels increased dressing percentage and carcass lean percentage, and decreased carcass fat percentage (Crenshaw et al., 1987; Hancock et al., 1987; Prince et al., 1987; Watkins et al., 1990). See et al. (2004) report ed an increase in percen tage lean using step-

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43 up, step-down, and constant feeding regimens. Ractopamine supplementation also increased estimated fat free muscle when fed at these levels (Watkins et al., 1990). Loin eye area is also used to demons trate increased lean deposition with RAC supplementation. Various studies feeding RAC at levels of 2.5, 5, 10, 20, and 30 ppm found that loin eye area increased linearly with increasi ng RAC supplementation level (Stites et al., 1991; Watkins et al., 1990) and increased overall when compared to non-supplemented pigs (Crenshaw et al., 1987; Hancock et al., 1987; Se e et al., 2004). By contrast, Sa inz et al. (1993) did not find a difference between the loin eye area of non-supplem ented pigs and pigs fed RAC at various rates (constant, step-up, step-dow n, and alternating). Carcass fat content is reduced by RAC supplem entation. Ractopamine fed at levels of 2.5, 5, 10, 20 and 30 ppm reduced the 10th rib back fat depth when compared to controls (Crenshaw et al., 1987, Prince et al., 1987; Watkins et al., 1990; See et al., 2004) Watkins et al. (1990) also reported lower average back fat depths for RA C supplemented animals, while Hancock et al. (1987) found that RAC supplementation only tended to decrease average back fat depth. Again, Sainz et al. (1993) found that average b ack fat thickness was not affected by RAC supplementation. Leaf fat deposited in the body cav ity was reduced in RAC fed pigs compared to controls (Prince et al., 1987; Wa tkins et al., 1990; See et al., 2004). Stites et al. (1991) reported that pigs fed 0, 5, 10, and 20 ppm of RAC had heavier loins and hams than controls. The percent weight of trimmed ham and loin was increased by RAC supplementation, while there was no effect on the percentage of trimme d shoulder, belly or boneless ham. These differential effects of RAC c ould indicate that RAC affects distinct muscle groups differently.

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44 Moreover, a minimum time on feed with RAC is required to observe an effect on carcass measures. Ractopamine did not improve hot carcass weights at any level of RAC supplementation (5, 10, or 20 ppm) at 6 days of supplementation. However, after 13 days of supplementation, all levels of RAC supplementa tion improved hot carcass weights. After 20 days of feeding, RAC only improved the hot ca rcass weights in pigs supplemented 10 and 20 ppm. When fed past 27 days, the 20 ppm level of supplementation ha d increased hot carcass weights when compared to the other levels of supplementation. Pigs supplemented 20 ppm of RAC for 6 and 13 days demonstrated an incr ease in dressing percentage. Ractopamine supplemented for 20, 27, and 34 days at 10 and 20 ppm resulted in increases in dressing percentage. Ractopamine supplementation did not affect loin eye area when fed for 6, 13, and 20 days. However, pigs supplemented with 10 a nd 20 ppm for 27 and 34 days possessed increased loin eye areas. Percentage of fat free lean was only increased by RAC supplementation at 10 and 20 ppm for 27 and 34 days. There was no effect on 10th rib back fat depth at any level of RAC supplementation for any of the time pe riods (Armstrong et al., 2004). As previously stated, the response of growth performance to RAC supplementation is not constant over time, which may be attributed to receptor desensitization (Dunshea et al., 1993; Williams et al., 1994). This trend is reflected in carcass characteristics. When comparing RAC step-up, step-down, and constant feeding programs, See et al. (2004) found an improvement in hot carcass weight and percent yi eld for pigs fed the RAC stepup and constant program. The step-down program was not different from cont rols. Weights of the boneless-trimmed shoulder and loin were increased when the RAC stepup and constant feeding regimen were followed, while the RAC step-down program resulted in no effects on these cuts. Sainz et al. (1993) found that dressing percentage wa s increased by the constant RAC supplementation method, RAC

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45 supplementation during the final three weeks of feeding, and an alternative weekly RAC supplementation method. The authors concluded that attenuation in growth and carcass characteristics observed during prolonged supplementation can be prevented by feeding RAC during alternating weeks. Unlike growth performance data, the magnitude of response to RAC supplementation in pig carcasses is not affected by genotype. Yen et al. (1991) reported that 20 ppm of RAC fed to three diverse genotypes during the final 52 days of feeding incr eased hot carcass weight and dressing percentage regardless of genotype. In agreement, Gu et al. (1991) found that dressing percentage was increased by 1.28 percent in pigs of five different ge netic backgrounds. In addition, loin eye area was also increased by RA C supplementation. Stoller et al. (2003) also reported improved loin eye area by 1.4 cm2 when feeding RAC, but did not find an effect of genetic line on this improvement. Uttaro et al. (1993) found that feed ing 20 ppm of RAC to crossbred swine decreased 10th rib back fat depth, and increa sed 10th rib lean depth, which resulted in an increase in pe rcent lean yield, regardless of ge netic background. Also in this study, the weight of trimmed shoulders from RA C supplemented animals was decreased due to less trim needing removal. Loin weights of supplemented pigs were increased, which also resulted in an increase in salable lean. Longissimus muscle area of supplemented pigs was greater than controls, du e to 13% more lean and 12% less fat being present. Trimmed hams from RAC fed animals had increased weight and decrease d fat content, while belly lean was increased and fat content decreased. In contrast to these studies, Stoller et al. (2003) reported th at supplementation of 10 ppm of RAC to pigs of different genetic background reduced 10th rib back fat in the high lean genetic line only. Yen et al. (1991) al so found that RAC increased the Longissimus muscle area of high

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46 lean yield and medium lean yield genotypes, while having no effect on the low lean yield genotype. In the same study, it was also determin ed that the high lean yield genotype had more muscling than the medium lean yield genotype, wh ich had more muscling than the low lean yield genotype. These two studies show that the pigs genetic capacity to deposit lean has an effect on carcass characteristic s in response to RAC supplementation. Yen et al. (1990) compared the effect of RAC on obese and lean lines of pigs and found that in both obese and lean lines, RAC was effective in reducing body fat deposition and increasing carcass leanness. Hot carcass weight and dressing percentage were increased by RAC supplementation, while loin eye area at the 10th rib was increased. The predicted amount of muscle was increased by RAC and the weights of the untrimmed picnic and trimmed Boston butt, picnic, and shoulder also were increased. The weight of the untrimmed or trimmed ham was unaffected by RAC supplementation, but proxim ate analysis for the ham showed decreased fat content and increased crude pr otein and moisture contents. In a study by Mimbs et al. (2005), pigs were separated phenotypically into fat and lean groups and supplemented with 10 ppm of RAC. Ultr asound carcass measurements revealed that during the first three weeks of the trial, RAC did not affect deposited back fat. However, after the fourth week of the trial, the RAC supplemente d pigs had lower ultrasound back fat measures. During the trial, there was a si gnificant phenotype x RAC interaction, which indicates that the phenotypes responded to supplementation differently. Longissimus muscle area measured by ultrasound also was increased also by RAC supp lementation in both groups, but no interaction was reported. Finally, Bark et al. (1992) supplemented 20 ppm of RAC to high and low lean tissue growth genotypes of pigs and found that RAC reduced backfat thickness in both genotypes by a

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47 similar magnitude. Ractopamine supplementation increased Longissimus muscle area in both genotypes, with the high genotype experiencing a greater magnitude of increase. Pigs of the high genotype tended to show increased estima ted muscle and increased dissected muscle weights. Ractopamine also decreased the amoun t of dissectible fatty tissue more in the high genotype than in the low genotype, and the high ge notype had more muscle in the ham, belly and shoulder. Despite the fact that RAC had no e ffect on the intramuscular fat content of the longissimus muscle of either genotype, RAC decreased the intr amuscular fat content of the triceps brachii of the low genot ype, with no effect on the high genotype. Therefore, it was determined that RAC included in the diet of a high lean yielding genotype and a low lean yielding genotype increases muscle accretion and decreases fatty tissue accretion to a greater degree in the high lean yielding genotype. Rapid and low lean growth lines of pigs we re fed 12% or 24% crude protein supplemented with 20 ppm of RAC from 60 to 90 kg of weight. Tenth rib back fat was reduced by 25% in the low lean pigs supplemented RAC and fed 12% crude protein, 16% in the high lean pigs supplemented RAC and fed 12% crude protein, and 7% in the high lean pigs supplemented RAC and fed 24% crude protein (Mitchel l et al., 1990). Over all, carcass lipid wa s reduced the most by RAC supplemented to the low lean pigs supplem ented RAC and fed 12% crude protein, than low lean pigs supplemented RAC and fed 24% crude protein, high lean pigs supplemented RAC and fed 12% crude protein, and high lean pigs supplemented RAC and fed 24% crude protein. There was a Line x Diet x Treatment interaction for back fat, backfat growth, and dissectible fat growth. For carcass and muscle protein, the low lean pigs supplemented RAC showed increases in carcass protein growth by 60% while the high lean pigs supplemented RAC increased carcass protein growth by 15%. This trend was true for Longissimus muscle area where low lean pigs

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48 supplemented RAC pigs responded more than high lean pigs supplemented RAC pigs. A Line x Diet x Treatment interaction was observed for Longissimus muscle area, which indicates that the genetic background of the pigs dete rmines the magnitude of the res ponse, but protein level of the diet is important. As demonstrated by the previous study, dietary factors such as crude protein content can affect the magnitude of the response to RAC supplementation. Additional studies examined dietary energy effects on RAC response. In a study by Williams et al. (1994), dietary energy intake has an effect on the magnitude of carcass ch aracteristic response to RAC supplementation. In this study, four energy intake levels were used in combination with 44.7 mg/d of RAC. Ractopamine reduced backfat, increased Longissimus muscle area and increased dressing percentage. For dressing percen tage, there was a linear trend toward an increase in dressing percentage with increasing energy intake level for RAC supplemented pigs. There was a RAC x Energy interaction for backfat thickness, and fat depth was increased for pigs supplemented with RAC at lower energy levels. A linear relati onship between increased energy intake and increased fat thickness for RAC supplemented pigs also was noticed. Longissimus muscle area also had an Energy x RAC interaction and area s were greater for pigs supplemented RAC at lower energy intakes. These interactions, ther efore, indicate that RA C is able to produce maximum gains in lean at lower energy intakes. Dunshea et al. (1998) studied th e effects of feeding five diffe rent energy levels on carcass characteristics of RAC supplemented pigs and f ound that RAC did not have an effect on average backfat thickness and dressing perc entage, but increased loin eye area independent of dietary energy content. Empty body protein and water deposition was increased by RAC supplementation, while empty body fat and fa t:protein ratio decreased with RAC

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49 supplementation. For fat deposition, there was no effect of RAC supplementation, but the protein:fat ratio was increased by RAC. All thes e measurements were a ffected independent of dietary energy content, but RAC supplementation did increase pr otein deposition. As dietary energy increased, the response to RAC supplemen tation also increased. Unlike energy content of the diet acting independent of RAC supplementation, Dunsh ea et al. (1993) looked at six levels of dietary protein al ong with 20 ppm of RAC supplem entation and found that empty body protein deposition was increased by RAC supplementation at the highe r dietary protein levels. Mitchell et al. (1991) fed thr ee different levels of dietar y protein and energy and two different level of RAC supplementation and found that even though crude protein levels had an effect on backfat thickness, RAC supplementation did not have an effect. There was a reduction in the amount of carcass lipid present in the st udy, with pigs fed a 12% (3.68 Mcal of DE/kg) crude protein diet having a gr eater reduction than the 18% (3.52 Mcal of DE/kg) crude protein diet. Likewise, RAC supplementation reduced th e rate of lipid deposition and once again the 12% crude protein diet ha d the greatest reduction. Longissimus muscle area also was increased by RAC supplementation with the 18% crude protein diet having the greatest increase compared to the 12% crude protein diet. For carcass protein, the 12% diet had more protein than the 18% diet, and the rate of protein accreti on was greater for the 12% diet. Adeola et al. (1990) supplemented 20 ppm of RAC in 13 and 17% crude protein diets and found that RAC reduced leaf fat, increased mu scle depth, increased primal and subprimal weights, and promoted rate of protein deposition in the Longissimus and Biceps femoris regardless of protein level in the diet. The authors concluded that RAC supplementation increases carcass lean at both high and low dietary protein levels. Carcass weights were the only

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50 characteristic where crude protein affected the magnitude of response to RAC. Weights were lower for RAC pigs fed the 13% protein diet when compared to the 17% protei n diet pigs. Few reports documenting the effect of RAC supplementation on fresh meat characteristics exist. Researchers have determined that RAC supplementation does not aff ect color, texture, or marbling (Hancock et al., 1987; St ites et al., 1991; Watkins et al ., 1990; Sainz et al., 1993). In contrast to these studies, Wa tkins et al. (1990) reported incr eases in color, firmness, and marbling when pigs were fed 5, 10, or 20 pp m of RAC. Armstrong et al. (2004) found no difference in marbling between supplemented and control pi gs at 6, 13, or 20 days of supplementation. However, at 10 and 20 ppm of supplementation, marbling was decreased at days 27 and 34. Color scores were not affect ed by supplementation on days 6, 13, or 34, but were lower for pigs supplemented 20 ppm for 20 a nd 27 days. This same trend was observed for L* and a* values. However, b* values were unaffected by dietary RAC concentration and length. Therefore, marbling and meat color of RAC supplemented animals can be affected by RAC supplementation. Effect of Beta-Adrenergic Agonists on Cattle Grow th Performance and Carcass Characteristics Because RAC was just recently approved for use in beef cattle, few publications exist evaluating its effects on beef cattle. Historically, clenbuterol was the main -agonist studied in cattle (Miller et al., 1988; Ricks et al., 1984b). One of the earliest RAC studies involving cattle was conducted by Anderson et al. (1989). In th is study, six levels of RAC (0, 10, 20, 40, 60, and 80 ppm) were supplemented at tw o different dietary protein leve ls (11 and 14%). There was no RAC x Diet interaction and average daily ga in was improved at 80 ppm of supplementation, while F/G was decreased at 20, 40, 60, and 80 ppm of RAC supplementation. For carcass characteristics, yield grade was decreased by 20, 40, 60, and 80 ppm of RAC supplementation;

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51 percent protein was increased by 40, 60, and 80 ppm of RAC supplementa tion; and percent fat was decreased by 60 and 80 ppm of RAC supplementation. Commercially RAC was approved by the Food and Drug Administration ( FDA ) in 2003. Elanco sells this product unde r the trade name Optaflexx for cattle fed in confinement during the last 28 to 42 days of feeding. The first data involving the use of Optaflexx in beef cattle was a summary of the results of live performance and carcass characteristics for Elancos FDA registration trials. The data pr esented represented 10 research tr ials evaluating the effects of RAC on both steers and heifers. Ractopamine was fed at approximately 100, 200, and 300 mghd-1d-1 for either 28 or 42 days prio r to harvest. All data for th e 10 trials were pooled and a total of 220 steers and 215 heifers were examined. Ractopamine did not affect feed intake in either steers or heifers. In steers, RAC improved ADG by 17.1, 19.6, and 25.7% in the 100, 200, and 300 mghd-1d-1 treatments, respectively. This resulted in feed efficiency improvements of 13.6, 15.9, and 20.5%. However, RAC does not seem to act as efficiently in heifers as it does in steers. While still significantly different, heifers fed 100, 200, and 300 mghd-1d-1 of RAC had improved ADG of 8.0, 17.5, and 20.4%, respectively. Feed efficiency was improved by the treatment levels by 6.9, 14.0, and 17.1%, respectively. Any improvements in carcass characteristics due to RAC supplementation also indicated that RAC has more of an effect on steers than heifers. Of the carcass characteristics evaluated, RAC significantly affected hot carcass weight, dressing percentage, ri beye area, and yield grade. Ractopamine improved hot carcass weight by as much as 18.2 lbs in steers and 11.3 lbs in heifers. Dressing percent was improved by 0.3 and 0.4 percentage points in the 200 and 300 mghd-1d-1 treatment groups of steers. The dressi ng percent of heifers was unaffected by RAC supplementation. Ribeye area, an indicator of muscling, improved by as much as half an inch in

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52 both steers and heifers fed 300 mghd-1d-1. Ractopamine supplementation tended to decrease the yield grade of steers fed 200 mghd-1d-1 and significantly decreased the yield grade of steers fed 300 mghd-1d-1. However, in heifers RAC, only tende d to decrease the yield grade in the 300 mghd-1d-1 treatment group. Producers are often concerned about feeding BAAs, including RAC, because of their effects on fat deposition. Acro ss all ten studies for both steers a nd heifers, all fat measurements including KPH, 12th rib fat thickness, and marbling were unaffected by RAC supplementation. In addition, quality grades of both steers and heifers were unaffected by RAC. Other meat quality parameters such as color, firmness, and texture of meat from steers were unaffected by RAC. In heifers, color was improved by RAC, wh ile the other two meat quality parameters were unaffected (Schroeder et al., 2004a,b). Following the release of this data, researcher s from Elanco published the results of the 10 individual studies in a series of abstracts. In the first ab stract, a dose titr ation study was conducted on feedlot steers supplemented 0, 10, 20, or 30 ppm of RAC. Growth performance data indicated that RAC did not change feed intake, but improved ADG, F/G, G/F, HCW, and dressing percentage (Schroeder et al., 2005a). For the same animals at the same supplementation levels, Longissimus muscle area was increased. Yield gr ade tended to be improved at the 20 ppm supplementation level and was significantly improved at the 30 ppm supplementation level. Twelfth rib fat thickness and KPH were unaffected by RAC as well as muscle color, firmness, and texture (Schroeder et al., 2005b). Protein content also was increased at the 20 and 30 ppm supplementation levels (Schroeder et al., 2005c). Schroeder et al. (2005d) analyzed these same levels of RAC supplementation fed to feedlot heifers and determined that feed intake was unaffected. However, RAC supplementation

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53 improved ADG and G/F, and decreased F/G. Hot carcass weight was increased in heifers fed 20 and 30 ppm of RAC, while dr essing percentage was unaffect ed by RAC supplementation. Longissimus muscle area was the largest for heifer s fed 30 ppm of RAC and yield grade was improved by 30 ppm of RAC. Muscle color was improved with RAC supplementation. Twelfth rib fat thickness, KPH, muscle firmness, and muscle texture we re unaffected by RAC supplementation (Schroeder et al., 2005e). The carcass composition of th ese heifers indicated that carcass protein and moistu re were increased by 30 ppm of RAC supplementation (Schroeder et al., 2005f). Laudert et al. (2005a) conducted a large pe n study to examine the effects of RAC supplementation (0, 100, and 200 mghd-1d-1) during the final 28 to 32 days of finishing in a feedlot setting. Ractopamine did not increase dry matter intake, but improved ADG, F/G, and G/F. These same feedlot finishing steers had increased dressing percentages when supplemented with RAC. Carcass dr essing percentage was higher for the 200 mghd-1d-1supplemented group compared to the 100 mghd-1d-1supplemented group. Loin muscle area was increased with increased supplementation level of RAC, while marbling score was unaffected by supplementation. Finally, 12th rib fat thickness, KPH, yield grade, and carcass maturity were unaffected by RAC supplementati on (Laudert et al., 2005b). Supplementation (0, 200, or 300 mghd-1d-1) to calf-fed Holstein steers did not affect ADFI, while ADG and F/G were improved by suppl ementation (Vogel et al., 2005a). Carcasses from the supplemented steers were heavier than the controls. Longissimus muscle area was increased by supplementation, and yield grad e was decreased by RAC supplementation. Marbling scores were decreased for steers fed the 200 mghd-1d-1 of RAC, while the scores for

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54 the 300 mghd-1d-1 supplementation were not affected. Feeding RAC did not affect KPH, carcass maturity, or the incidence of dark cutting (Vogel et al., 2005b). Beginning in 2006, university sponsored research st udies began to appear in the literature with most studies reporting similar results as the Elanco data. Walker et al. (2006) evaluated the effects of RAC (200 mghd-1d-1 for 28 days) and protein source (688, 761, and 808 g/d) on the growth performance and carcass ch aracteristics of heifers. F eed efficiency was improved by RAC by 17%, while ADG increased by 18%. Howeve r, increasing metabolizable protein did not improve these growth performance measurements in RAC fed animals. Modest improvements in some carcass characteristics were reported. Final body weights of RAC supplemented animals were increased by 8.3 kg and HCW was increa sed by 6.9 kg. As was found with growth performance parameters, increasing metabolizab le protein did not im prove HCW or final body weight. Other carcass characteristics incl uding dressing percentage, ribeye area, 12th rib fat, yield grade, and marbling score were unaffected by RAC supplementation or protein source. The authors concluded that while RAC impr oves some growth performance and carcass characteristics, the level of metabolizable protein does not need to be adjusted. During 2007, numerous studies were publishe d evaluating the eff ects of different management strategies and RAC supplementation on growth performance and carcass characteristics. Initially, resear chers studied the effect of the ag e of beef cattle on the response to RAC. The goal of the study was to determine if di fferences in age affect the ability of a BAA to repartition nutrients. Yearli ng steers were serial harveste d at 150, 171, or 192 days on feed. Within each harvest group, steers were fed 0 or 200 mghd-1d-1 of RAC for the last 28 days of feeding. The inclusion of RAC in the di et increased ADG and G:F ratio by 4.6 and 3.8%, respectively. During the last 28 days of feeding, RAC increased the dry matter intake compared

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55 to controls by 3.5%. Ractopamine te nded to increase ribeye area by 1.74 cm2 and significantly increased HCW by 8 kg. All other carcass charac teristics were unaffected by the inclusion of RAC in the diet. The authors reported no RAC da ys on feed interaction, which indicates that RAC improves the aforementioned characteristics regardless of feeding length (Winterholler et al., 2007). The influence of biological type on the RAC response was examined next. Steers of British, Continental, and Brahman biological type were fed either 0 or 200 mghd-1d-1 for the final 28 days of feeding. Mirro ring previous studies, RAC incr eased ADG and G:F ratio and did not affect dry matter intake. The only carca ss characteristics signi ficantly improved by RAC were HCW and ribeye area, which were increased by 2 and 4%, respectively. Ractopamine tended to lower marbling, but marbling was not lo wered enough to affect the distribution of quality grade. However, there was no RAC bi ological type interaction which led authors to conclude that RAC affected gr owth performance and carcass characteristics in a similar manner across these three distinct biolog ical types (Gruber et al., 2007). While biological type may not affect the response of growth performance and carcass characteristics to RAC supplementation, implant stra tegy may play a role in the response. In a series of experiments, aggressi ve and conservative implant strategies were followed when RAC was supplemented to heifers. When the c onservative protocol was administered, RAC supplementation increased ADG, G: F ratio, HCW, and ribeye area. In addition, RAC decreased 12th rib fat and yield grades. When the aggr essive protocol was combined with RAC supplementation, RAC improved G:F ratio but did not affect other growth performance or carcass characteristics. The au thors hypothesized that steroid implants could affect the sensitivity of BAA receptors and interfere with the response to RA C. Therefore, they concluded

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56 that implant strategy in addition to other management strategies must be considered to maximize the response to RAC (Sissom et al., 2007). The effects of RAC supplementation level (0, 100, 200 mghd-1d-1) and duration of supplementation (28, 35, 42 days) on growth pe rformance and carcass ch aracteristics were evaluated next. Data found that there was no RAC dose duration interaction. However, as RAC dose was increased, final body weight, ADG, and G:F ratio increased. Ractopamine duration tended to cause quadrat ic increases in dry matter inta ke, G:F ratio, and final body weight. There was a significant quadratic effect for ADG and supplementation duration with increases detected up to day 35, but no further in creases thereafter. When the two RAC dosage levels were separated and analy zed independently of one anothe r, data indicated that the 100 mghd-1d-1 dosage group had linear increases in dry matter intake, ADG, and G:F ratio. However, the 200 mghd-1d-1 supplementation group had quadrat ic increase in the same parameters. The authors concluded that the maximum benefit for growth performance was reached at day 42 for the 100 mghd-1d-1 supplementation group and day 35 for the 200 mghd1d-1 supplementation group. The authors hypothesized that the differences between the two groups was due to receptor desens itization experienced when feed ing high levels of BAAs. Ractopamine had only a modest effect on carcass ch aracteristics. In agreement with the growth performance data, there were no RAC dose durat ion interactions for carcass characteristics. Hot carcass weight increased linearly as RAC dose was increased, but tended to act quadratically as duration of supplementation incr eased. A tendency for linear incr eases in ribeye area due to increases in dosage was the only other carcass ch aracteristic affected by RAC. The authors concluded that RAC improves growth performan ce and carcass weight, and should be fed for a shorter period if fed at a high dose (Abney et al., 2007).

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57 Data from Schroder et al. (2004a,b) and Sisso m et al. (2007) reporte d that heifers do not respond to RAC in a similar manner to steers. Therefore, two experiments were conducted examining the response of heifers to RAC. In the first experiment, RAC was fed for 28 days prior to harvest at the manufactur ers recommended dose of 200 mghd-1d-1. Feeding RAC to heifers in this manner did not affect growth pe rformance except for a tendency to increase G:F ratio. All carcass characteristics and meat quality characteristics, including Warner-Bratzler shear force, purge loss, cook loss, and L*a*b* co lorimetric values during retail display were unaffected by RAC supplementation. Due to the lack of response by the heifers when RAC was fed according to the manufacturers recommendation, three alternate feeding regimens were tested. These regimens included feeding 300 mghd-1d-1 for the last 28 days, feeding 200 mghd-1d-1 for the last 42 days, and a step-up met hod that involved increasing the dosage rate 100 mghd-1d-1 every 14 days for the last 42 days. Feeding RAC using these protocols had minimal effects on growth performance. The 300 mghd-1d-1 dosage level reduced dry matter intake. Ractopamine supplementation impr oved G:F ratio and ADG, but all RAC feeding regimens were not different. While differences between the feeding regimens did not occur for growth performance, there were numerical di fferences between the 28 day and 42 day feeding periods. The authors also noted that feeding the step-up method a ppeared to be more effective than feeding constant doses for 28 days. However, this method did not cause greater improvements over feeding 200 mghd-1d-1 for 42 days. Therefore, the authors suggested that feeding RAC for a longer period, not a higher dosage, may be needed to elic it a greater response. In agreement with the first experiment, all carcass characteristics were unaffected by RAC supplementation (Quinn et al., 2008).

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58 The most recent study published involving th e supplementation of RAC to beef cattle examined the effects of management system on the RAC response. In two experiments, conventionally raised cattle and naturally raised cattle were compared in a 2 2 factorial arrangement. Conventionally ra ised cattle were admi nistered a Revalor-S implant and fed a monensin/tylosin feed additive. Naturally raised cattle were neither implanted nor fed the feed additive. In experiment one, RAC was supplemen ted for 37 days and increased ADG. Gain to feed ratio was greater in conven tionally raised steers fed RAC than conventionally raised steers not fed RAC. Gain to feed ratio also was gr eater in naturally raised cattle fed RAC than naturally raised cattle not fed RAC. For carca ss characteristics, RAC lowered yield grade and quality score and tended to increas e ribeye area. These findings le d the authors to conclude that a synergistic effect may occur between the gr owth promotants employed. In addition, steers implanted before RAC supplementation had a gr eater ADG and G:F ratio, which indicated that management practices utilized before RAC ad ministration affected the RAC response. In the second experiment, a management syst em RAC interaction was detected for ADG, G:F ratio, and HCW. During that last 28 days of feeding, th e conventionally raised cattle supplemented RAC had a greater ADG and G:F ra tio, and heavier carcasse s than the naturally raised cattle not fed RAC. The authors concl uded that that the management system and RAC worked synergistically to regulate growth and the management system followed in time period before RAC supplementation caused the observed differences. The authors hypothesized that this was caused by the ability of the implants in the conventional treatments increased satellite cell proliferation and enhanced muscle fiber hypertrophy (Winter holler et al., 2008). Effect of -Adrenergic Agonists on Fresh Mea t Shelf-Life Consumers visually evaluate numerous f actors when considering which product to purchase in the retail display case (MacKinney et al., 1966). These factors may include portion

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59 size, leanness, ease of preparation, and color. Hedrick et al. (1994) a nd Kropf (1980) identified color as the single most important visual component that determines if a consumer will purchase a meat product. Because BAAs, including RAC, sh ift muscle fiber isoforms, supplementation of RAC may affect color and shelf-life. Currentl y, little data exists documenting the effects of RAC on fresh meat color and shelf-life. Ractopamine supplemented to pigs has little to no effect on the color or shelf-life of pork products. Watkins et al. (1990) re ported mixed results when RAC was fed to finishing pigs at dosage levels of 2.5, 5, 10, 20, 30 ppm. In their initi al experiment, RAC did not affect the color of the loin. However, in their second experiment, RAC fed at 10 and 15 ppm caused loins from these pigs to become darker. In agreement with the Watkins et al. (1990) first experiment, Stites et al. (1991) found that the RAC had no effect on loin color when fed at 5, 10, and 15 ppm. Stites et al. (1994) also examined these levels of supplementation and their effect on cured ham color. The researchers found that RAC did not aff ect cured color, cured color uniformity, or the amount of discoloration. In the same study, RAC did not affect loin c hop lean color, surface discoloration, or overall a ppearance during a four day retail display study. Recently, Apple et al. (2008) studied the e ffect of RAC supplementation on the shelf-life properties of loin chops. Pigs were fed 10 mg/kg of RAC during the final 35 days of feeding and loin chops were displayed under re tail conditions for five days. Across the five days of display, RAC chops received higher subjective color scor es, which indicates that RAC caused the chops to become darker. This finding was in disagreement with Armstrong et al (2004) and Carr et al. (2005), who each found that RAC did not affect eith er Japanese of American pork color scores. In addition, RAC caused chops to become darker less red, less yellow, and less vivid in color than controls. Others have also found th at RAC supplemented between 5 and 20 mg/kg

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60 decreases redness and yellowness in loin chops (Armstrong et al., 2004; Carr et al., 2005). These findings led the authors to conclude that over a five day reta il study, RAC fed at 10 mg/kg can enhance loin chop muscle quality. Color and shelf-life data from studies with RAC and the ot her beef cattle approved BAA, zilpaterol, differ from study to study. Dietzel (1990) reported that supplementing RAC at 30 ppm during the last 45 days of feeding produced steaks with a subjectivel y brighter cherry red lean color after four days of display when compared to untrea ted steaks. Additionally, steaks from RAC fed steers decreased in overall appear ance slower than steaks from non-treated steers. In agreement with this study, Avendano-Reyes et al. (2006) compared steaks from ractopamine (300 mghd-1d-1) and zilpaterol (60 mghd-1d-1) supplemented animals to control steaks and found that control steaks became darker than st eaks from BAA supplemented animals on day 5 of a retail display period. However, Quinn et al. (2008) supplemented heifers with 200 mghd1d-1 of RAC for 28 days prior to harvest and found that the five day reta il L*a*b* values of longissimus muscle steaks were unaffected. Theref ore, this could indicate that larger dose or longer supplementation time may be needed to affect beef steak color and shelf-life. Other studies conducted with the BAA zilp aterol-HCl have c ontradictory findings concerning its effect on color and sh elf-life. Brooks et al. (2008) summarized a series of studies concluding that this agonist ha s no detrimental effect on color scores and can improve color stability and shelf-life. In thei r summary, Brooks et al. (2008) re ported that several studies found that zilpaterol-HCl improved carcass color scores and resulted in muscles that were more cherryred in color. Other studies cited indicate that zilpaterol-HCl improved shelf-life and reduced the levels of metmyoglobin accumulation on the surface of steaks. Additional research found that zilpaterol-HCl supplementation produ ced steaks with a more desirable lean color. In contrast to

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61 this report, Neill et al. (2008) found that steaks originating from the knuckle of implanted cull cows fed zilpaterol-HCl were da rker and had more discoloration on day 5 of display than cows that were grass fed, concentrate fed, and concentr ate fed with the inclusion of zilpaterol. This could suggest that zilpaterol-HCl has a differential effect on the color and shelf-life of various muscles. While certain hypothesis and conclu sions can be drawn from the data already published, more research is needed to fully und erstand the effects of BAAs on the color and shelf-life of different muscles of the beef animal.

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62 CHAPTER 3 EFFECT OF RACTOPAMINE-HCl AND TR E NBOLONE ACETATE ON LONGISSIMUS MUSCLE FIBER AREA, DIAMETER, AND SATELLITE CELL NUMBERS IN CULL BEEF COWS *Used by permission of the Journal of Animal Science 2007, volume 85, pages 18931901. Introduction Ractopam ine HCl ( RAC ), a beta-agonist with establishe d nutrient partitio ning capabilities, promotes skeletal muscle accretion at the expe nse of fat deposition (for review see Mersmann, 1998). Many studies exist detailing the posit ive effects of RAC on swine performance and carcass composition (Dunshea et al ., 1993, 1998; Sainz et al., 1993). At the cellular level in pigs, beta-agonists increase the size of type IIB mu scle fibers. Cross-sectional area and fiber diameters were larger in pigs fed RAC (Aalhus et al., 1992) and an increase in the relative amount of myosin IIB was apparent (Depreux et al., 2002). A second effective means of altering feed efficiency and carcass composition is the use of steroid implants. In growing steers, treatment with Revalor-S (trenbolone acetate + estradiol; TBA) resulted in an increase in type IIB fibers without a change in th e size or number of type I muscle cells (Fritsche et al., 2000). Satellite cells, or muscle stem cells, are res ponsible for postnatal muscle fiber growth and repair (Mauro, 1961; Schultz et al., 1978). Th is typically quiescent population of cells lies adjacent to the muscle fiber under th e basal lamina and is identified in vivo by their expression of Pax7. Pax7 is a member of the paired-box family of transcriptional medi ators and is implicated in the establishment of the satellite cell lineage (Seale et al., 2000). Mice devoid of Pax7 exhibit severe muscle size and functional deficits that ar e due to an absence of satellite cells (Mansouri et al., 1996, Seale et al., 2000). The animals die within 3 to 4 wk of age. Pax7 does not alter

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63 proliferation rates but does inhibit satellite cell differentiation and apoptosis (Olguin and Olwin, 2004; Relaix et al., 2006; Zammitt et al., 2006). Due to limited information regarding aged bovine muscle fiber size, satellite cell numbers and growth capabilities, we examined LM fi ber morphometrics and myonuclei numbers in cull cows fed RAC and TBA. Materials and Methods Animals and Diets This experiment was approved by the University of Florida Institutional Animal Care and Use Committee. Ninety-two crossbred beef co ws (11 yr 1.8) culled from a commercial cowcalf operation in south Florida (Lykes Bros., Okeechobee, FL) we re shipped by two different truck loads to a feeding facility near Gainesville, FL on the same day. Upon arrival, the cattle were weighed and the general h ealth was evaluated. Cows we re given individual ID tags, dewormed with a generic anthel mintic (Agri Laboratories, Lt d., St. Joseph, MO), and tail switches were trimmed. Cows were blocked by BW on arrival into two replicates (heavy and light) and randomly assigned to treatments accordi ng to a 2 x 2 factorial arrangement. At the beginning of the study light cows weighed 370 kg and heavy cows weighed 418 kg. Cows were fed in four pens with implant status and dietary treatment as the main effects. All diets were fed ad libitum in self feeders. The BCS of all cows on arrival was uniform (4.3 0.03; Carter et al., 2006). One half of the cows in each pen were implanted with Revalor-IS (80 mg trenbolone acetate plus 16 mg estradiol; Intervet, Millsboro, DE), while the remainder received no implant. The basal diet was fed to one half of the cows (two pens) for the duration of the 92 d on feed. The remaining one half (two pens) were fed the same basal diet from d 0 to 55. On d 56, a pelleted supplement containing RAC was added to the basal diet, delivered to empty self-feeders, and fed ad libitum for the re maining 35 d on feed. The basal diet consisted

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64 of (DM basis) soybean hulls (21.1%), citrus pulp (19.7%), cracked corn (14.4%), wheat middlings (14.2%), cottonseed hulls (12.7%), co ttonseed meal (7.0%), liquid molasses (7.0%), vitamins and minerals (included sodium bicarbonat e) (2.1%), tallow (1.3 %), and urea (0.4%). The diet provided 87.6% DM, 14% crude protein (DM basis) a nd 79.5% TDN and was formulated to meet the nutrient requireme nts of a non-pregnant, non-lactating, beef cow predicted to gain 2.06 kg/d. The pelleted, prem ixed supplement (Type B premix) consisted of wheat middlings (97.6%) and ractopamine HCl (2.4%) (Optaflexx 45; Elanco Animal Health, Greenfield, IN) and was formulated to provide approximately 15 ppm when combined at the proper rate in our basal diet depending on DMI (projected to be approximately 13.6 kghd-1d-1). This study was designed to mimic feedyard st andard housing and management practices. Individual animal intake was not monitored and it is acknowledged that the implanted cows may have had a greater intake. Thus, the projected range of RAC was betwee n 15 ppm and 16.5 ppm. The type B premix with RAC was randomly sample d and analyzed for the concentration of the experimental compound before blending and feedi ng to ensure accurate delivery of the formula at the prescribed rate. Analytical results in dicated that the B-premix contained on average 2.15 g/kg of RAC (As-fed basis) a nd would adequately provide th e target level of RAC. The basal diet also included an ionophore [Rumensin 80 (monensin granulated); Elanco Animal Health, Greenfield, IN] form ulated at the rate of 22 mg/kg of feed. Feed samples were collected randomly over the feeding period and analyzed for monensin concentration, which averaged 22.22 mg/kg. Harvesting and Sample Collection On d 92, cows were harvested in a commercial slaughter facility located in Center Hill, FL under USDA inspection. Preharvest BW were 502 kg for light cows and 522 kg for the heavy replicate. Following a 48 h chill period and carcass data collection, 10 wholesale ribs were

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65 randomly selected from each treatment group (n = 40). The sixth rib steak from each wholesale rib section was removed. Two 1 cm x 1 cm x 1 cm portions of the sixth rib LD were suspended in OCT tissue freezing medium (Fisher Scientific, Hampton, NH), frozen by submersion in super-cooled isopentane, and stored at -80C. Immunohistochemistry Three serial cryosections (12 m), one for each fiber isoform, were collected on frost resistant slides (Fisher Scientific, Hampton, NH) for each LD sample. Two sets of serial cryosections were collected for each animal a nd the protocol of Watson et al. (2003) was followed with modifications. Non-specific antigen sites were blocked in 5% horse serum in PBS for 20 min at room temperature. Cryosections we re incubated for 60 min at room temperature in primary antibodies. Antibodies and dilutions were: -dystrophin (Abcam, Cambridge, MA) 1:50; undiluted supernatant myosin heavy ch ain type 1 (BAD.5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa Cit y, IA); myosin heavy chain type 2A (SC.71, Developmental Studies Hybridoma Bank); myosin heavy chain type 2B (BF.F3, Developmental Studies Hybridoma Bank); Pax7 (Developmental Studies Hybridoma Bank) 1:5 cultured supernatant. The myosin heavy chain antibodies were directed toward bovine skeletal muscle myosin (Schiaffino et al., 1989). After extensive washing with PBS, tissues were incubated for 40 minutes with goat antimouse Alexa Flour 568 (1:500; Invitrogen, San Diego, CA) for -dystrophin or goat anti-mouse biotin (1:100; Vector Laboratories, Burlinga me, CA) followed by steptavidin Alexa Flour 488 (1:500; Invitrogen) for Pax7 and myosin heavy chain isoform detection. Following Pax7 immunostaining, Hoechst 33245 (1 g/ml in PBS) was used to identify total nuclei (Example

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66 microphotograph in Appendix A). Fiber associated nuclei ( FAN ) were visualized with propidium iodide (1 g/ml; Invitrogen ; Example microphotograph in Appendix B). After a final PBS wash, slides were cover slipped and immunostaining was evaluated using a Eclipse TE 2000-U stage microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120 epifluorescence illumination system (EXFO, Mississauga, Ontario, Canada). Images were captured at 100X magnification using a DXM 1200F digital camera (Nikon) and analyzed for individual muscle fiber area and diameter and total number of fibe r associated nuclei per field using the NIS-Elements computer system (Nikon). For each set of serial cryosections, 4 images from the same area of each cryosection was co llected for each myosin heavy chain isoform (Figure 3-1). Fibers that were reactive with the antibody to the specific myosin heavy chain isoform were counted, and fiber area was defined as the region constrained by -dystrophin immunostaining. Diameter was measured by th e computer system rotating every 90 degrees around the fiber, taking a diameter measurement, and averaging the measurements. For each animal, a minimum of 475 fibers were measured and used for analysis. Fiber associated nuclei was defined as propidium iodide stained cells contained within a -dystrophin boundary. Nuclei that were identified with Hoechst dye labeled as Pax7 positive were counted as being a satellite cell. To ensure collecting samples 48 h postmortem did not have an effect on satellite cell detection, a validation study was conducted. Satell ite cell numbers were measured in 3 LD immediately after slaughter and at 24, 48 and 72 h of chill. No differences in Pax7 immunopositive cells were observed (Figure 3-2). Statistics The study was designed as a randomized comple te block design with individual carcasses of the 4 different feeding regime ns as the experimental unit (Mat ulis et al., 1987; Cranwell et al.,

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67 1996b; Schnell et al., 1997). Fiber frequencies we re tabulated and compared by chi-square analysis using PROC FREQ of SAS (SAS Inst. Inc., Ca ry, NC, 2002). Treatment group frequencies within a fiber type were compared to one another by a two sample t-test for proportions. Data for fiber area a nd diameter were sorted and anal yzed by individual fiber type, while fiber-associated nuclei a nd Pax7 nuclei were not sorted. Data were analyzed with the PROC MIXED procedure of SAS where implant st atus, dietary treatment and their interaction were the fixed effects. Random effects included BW replicate, truck load, and animal within treatment. Each combination of BW replicate and truck load were grouped and used in the random statement. Pair-wise comparisons between the least square means of the factor levels were computed by using the PDIFF option of the LSMEANS statement. The PROC UNIVARIATE procedure of SAS was used to generate histograms and analyze distributions of fiber diameter and area within each treatment group for each fiber type. Results Muscle fiber types were measured using anti bodies specific to myosin heavy chain type I, IIA and IIB isoforms. No t ype IIB immunoreactivity was observed suggesting that LM was comprised solely of type I and IIA fibers (Figur e 3-1). Muscle fiber diameters were measured following co-localization of myosin and dystrophin. There was a significant ( P < 0.01) RAC x TBA interaction for type I fibers. The main effects of TBA and RAC increased ( P < 0.05) the diameter and computed cross-sectional area ( CSA ) of type I fibers (Table 3-1). Feeding RAC to TBA implanted cull cows did not further increase ( P > 0.05) the LM fiber di ameter or CSA. No change ( P > 0.05) in the CSA or diameters of type II fibers occurred in response to TBA, RAC or TBA + RAC. No differences ( P > 0.05) in the percentage of t ype I and type II fibers were observed across the CON, RAC, and TBA treatments (Table 3-1). There was an increase ( P < 0.001) in the percentage of type II fibers in the LM of cull cows treated with RAC/TBA.

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68 Size-frequency histograms of CSA were generated for type I and type II LM fibers. Trenbolone acetate and TBA + RAC caused a shift in numbers of type I fibers with larger CSA than CON (Figure 3-3). Curve of best fit for type II muscle fiber histograms do not differ ( P = 0.18) between the groups (Figure 3-4). The numbers of nuclei contained within the dystrophin boundary were measured as an index of hypertrophy. Fiber associated nuclei were not affected ( P > 0.05) by any of the treatments given (Table 3-2). Satellite cell number, as measured by anti-Pax7 at 48 h postmortem, did not change ( P > 0.05) in response to any treatment (Table 3-2). Discussion Growing pigs fed RAC dem onstrate an increase in loin eye area that is a result of increased type II fiber size. Coincident w ith the larger fiber diameter and CSA is a shift from the fast oxidative (type IIA) to the fast glycolytic (type IIB) metabolic phenotype (Aalhus et al., 1992). A two-fold increase in the numbers of type IIB fi bers, at the expense of the type IIA fibers, was recorded in RAC supplemented hogs (Depreux et al., 2002). In the present study, neither type IIB nor type IIX muscle fibers were detected by immunocytochemistry. The population of type IIA muscle fibers remained constant at approxim ately 68% of the total fibers in cattle fed RAC or implanted with TBA. A greater percentage of type IIA fibers at the ex pense of type I fibers was observed in TBA implanted cows fed RAC. This result is intriguing in light of our inability to detect a change in the proportion of type II fi bers in cows fed RAC only. In cattle and mice, it is well established that beta agonists increase the percentage of type IIA fi bers at the expense of type I fibers (Vestergaard et al ., 1994; Rajab et al., 2000; Bricout et al., 2004). By contrast, anabolic agents have no effect on the distribution of type I or type IIA fibers (Ono et al., 1996; Fritsche et al., 2000). The underlying mechanism of action of RAC/TBA that elicits a change in myosin isoforms remains unknown and warrants further investigation.

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69 The type IIA antibody used in th is study reportedly cr oss reacts with myosin IIX and IIB in cattle (Duris et al., 2000) and type IIX fibers in swine (Depreux et al., 2000). Based on the assumption of that all type II fibers are immunoreactive, 60% of the LM fibers in cull cows are classified as fast, a value in agreement with prior publications. Usi ng enzymatic techniques, Brandstetter et al. (1998) identified the percentage of t ype I, type IIA and type IIB fibers as 25%, 25% and 50%, respectively, in the LM. Fritsche et al. (2000) reported a si milar percentage with 20 to 30% of fibers classified as oxidative and ap proximately 55% of the total fibers present as fast glycolytic fibers. In a similar manner, th e inability to detect type IIB fibers may be a limitation of the immunodetection method. Watson et al. (2003) failed to de tect type IIB fibers in harbor seals using the same antibody. Duris et al. (2000) re ported this antibody demonstrates a low specificity for type II myosin heavy chain in bovine tissues and indicated that anti-myosin IIB (N3.36) is a suitable alternative. However, monoclonal N 3.36 failed to detect type IIB fibers in the LM of cull cows fed RAC. Our inability to detect myosin type IIB fibers agrees with Tanabe et al. (1998) and Toniol o et al. (2005), who were unable to establish th e presence of myosin IIB in bovine LM by semi-quantitative RT -PCR. Chikuni et al. (2004) also reported a lack of myosin IIB mRNA as measured by real-time PCR, and hypothesized the absence of this isoform may explain the differences between beef and pork. Thus, the inability of RAC to shift myosin expression to the fastest isoform may be uni que to cattle. Alterna tively, fiber type shifts in response to RAC may occur only in muscle that normally expresses limited amounts of myosin IIB. Quartile analysis of all type II fibers demonstrates no apparent difference in the percentage of fibers present with a larger diameter in RAC cull cows by comparison with CON suggesting that the response of IIA fibers was minimal. Indeed, CON animals appear to have the greatest

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70 percentage of large diameter fibers that may repr esent type IIB. The unresponsive nature of type II fibers of cull cows to RAC in the present stud y may reflect the muscle environment. Finishing hogs fed normal or supraphysiological levels of RAC contained an equivalent amount of myosin IIA in the LM as control animals. By contrast, the semitendinosus muscle contained less myosin IIA in response to RAC (Depreux et al., 2002). Alternatively, the reside nt type II population may be refractile to growth enhancing agents due to the advanced age of the animal. The differential response of bovine and porcine type II muscle fibers to the beta agonist is intriguing and warrants further investigation. A substantial increase in the size of type I fibers was evident in cows treated with RAC or TBA. Administration of TBA to cull cows increased LM area and carcass fat-free lean (Cranwell et al., 1996a). In a si milar manner, implantation of fe edlot steers with TBA increased type I and IIA CSA in the LM (H ughes et al., 1998). Thus, the larg er diameter type I fibers in cull cows receiving TBA reflects previous reports. Conversely, an increase in LM type I CSA by RAC supplementation is novel. No change in the si ze of type I fibers is apparent in finishing hogs supplemented with RAC (Aalhus et al., 1992) Longissimus muscle type I fibers from lambs fed cimaterol showed no change to a modest 15 percent increase in fiber CSA (Beermann et al., 1987; Kim et al., 1987). The thirty pe rcent increase in type I CSA found in RAC supplemented cows suggests that this population of cells are two to three times more responsive in cattle than other species. Further support for specie differences is reflected by a 35 percent larger type I CSA in bulls fed cimaterol (Veste rgaard et al., 1994). However, cull cows are unique in that their type II fi ber population is completely refractile to RAC induced hypertrophy whereas cimaterol stimulated hypertrophy in bulls (Vestergaard et al., 1994). The mechanism behind the differential response in cattle may be a reflection of the age of the animal. During

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71 aging in humans, the numbers of type IIA/X muscle fibers and size are reduced (Lee et al., 2006; Verdijk et al., 2007). Type I fiber CSA remains unchanged largely but the percentage of type I fibers are increased. In addition, the ability of the muscle to respond to hypertrophic events is altered in extreme age (for revi ew see Carmeli et al., 2002). Adva nced age in rodents, birds and humans demonstrate an impaired ability to increase in size that is associated with reduced type II muscle fiber numbers (Carson et al., 1995; Blough a nd Linderman, 2000; Shor t et al., 2005; Lee et al, 2006). The percentage of type I and II fi bers is established by 24 mo of age in cattle, irrespective of bree d, and the LM is comprised predominantl y of type I and IIB (Wegner et al., 2000; Kirchofer, et al., 2002). In the event that t ype II fibers are limited in their protein synthetic capacity and are intolerant to hype rtrophic stimuli, the nutrients supplied by diet coupled with the partitioning agents leads to an excess of available substrate for type I fiber growth. One of the primary reasons for supplementing livestock with RAC or TBA is to improve carcass value. Schroeder et al ( 2005b,f) reported an increase in ribe ye area and fat-free lean in steers and heifers fed RAC. Therefore, based on the findings above we predicted that cull cows receiving 15 ppm RAC daily for 35 d would posse ss a larger REA. However, no improvement in carcass characteristic s, including REA, due to RAC or TBA supplementation were detected (Carter et al., 2006). This may be a reflecti on of an unresponsive ty pe II fiber population. Approximately 30% of the total number of fibers was present as type I. Based on the 30% increase in type I size found in young bulls (V estergaard et al., 1994), we would predict a minimal 9% increase in REA. This small change may require more animals to reach statistical significance. Postnatal skeletal muscle growth is acco mplished through the sate llite cell population. These normally quiescent muscle stem cells become mitotically active, proliferate and fuse into

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72 existing muscle fibers (for review see Collins, 200 6). The number of satellite cells declines with age and the activation potential of these cells are reduced in older i ndividuals (Collins and Partridge, 2005). In aged rats and elderly humans, satellite cells represen t one to two percent of the total myonuclei (Gallegly et al., 2004; Sajko et al., 2004; Brack et al., 2005). The number of Pax7 immunopositive satellite cells in cull cows represents approximately 1% of the total number of myonuclei, in close ag reement with rodent data. Sa tellite cells isolated from TBA implanted steers exit the dormant state sooner than their cont emporaries suggesting that the anabolic steroid affects self-rene wal and subsequent proliferation. In addition, these cells fused into larger muscle fibers in vitro (Johnson et al., 1998). The enhan ced myogenic capabilities may account for the larger fiber sizes found in TBA implanted cull cows Alternatively, TBA increased circulating levels of IGF-I and autocrin e synthesis of the growth factor (Thompson et al., 1989; White et al., 2003; Kamang a-Sollo, et al., 2004). It is possible that the increased type I fiber diameters and calculated CSA are a product of elevated IGF-1 activities. Satellite cells proliferate, differentiate and fu se with the muscle fibers to provide FAN for increased contractile gene expression and mainte nance of the myonuclear domain (Aberle et al., 2001). The lack of an increase of FAN found in all the supplemented cows indicates that the two growth promotants may act through a similar pathway of altered prot ein synthesis and/or degradation rates. It was repor ted that RAC stimulates protein synthesis without an apparent effect on satellite cell cycle ki netics or fusion (Shappell et al., 2000). Beta-agonists may also augment nutrient supply to the muscle cell by in creasing blood flow. Muscle accretion is supported and possibly bolstered by the enhanced delivery of substrates and energy needed for protein synthesis (Mersmann, 1998). In several swine and cattle studie s, the mechanism of muscle growth due to ractopamine supplementation was attributed to enha nced protein synthesis

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73 (Smith et al., 1987; Dunshea et al., 1993, 1998; Williams et al., 1994). Beermann et al. (1987) and Kim et al. (1987) reported th at DNA concentration per gram of protein was less in betaagonist supplemented lambs and both groups co ncluded that muscle growth was due to a reduction in protein degradation and independent of satellite cell activity. A similar observation was reported for rats (Maltin et al., 1986) and lambs (Bohorov et al., 1987) fed clenbuterol. The discrepancies between increased protein synthe sis or reduced degradation rates may be a reflection of the beta agonist fed a nd the beta-receptor isoform activated. Conclusion Supplementing cull cows with either RAC or TBA alone, or the combination increased LM fiber CSA and diameter for type I fibers, while having no effect on type II fibers. There were no fiber type shifts between the different myosin heavy chain isoforms due to the presence of TBA or RAC. Fiber associated or satellite cell numbers were not affected by the RAC or TBA treatments. The lack of affect on FAN and c onstant satellite cell numbers suggests that any hypertrophy occurred due to changes in protein synthesis and/or degrad ation rates. It is hypothesized that because protein synthesis is limited in older animals, this prevented the response to both TBA and RAC normally seen in younger animals.

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74Table 3-1. Longissimus muscle type I and t ype II fiber percentage and least square mean s of fiber cross-sectio nal area and diameter from cull cows fed four different feeding regimens Treatment Type I Type II Percentage1 Area (m2) Diameter (m) Percentage1Area (m2) Diameter (m) CON2 32.35a 2432.97 73.56a 54.61 0.73a67.65a5087.12 261.30 79.70 1.56 RAC3 31.54a 3191.89 99.18 b 62.62 0.98 b 68.46a3626.91 253.48 66.55 1.52 TBA4 32.35a 3678.72 190.40c 66.89 .78c 67.65a4690.68 446.62 75.23 3.60 RAC/TBA5 28.78 b 3615.42 96.36c 66.58 0.93c 71.22 b 4830.49 241.12 76.13 1.50 Means within a column with a differe nt letter are significantly different ( P < 0.05). 1Percent fiber type frequency detected by monoclonal antibody immunohistochemistry. 2 Cull cows fed the control diet (n = 10). 3Cull cows fed the control diet plus Opta flexx (15 ppm ractopamine-HCl) supplement during the last 35 days on feed (n = 10). 4Cull cows implanted with Revalor-IS (80 mg trenbolone acetate plus 16 mg estradiol) and fed the control diet(n = 10). 5Cull cows implanted with Revalor-IS, fed the control diet pl us Optaflexx supplement during the last 35 days on feed (n = 10).

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75 Table 3-2. Least squares means of LM fiber associated nuclei per fiber1, and satellite cells per hundred fibers2 of cull cows fed four different feeding regimens Treatment Fiber Associated Nuclei Satellite Cells CON3 4.69 0.75 4.63 0.91 RAC4 4.62 0.77 3.20 0.88 TBA5 5.16 0.71 5.89 0.70 RAC/TBA6 4.97 0.76 4.24 0.64 1Total nuclei counted in a field divided by number of fibers counted per field (field equals 381 mm2). 2 Total satellite cells counted in a field divided by number of fibers counted per field mu ltiplied by 100(field equals 381 mm2). 3Cull cows fed the control diet (n = 10). 4Cull cows fed the control diet plus Opta flexx (15 ppm ractopamine-HCl) supplement during the last 35 days on feed (n = 10). 5Cull cows implanted with Revalor-IS (80 mg trenbolone acetate plus 16 mg estradiol) and fed the control diet(n = 10). 6Cull cows implanted with Revalor-IS, fed the control diet pl us Optaflexx supplement during the last 35 days on feed (n = 10).

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76 Figure 3-1. Representative photomicrographs of LM fibers immunostained for type I and type II fibers from (CON) control diet fed cows, (RAC) control diet + RAC fed cows, (TBA) control diet + impl ant fed cows, and (RAC/TBA) control diet + RAC + implant fed cows. Scale bar equals 100 m.

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77 Figure 3-2. Number of Pax7 positive nuclei counted per field during a 72 h period postmortem. Samples were taken at 0, 24, 48, and 72 hours postmortem and subjected to the Pax7 staining protocol us ed in the present study. Area of field equals 41.5 mm2. 0 h 72 h 48 h 24 h 0.00 0.50 1.00 1.50 2.00 Time, h Pax7 Positive Nuclei/Field

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78 Figure 3-3. Histograms of LM fiber cross-sectiona l areas of all type I fibers sampled from (Control) control diet fed cows, (RAC) control diet + RAC fed cows, (TBA) control diet + implant fed cows, a nd (RAC/TBA) control diet + RAC + implant fed cows.

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79 Figure 3-4. Histograms of LM fiber cross-sectional areas of all type IIA fibers sampled from (Control) control diet fed cows, (RAC) control diet + RAC fed cows, (TBA) control diet + implant fed cows and (RAC/TBA) control diet + RAC + implant fed cows.

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80 CHAPTER 4 DIFFERENTIAL RESPONSE OF CULL COW MUSCLES TO THE HYPERTROPHIC ACTIONS OF RACTOPAMINE-HCl *Used by permission of the Journ al of Animal Science 2008, doi:10.2527/jas.20081049. Introduction Ractopam ine-HCl (RAC ) is a beta-adrenergic agonist ( AR ) approved for use in beef cattle in the United States. Be ta-adrenergic agonists improve feedlot performance in growing beef steers and heifers as evidenced by increased average daily gain and feed efficiency (Avendano-Reyes et al., 2006; Walk er et al., 2006). However, ca ttle fed RAC demonstrate only modest improvements in carcass traits (Grube r et al., 2007). Heavie r hot carcass weights ( HCW) with no changes in loin eye area ( LEA), yield grade or measures of fat deposition were observed in steers fed RAC (Winter holler et al., 2007). Others repo rt an increase in both HCW and LEA in RAC-fed steers (Grube r et al., 2007). A similar temp ered response is evident in heifers. Ractopamine augmented heifer feed lot performance measures, HCW, LEA and yield grade (Sissom et al., 2007). By contrast, Walker (2006) found no improvement in HCW, LEA or yield grade in heifers receiving RAC. The disparity in RAC effects in fed cattle remains unresolved. Individual tissue responses to RAC are a consequence of AR isoform expression and numbers. Swine adipocytes express an equal percentage of 1 and 2 ARs while muscle fibers express predominately the 2 AR (Liang and Mills, 2002; Sillen ce et al., 2005; Spurlock et al., 1994). Interestingly, AR density is reduced in backfat depots in pigs fed RAC but not in skeletal muscle (Spurlock et al., 1994). Down-regulation of AR may account for the loss of a lipolytic effect over time of RAC feeding. In cattle, transc ripts for all three AR isoforms are

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81 present in skeletal muscle (Walker et al., 2007) RAC supplementation to steers and heifers causes a reduction in 2AR mRNA with no effect on either 1 or 3-AR expression (Sissom et al., 2007; Winterholler et al., 2007). The ability of RAC supplementation to down-regulate muscle ARs in cull-cows is unknown. Du e to the subtle improvements in lean deposition in cattle, a more thorough investigation of RAC effects on muscles of vary ing fiber type composition is required. The objective of this st udy was to examine the effects of varying concentrations of RAC on fiber hypertrophy and AR gene expression from musc les located in the foreand hindquarter of cull beef cows. Materials and Methods Animals and Diets Eighty eight Beefm aster and Angus-type cull co ws were stratified by breed and weight to one of four RAC supplementation treatments. Cows consumed a concentrate diet (Table 4-1) ad libitum for 54 days prior to harvest. Ractopamine-HCl (0, 100, 200, and 300 mghd-1d-1) was supplemented during the final 28 days on feed as a pelleted Type B premix that consisted of wheat middlings (97.6%) and ractopamine-HCl (2.4%) (Optaflexx 45; Elanco Animal Health, Greenfield, IN). The appropriate concentrati on of RAC of each treatment group premix was formulated based on a projected DMI of approximately 13.6 kghd-1d-1. The initial body weights of the four treatment groups were 426.3 kg, 436.9 kg, 418.8 kg and 439.0 kg for the 0, 100, 200, and 300 groups, respectively. At the end of the feeding portion cows weighted 490.0 kg, 483.1 kg, 466.7 kg, and 497.8 kg for the 0, 100, 200, and 300 groups, respectively. Harvesting and Sample Collection Cows were slaughtered at a comm ercial USDA-inspected facility. Within 60 minutes of exsanguination, portions of the longissimus ( LM ) and semimembranosus ( SM ) muscles from

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82 four randomly selected animals pe r group (n = 16) were collected and frozen in liquid nitrogen for RNA extraction. Twenty-four hours pos tmortem, whole muscles of the LM, SM, Infraspinatus ( INF), and Vastus lateralis ( VL) were transported to the University of Florida Meats Laboratory. Two 1 cm3 portions of each muscle from 10 randomly selected cows per group (n = 40) were suspended in OCT tissue freezing medium (Fisher Scientific, Hampton, NH), frozen by submersion in super-cooled isopentane, and stored at -80C. Immunohistochemistry The m ethods used by Gonzalez et al. (2007) were followed for immunohistochemical staining. Briefly, two 12 micromet er serial cryosections were colle cted on frost resistant slides (Fisher Scientific, Hampton, NH). Non-specific antigen sites were blocked with 5% horse serum in phosphate buffered saline ( PBS). Cryosections were incubated in primary antibodies for 60 minutes at room temperature. Primary antibodies and dilutions were -dystrophin (Abcam, Cambridge, MA) 1:50, myosin heavy chain type I (BAD.5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) hybridom a supernatant, and myosin heavy chain type IIA (SC.71, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) hybridoma supernatant. After washing with PBS, tissues were incubated in secondary antibodies for 45 minutes at room temperature. Labeled s econdary antibodies incl uded rabbit anti-mouse AlexaFluor 568 (Invitrogen, San Diego, CA) for -dystrophin detection and goat anti-rat biotin (Vector Laboratories, Burlingame, CA ) followed by steptavidin AlexaFluor 488 (Invitrogen, San Diego, CA) for myosin heavy chain isoform detection. Hoechst 33245 was used to detect nuclei. After a final PBS wash, slides were co ver-slipped and fluorescence was visualized using an Eclipse TE 2000-U microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120 epifluorescence illumination system (EXFO, Mississauga, Ontario, Canada). Images were captured using a DXM 1200F digital camera (N ikon, Lewisville, TX) and analyzed for

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83 individual muscle fiber area a nd diameter using the NIS-Elemen ts software (Nikon, Lewisville, TX). For each animal, a minimum of 1,000 fibers were measured and analyzed. The region constrained by -dystrophin immunostaining defined individual fibers for cross-sectional area ( CSA) and diameter measurement. Fiber associated nuclei ( FAN ) were identified as Hoechst 33245 labeled nuclei lying adjacent to the -dystrophin border. The num ber of fibers located within each micrograph was counted to dete rmine the number of nuclei per fiber CSA. RNA Extraction and Real -Time PCR Analysis Five hundred m illigrams of muscle was hom ogenized in 10 mL STAT-60 (Tel-Test Inc., Friendswoods, TX) with a mechanical tissue disruptor. Two mL of chloroform was added and the upper aqueous layer contai ning nuclei acids was collect ed by centrifugation. RNA was precipitated by isopropanol and cen trifugation. The nucleic acid pellet was washed with 70% ethanol and air-dried. Pe llets were resuspended in sterile -filtered, double distilled water and further purified using the PureLi nk Micro-to-Midi Total RNA Purification System (Invitrogen, San Diego, CA). Purity of the RNA was eval uated by spectroscopy with all samples exhibiting an OD260:280 greater than 1.9. Integrity of R NA was verified by the presence of intact ribosomal RNA bands following el ectrophoresis through ethidium bromide impregnated agarose gels. Aliquots of RNA we re stored at -80 C. One g of total RNA was treated with RNase-free DNAse (Promega, Madison, WI) to remove trace genomic DNA contamination. Su bsequently, the RNA wa s reverse transcribed with MMLV-Reverse Transcriptase (Ambion, Aus tin, TX) and random hexamers at 42C for 60 minutes. cDNA from 50 ng of RNA was am plified with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the a ppropriate forward and reverse primers (20 pM; Table 4-2) in an ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). Thermal cycling parameters included a denature step of 95C for 10 minutes and 40 cycles of

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84 15s at 95.0C and 1 minute at 55.0C. A final dissociation step included 95C for 15 seconds, 60C for 30 seconds, and 95C for 15 seconds. Fo llowing the procedures of Castellani et al. (2004) serial dilutions of pooled samples were us ed to generated standard curves to ensure generation of Ct values were within the linear range of amplification. Statistics Use of carcass as the experimental unit and data was statistically analyzed similar to Wheeler et al. (1990) and Gonzal ez et al. (2007). Data for gene expression and FAN numbers were analyzed as a split-plot design using the PROC MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, 2002). The whole plot consisted of br eed type, RAC treatment, and whole plot error. The whole plot error consisted of breed type x RAC treatment. The subplot was muscle, breed type x muscle, RAC treatment x mu scle interaction, and the subplot error. The subplot error was comprised of the three-way interaction between breed type, RAC treatment, and muscle. Muscle fiber CSA and diameter data was analyzed using a split-split-plot desi gn. The whole plots and subplots were the same as the split-plot analysis. The sub-subplot consisted of muscle fiber type and the remaining interactions. The four factor interaction betw een breed type, RAC treatment, muscle, and fiber type were used as the sub-s ubplot error. Pair-wise comparisons between the least square means of the factor levels we re computed by using the PDIFF option of the LSMEANS statement. Results Cryosections from each of the four muscles were immunostained for MyHC type I and IIA isoforms. Morphometrics for type I and II fibers from the LD, VL, SM and INF were measured (Table 4-3). Ractopamine fed at a rate of 100 or 300 mghd-1d-1 (RAC-100 and RAC300, respectively) altered ( P < 0.05) the fiber type composition in all muscles examined. Ractopamine fed at 200 mghd-1d-1 (RAC-200) caused an increase ( P < 0.05) in the percentage

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85 of type IIA fibers in the LD, SM and VL. By comparison to the LD, the SM and VL had the largest fiber type distribu tion shifts. By contrast a shift toward more ( P < 0.05) type I fibers in the INF was measured in cull cows fed RA C-200 and 300. RAC-200 tended to increase ( P = 0.14) the diameter and CSA of the type I fibers within the VL and significantly increase ( P = 0.05) type IIA fibers within the SM. An increase (P < 0.05) in the dimensions of both type I and IIA fibers were observed within the INF of cull cows fed RAC-100; no changes ( P > 0.05) in fiber CSA or diameter were obser ved within the LD, SM or VL. As reported previously, RAC-200 does not increase the calculated LD myonuclear domain (Gonzalez et al., 2007). Fewer ( P < 0.05) myonuclei per fiber we re observed in the LD and VL of cull cows fed RAC-200; no changes ( P > 0.05) were found in the INF or SM (Table 4-4). Cows fed RAC100 contained fewer ( P < 0.05) FAN in the INF, LD and VL than controls. RAC-300 cull cows contained fibers with fewer FAN within the LD and VL. Semi-quantitative RT-PCR indicated that RAC supplementation at any level did not change ( P > 0.05) the amount of detectable 2-AR, MyHC type I, IIA or IIX mRNA in the LD (Table 4-5). Compared to controls, RAC decreased ( P < 0.05) 2-AR, MyHC type I and IIX mRNA content in the SM when fed at 100 mghd-1d-1 (Table 4-5). RAC supplementation at 200 mghd-1d-1 tended to increase both 2-AR and MyHC IIX mRNA abundance in the SM. Discussion RAC supplem entation to growing cattle offe rs an advantageous improvement in performance parameters including increased averag e daily gains and lower feed to gain ratios (Walker et al., 2007). However, these perfor mance measures translate into only modest improvements in carcass traits. The tempered responses to RAC are further confounded by sex. Heifers fed RAC (200 mghd-1d-1) require less feed per unit of we ight gain but do not differ from

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86 control heifers with regard to ADG, DMI or car cass parameters (Sissom et al., 2007; Walker et al., 2006). In a similar manner, non-impla nted heifers fed both 200 and 300 mghd-1d-1 of RAC during the final 28 days of a 42 day feeding tr ial demonstrated no improvements in live performance parameters or carcass weight (Quinn et al., 2008). The beef cull cows fed RAC in the current study demonstrated no improvements in either feedlot pe rformance (Carter and Johnson, 2007) or carcass traits (Dijkhuis et al., 2008). Thus the lack of an improved performance response is not due to the age of our animals but may be related to animal gender. Ractopamine concentration was sufficient to cause a biological response in cull cows as evidenced by an increase in muscle fiber CSA (Gonzalez et al., 2007). Ho wever, larger muscle fiber CSA stimulated by RAC fed at the manufacturers recommended dose (200 mghd-1d-1) does not translate into larger REA (Carter et al., 2006). The pres ent study addressed the possibility that a higher concentration of RAC may be required to elicit a global improvement on muscle cell size in aged cows. Ractopami ne supplementation at a rate of 300 mghd-1d-1, a concentration within the recomm ended feeding guideline, provoke d a response no different than the conventional 200 mghd-1d-1 feeding rate. The measured CSA of LD type I and II fibers did not differ between controls, RAC-100 or RAC-200. The shift in fiber type in RAC treated animals indicates that the feed additive is bioac tive. Assuming an equivalent rate of absorption and cellular delivery, no change in fiber morpho metrics would suggest that the limitation to further increases in muscle growth are not a c onsequence of insufficient RAC concentration. The lack of a robust increase in fiber size is not likely attr ibutable to low numbers of AR. 2AR is the preferred receptor for RAC in swine ti ssues (Mills et al., 2003) and the receptor is present in skeletal muscles of cattle (Bridge et al., 1998). Our results indicate that the LD and SM transcribe the 2 adrenergic receptor gene and transc ript abundance is not diminished in

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87 response to RAC. Indeed, SM 2AR mRNA levels tend to be increased by RAC-200, as reported by others (Sissom et al ., 2007; Winterholler et al., 2007). Because receptor protein expression was not determined in any of these st udies, it remains possible that female cattle are refractile to the positive e ffects of RAC at the cellular level due to low abundance AR numbers and/or deficits in the sign al transduction system. As reported in swine (Depreux et al., 2002; Guna wan et al., 2007), RAC initiated a shift in fiber types from slow to fast. A reduction in the pe rcentage of type I slow fibers is evident in the LD, SM and VL with the relative decline betw een muscles variable. In the LD, RAC-100 is sufficient to elicit a reduction in the percentage of type I fibers with no further decline with increased RAC consumption. By contrast, the SM and VL are highly vari able in their response to RAC. In both muscles, RAC-200 elicited the greatest effect with approximately 30% of the type I fibers transitioning to a fa st isoform. In the SM, the increased number of type II fibers is associated with greater CSA and a tendency toward higher amounts of MyHC-IIX mRNA. Further increases in RAC did not exacerbate th e shift but caused a dampened response. The slight increase in type I fibers in the SM and VL of cows receiving RAC-300 versus RAC-200 may indicate a down-regulation of key mediator(s ) of RAC effects. The identity of the intracellular mediators of RAC signals in bovine muscle fibers is unknown at this time. The divergence in responses to RAC amongst mu scles is further exemplified by fiber type shifts in the INF. The INF, a forelimb muscle, is nearly a 50:50 mix of slow and fast fibers. RAC-100 caused a reduction in the numbers of oxidative type I fibers, as expected. However, RAC-200 behaved in the opposite manner with a significant increase in the numbers of slow fibers. RAC-300 did not differ from RAC-200 in these measured parameters. Feeding RAC-100 also caused a 65-70% increase in the size of the INF muscle fiber independent of metabolic

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88 enzyme classification. The unexpected shift from fa st to slow fiber types also was noted in the SM of cows fed RAC-100 without a change in CSA. Interestingly, the larger SM fiber CSA in RAC-100 cows is associated w ith a reduction in MyHC gene expression. The dichotomy of larger fibers with less MyHC mRNA suggests that the increased size may reflect a reduction in protein degradation or prolonged half-life of the contractile protein. At the very least, these results underscore the complexity of RAC effects in cattle. The results presented in this study support our previous work de monstrating that fiber size increases without an increase in myonuclei numbers (Gonzalez et al., 2007). Each fiber contains hundreds of myonuclei, and the ratio of nuclei/cyt oplasm, or myonuclear domain, must remain constant (Aberle et al., 2001). OConnor and Pavlath (2007) described muscle fiber growth as having an initial phase characterized by enhanc ed transcription and translation, leading to increased protein accretion and a small expansion of the myonuclear domain. Following this initial growth, fusion of satellite cells must occur to combat the threshold or ceiling established by myonuclear domain and facilitate s additional increases in fiber CSA. However, O'Connor et al. (2007) concluded that increase s in nuclear content are not need ed to induce skeletal muscle growth, and strong evidence is provided by the ability of -agonists to promote protein synthesis without the addition of myonuclei. The mechanism underlying the increase in muscle fiber size in response to RAC has been linked to altered protein turnover. In pigs, poultry, lambs, and cattle, numerous -agonists including RAC, cimaterol, and clenbuterol increased skeletal muscle hypertrophy in the absence of increases in either DNA content or myonuclear number (Beermann et al., 1987; Dunshea et al., 1993; Gwartney et al., 1992; Smith et al., 1987). Pigs fed RAC demonstrate an increase in fractional protein synthesis rate s (Dunshea et al., 1993; Dunshea et al., 1998; Williams et al., 1994). To date, dire ct measurement of protein turnover rates in

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89 cattle receiving RAC have not been reported. T hus, in cattle RAC likely stimulates muscle fiber growth by a change in protein synthe sis and/or degradation rates. Conclusion Ractopamine supplementation to cattle causes a biological effect on muscle fiber isoform distribution and size at concentra tions ranging from 100 to 300 mghd-1d-1. Cull beef cows fed RAC-100 responded in a manner similar to conve ntional RAC-200 as measured by a shift in muscle fiber isotypes. Interes tingly, the lower concentration of RAC improved fiber size only in the INF, a muscle characterized by a higher proportion of red fibe rs. Cull cow feeding programs may consider supplementing RAC-100 as a means of adding value to cuts within the chuck, such as the INF.

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90 Table 4-1. Composition of basal diet1Item Amount, percent of DM Soybean hulls 21.1 Citrus pulp 19.7 Cracked corn 14.4 Wheat middlings 14.2 Cottonseed hulls 12.7 Cottonseed meal 7.0 Liquid molasses 7.0 Vitamins and minerals 2.1 Tallow 1.3 Urea 0.4 1 Diet designed to meet the nutrient requi rements of a non-pregnant, non-lactating, beef cow predicted to gain 2.06 kg/d and provide d 87.6% DM, 14% crude protein (DM basis), and 79.5% TDN (Abney et al., 2007). An ionophore [Rumensin 80 (monensin granulated); Elanco Animal H ealth, Greenfield, IN] was also in cluded in the basal diet at the formulated rate of 22 mg/kg of feed.

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91 Table 4-2. Sequence of bovine-specific PCR primers used for determination of the expression of mRNA for 1and 2-adrenergic receptors and myosin heavy chain isoforms Gene of interest Primer 18S Forward 5-GTAACCCGTTGAACCCCATT-3 Reverse 5-CCATCCAATCGGTAGTAGCG-3 2 Forward 5-TCATGTC GCTTATTGTCCTGG-3 Reverse 5-TCATGTC GCTTATTGTCCTGG-3 Myosin Heavy Chain MYO2a 5-ATCCAGGCTGCGTAAC GCTCTTTGAGGTTGTA-3 MYO111 b 5-CACTTGCTAACAAGGACCTCTGAGTTC-3 MYO209c 5-CTTTCCTCATAAAGCTTCAAGTTCTGACC-3 MYO405 d 5-TGCTGCTCTCAGGCCCCTGCCACCTT-3 a Common reverse sequence used for all thr ee myosin heavy chain isoforms (Tanabe 1998). b Forward sequence used for detection of MyHC IIA (Tanabe et al., 1998). c Forward sequence used for detection of MyHC IIX (Tanabe et al., 1998). d Forward sequence used for detectio n of MyHC I (Tanabe et al., 1998).

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92Table 4-3. Least squares means of muscle fiber myosin heavy chain isofor m distribution, cross-sectional ar ea and diameter from four muscles of cull-cows fed three levels of ractopamine-HCl Type I Fiber Type IIA Fiber Muscle1 Percentage Area ( m2) Diameter ( m) Percentage Area ( m2) Diameter ( m) I N F CON 51.1a 4545 384a74 4a48.9a2554 384a56 4a100 45.7c 6432 385 b 88 4 b 54.3c3871 385 b 68 4 b 200 54.3 b 3955 384a 69 4a45.7 b 2617 384a55 4a300 54.8 b 4927 384a 77 4a45.2 b 3100 384a, b 61 4aL D CON 34.4a 2109 385 49 4 65.6a3124 384 60 4 100 32.6 b 2753 57 4 67.4 b 3116 384 61 4 200 32.2 b 2833 385 58 4 67.8 b 3473 384 64 4 300 32.6 b 2090 385 50 4 67.4 b 3591 384 65 4 S M CON 34.0a 1980 385 49 4 66.0a3127 354a61 4a100 39.5 b 2067 386 50 4 60.5 b 3820 384a, b 67 4a, b 200 23.6c 2368 385 54 4 76.4c4175 384 b 71 4 b 300 27.5 d 1937 385 47 4 72.5 d 3674 384a, b 64 4a, b V L CON 35.3a 2284 384x52 4x64.7a3046 384 60 4 100 31.0 b 2722 385x,y 56 4x,y69.0 b 3554 384 65 4 200 24.6c 3044 385y61 4y75.4c3658 384 66 4 300 29.1 d 2222 385x51 4x70.9 d 3322 384 62 4 1 Muscles: INF = Infraspinatus ; LD = Longissimus dorsi ; SM = Semimembranosus ; VL = Vastus lateralis. Treatments: CON = 0 mghd-1d-1; 100 = 100 mghd-1d-1; 200 = 200 mghd-1d-1; 300 = 300 mghd-1d-1 of ractopamine-HCl supplemented during the final 30 days of feeding. a,b Means within a muscle and column with different letters are significantly different (P < 0.05). x,y Means within a muscle and column with different letter are different (P < 0.15).

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93 Table 4-4. Myonuclei per fiber cr oss-section from cull-cows fe d four different levels of ractopamine-HCl Muscle1 Fiber-associated nuclei INF 0 1.87 0.05a 100 1.70 0.06 b 200 1.78 0.06a, b 300 1.81 0.06a, b LD 0 1.35 0.06a 100 1.07 0.06 b 200 1.15 0.06 b 300 1.13 0.05 b SM 0 1.55 0.06 100 1.64 0.07 200 1.68 0.06 300 1.52 0.06 VL 0 2.06 0.06a 100 1.50 0.07 b 200 1.82 0.06c 300 1.86 0.06c 1 Muscles: INF= Infraspinatus ; LD = Longissimus dorsi ; SM = Semimembranosus ; VL= Vastus lateralis Treatments: CON = 0 mghd-1d-1; 100 = 100 mghd-1d-1; 200 = 200 mghd-1d-1; 300 = 300 mghd-1d-1 of ractopamine-HCl supplemented during the final 30 days of feeding. a-c Means within a muscle and column with diffe rent letters are significantly different (P < 0.05).

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94 Table 4-5. Real time PCR Ct values for 2-adrenergic receptor, myosin heavy chain isoform expression from the Longissimus dorsi and Semimembranosus of cullcows fed four levels of ractopamine-HCl1 Muscle2 2-receptor Ct Type I Ct Type IIA Ct Type IIX Ct Longissimus dorsi 0 10.24 11.71 14.55 20.95 100 10.11 11.50 13.10 22.10 200 9.84 9.94 12.27 20.19 300 10.62 10.34 12.74 21.55 Semimembranosus 0 10.35a,x 9.79a11.12x13.67a,x 100 13.11 b 13.17 b ,x14.59y21.01 b 200 8.80a,y 9.31a11.22x10.86a,y 300 8.63a,y 10.39a,y11.96x12.57a SEM 0.88 1.27 1.35 1.11 Slope3 -3.10 -3.45 -3.57 -3.22 a, b Means within a muscle and column with different letters are significantly different (P < 0.05). x-yMeans within a muscle and column wit hout common superscript tend to differ ( P < 0.10). 1 Bovine 18S gene expression used as house keeping gene for computation of Ct values. 18S gene expression did not differ betw een treatments of both muscle groups. Ct = Ct gene of interest Ct 18S. 2 Treatments: CON = 0 mghd-1d-1; 100 = 100 mghd-1d-1; 200 = 200 mghd-1d-1; 300 = 300 mghd-1d-1 of ractopamine-HCl supplemented dur ing the final 30 days of feeding. 3Slope of standard curve of pr imers sequences used for real-time PCR analysis. Bovine 18S gene slope = -3.22.

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95 CHAPTER 5 EFFECT OF RACTOPAMINE-HCl ON THE F IBER TYPE DISTRI BUTION AND SHELFLIFE OF SIX MUSCLES OF STEERS Introduction Feeding ractopam ine-HCl ( RAC ; 200 mghd-1d-1) to steers influences live and carcass performance in a positive manner. Schroeder et al. (2005a) reported that RAC improves both average daily gain and gain to feed ratio by 26 %, and Winterholler et al. (2007) reported that total gain can significantly improve by 6 percent. RAC increases hot carcass weight and ribeye area, decreases fat, and increases dressing per centage by as much as 3.6% (Schroeder et al., 2005b; Winterholler et al., 2008). Color represents the single most important visual com ponent that determines if a consumer will purchase a meat product (Hedrick et al., 1994). Several biochemical and physical factors affect meat color stability. The metm yoglobin reducing system relies heavily on NADH to chemically reduce metmyoglobin to deoxymy oglobin (Mancini and Hunt, 2005) depressing discoloration and promoting color stability (McKenna et al. 2005). NADH content in meat is determined by the energy metabolism profile of the individual muscle fibers (Howlett and Willis, 1998). NADH content is higher in type I slow fibers than in type II fast tw itch fibers. A loss of type I fibers may cause a reduction in metmyoglob in reducing activity thus, negatively impacting meat color. In both swine (Aalhus et al., 1992) and cattle (Gonzalez et al., 2007; 2008), data show that RAC can induce a fiber ty pe shift from type I slow-twitc h to type II fast-twitch fibers by as much as 30%. Therefore, the objective of this study was to evaluate the effects of RAC on MyHC isoform distribution and shelf-life prop erties of muscles of the loin and round.

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96 Materials and Methods Animals and Pre-Harvest Diets This exper iment was approved by the University of Florida Institutional Animal Care and Use Committee. Thirty-four crossbred steers were selected from steers housed at the University of Florida Beef Teaching Unit. Upon selection, steers were separated into four harvest groups (three groups of eight and one gr oup of ten). All cattl e were administered a Ralgro implant (36 mg Zeranol) followed by a Revalor-S implant ( 120 mg trenbolone acetate and 24 mg estradiol). Within each harvest group, steers were stratified by breed type and separated into two pens so that initial pen weight and visual backfat thic kness were similar. Pens were established two weeks prior to RAC supplementation to al low establishment of herd dynamics. Steers were fed daily a concentrate diet cons isting of 85% corn, 7.5% cottonseed hulls, and 7.5% commercially produced protein pellet. All st eers were supplemented with a hand mixed top dress (0 mghd-1d-1 of RAC; dehydrated alfalfa meal, corn meal, calcium carbonate, rice hulls, soybean oil, and mineral oil) at a rate of 0.45 kg per head per day for two weeks prior to the RAC supplementation period. Subsequently, the treatment pens received 0.91 kg per head per day of hand mixed top dress de signed to provide 200 mghd-1d-1 of RAC (Elanco Animal Health, Greenfield, IN) for 28 days prior to harvest. Control animals were fed an equivalent amount of top dress. The top dress provided 11% crude protein, 5% crude fat, and 16% crude fiber. Harvesting and Sample Collection Steers were harvested at the University of Florida Meat Laboratory under Federal Inspection following the Humane Methods of Slaughter Act of 1978. Seventy-two hours postmortem, the bone-in strip loin, knuckle, and top round were excised from the right side of each carcass. Whole muscles were re moved from the subprimals including the Longissimus

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97 lumborum ( LL) from the bone-in strip loin; the Adductor ( ADD ), Gracilis ( GRA ), and Semimembranosus ( SM ) from the top round; and the Rectus Femoris ( RF ) and Vastus lateralis ( VL) from the knuckle. A 1 cm3 portion was removed from each muscle, frozen in OCT tissue freezing medium (Fisher Scientific, Hamp ton, NH), and stored at -80C for immunohistochemical analysis. Whole muscles were placed in heat shrink vacuum bags (B2570; Cryovac, Duncan, SC) and vacuum packaged us ing a Multivac C500 (Multivac, Inc., Kansas City, MO). Muscles were wet aged for 13 days postmortem at 1 3C. Immunohistochemistry The m ethods used by Gonzalez et al. (2007, 2008) for immunohistoche mical staining were followed with slight modifications. Twelve mi crometer cryosections from each muscle were collected on frost resistant slid es (Fisher Scientific, Hampton, NH). Non-specific antigen sites were blocked with 5% horse serum in phosphate buffered saline (PBS). Cryosections were incubated in primary antibodies for 60 minutes at room temperature. Primary antibody solution consisted of anti-myosin heavy chain type I (BAD.5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA ) hybridoma supernatant with anti-dystrophin (1:50; Abcam, Cambridge, MA). After washing with phosphate buffered saline ( PBS), cryosections were incubated in goat anti-rat biotin (Vector Laboratories, Burlingame, CA) for 20 minutes at room temperature. After washing with PBS, cryosections were incubate d in rabbit anti-mouse AlexaFluor 568 (Invitrogen, San Diego, CA) and steptavidin AlexaFluor 488 (Invitrogen, San Diego, CA) for 45 minutes to det ect dystrophin and MyHC type I, respectively. After a final PBS wash, slides were visualized using an Eclipse TE 2000-U microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120 epifluores cence illumination system (EXFO, Mississauga, Ontario, Canada). Photomicrograp hs were captured using a Photom etrics Cool Snap EF digital camera (Nikon, Lewisville, TX) and analyzed for individual muscle fibe r cross-sectional area

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98 (CSA) using the NIS-Elements software (Nikon, Lewisville, TX). For each animal, a minimum of 500 fibers were measured and analyzed. All fi bers not labeled as MyHC type I were assumed to be type II fast-twitch. The region constrained by -dystrophin immunostaining defined individual fibers for CSA measurement. Steak Cutting, Packaging, and Display Following aging, m uscles were removed from th eir vacuum bags and cut into six 1.27 cm steaks. Steaks were cut from the same end of each muscle, perpendicular to the orientation of the muscle fibers. The first five steaks were used for reduction of nitric oxide metmyoglobin ( NOM ) analysis at days zero, one, two, three, and four. The sixth steak was used for daily visual panel evaluations and day five reduction of NOM analysis. Steaks from the LD and VL were placed on 17S Styrofoam trays (Genpack, Glens Falls, NY), steaks from the ADD, GRA, and VL were placed on 1S Styrofoam trays (Genpack, Gl ens Falls, NY), and steaks from the SM were placed on 10S Styrofoam trays (Genpack, Glens Falls, NY). Each tray contained a Dri-Loc 40 gram white meat pad (Sealed-Air Corporati on, Elmwood Park, NJ) and was overwrapped with polyvinylchloride film (23,250 cc O2/m2/24h C/%RH). Steaks were displayed in a Hill (Hill Refrigeration Div., Trenton, NJ) co ffin-style retail case at 2 3C for five days. Cases were illuminated with GE T8 Linear Fluorescent lamps (2800 lumens, 4100 K; General Electric Company, Fairfield, CT) that emitted a case average of 106.7 footcandle with a 12 hour on 12 hour off lighting schedule. Steaks were rotated daily to compensate for uneven temperature and light distribution within the case. Nitric Oxide Metmyoglobin Reducing Analysis The procedures of W atts et al. (1966), Sammel et al. (2002) and McKenna et al. (2005) were followed with minor adjustments. Each day, samples of each steak measuring 5 cm x 5cm x 1.27 cm were placed in 400 mL Pyrex beakers (Corning Inc., Acton, MA). Samples were

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99 oxidized in 50 mL of 0.3% sodi um nitrite (Fisher Scientific, Hampton, NH) at 25 2C for 30 minutes. The samples were removed from the s odium nitrite solution, bl otted of excess solution, and vacuum packaged in FoodSaver (Jarden Corp., Rye, NY) 27.94 cm bags with an oxygen transmission rate of 6.7 cc/m2/24 hr/23C/0% RH. Reflectance measurements ranging from 400700 nm were taken every 30 minutes for two hour s using a HunterLab MiniScan XE (HunterLab, Reston, VA) spectrophotometer with a 2.54 cm ap erture. Spectrophotometric measurements were captured using illuminant A and 10 standard observer. Before each data collection period, the MiniScan was calibrated on both a black and wh ite tile. Spectral data collected at 525 nm and 572 nm were used to calculate metmyoglobin percentage following American Meat Science Association (AMSA) procedur es (2003). Nitric oxide me tmyoglobin reducing ability was calculated as (observed decrease in metmyogl obin concentration initial metmyoglobin concentration) 100. Subjective and Objective Color Analysis A six to eight member experienced panel evaluated steaks for beef lean color (8 = extremely bright cherry red; 1 = extremely dark red), fat color (5 = yellow; 1 = white), and surface discoloration (7 = total [100%] discoloration; 1 = no [0%] discolorati on) daily for 5 days. Objective color measurements of these samples were taken using a HunterLab MiniScan XE. Illuminance and aperture settings, as well as ca libration procedures, were the same as described above. Spectral reflectance data from 400 to 700 nm was used to calculate ratios of metmyoglobin and oxymyoglobin present on the su rface of the steaks, according to AMSA procedures (2003). Following collection of spec tral data, absolute L*, a*, b* reflectance data were collected on the same sample s. Two measurements per steak to represent the color of the entire steak were averaged for spectral and absolute data.

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100 Statistics Data were analyzed using carcass as the e xperimental unit. Reduc ing activity, objective, and subjective color data was analyzed as a split-plot design with repeat ed measures. Kill group and treatment was considered the whole plot and muscle was considered the sub-plot. Kill group treatment was utilized as the random e rror for the whole-plot, and kill group the treatment/muscle interac tion was considered the random error for the sub-plot. Day was the repeated measure with animal within treatmen t as the subject. Muscle fiber CSA data was analyzed using a split-split-plot design. The whole plot and subplot variables were the same as in the shelf-life data. The sub-subplot cons isted of muscle fiber type and the remaining interactions. Kill group treatment was consider ed the random error for the whole-plot, kill group the treatment/muscl e interaction was considered the random error for the sub-plot, and the kill group the treatment/muscle/fiber type interaction was considered the random error for the sub-sub-plot. All measured variables were analyzed with the PR OC MIXED procedure of SAS (SAS Inst. Inc., Carry, NC, 2002). Pair-wis e comparisons between the least square means of the factor levels were computed using the PDIFF option of the LSMEANS statement. Fiber frequencies were tabulated and compared by chi-s quare analysis using PROC FREQ statement of SAS. Treatment group frequencies within a fibe r type were compared to one another by a two sample t-test for proportions. Differences were considered significant at an alpha = 0.05 and tendencies at an alpha = 0.10. Results Ractopam ine supplementation did not affect ( P > 0.05) the CSA of type I or II fibers in the six muscles analyzed (Table 5-1). Ractopa mine supplementation caused a significant type I to type II fiber shift in the ADD ( P = 0.0033), RF ( P < 0.0001), LL ( P = 0.0044), and VL (P <

PAGE 101

101 0.0001). The VL and GRA exhibited the largest percentage fiber shift. MyHC distribution within the SM remained unchanged by RAC. Nitric oxide metmyoglobin reducing ab ility was not significantly affected ( P > 0.05) by RAC supplementation in the GRA, RF, SM, or VL. The ability of RAC treated steaks from the ADD to reduce nitric oxide metmyoglobin was greater on day zero ( P = 0.05) and day one ( P = 0.01) of the display period, but was not different thereafter. The LL was the only other muscle affected by RAC supplementation. Longissimus lumborum NOM reducing ability was significantly greater ( P = 0.02) in RAC treated steaks on day 2. Ractopamine did not affect metmyoglobin or oxymyoglobin accumu lation or L*a*b* color scores (Table 5-2 and Table 5-3). Visual panel scores of the six muscles indi cate that RAC supplemen tation had little affect on either beef lean color or fat color (Table 5-4). R actopamine did not affect (P > 0.05) surface discoloration scores from d0 to d3 of the displa y period in the GRA, SM, LL, RF or VL. VL steaks from RAC animals had more surface discoloration on day four ( P = 0.05) and day five (P = 0.07). Similarly, RF and SM steaks from RA C steers exhibited higher surface discoloration (P = 0.0092 and P = 0.04, respectively) than CON at day fi ve. The ADD steaks discolored rapidly with RAC steaks scoring higher ( P = 0.05) than CON by day three. Discussion The benefits of feeding RAC to cattle on both liv e and carcass characteristics are well documented (Sissom et al., 2007; Winterholler et al., 2007, 2008). However, little data exists profiling the effects of RAC on st eak retail display color stability. The oxidation state of myoglobin, the heme-containing prot ein that stores oxygen in the muscle, determines the color that a consumer sees. Before myoglobin is e xposed to oxygen, it is in the deoxymyoglobin state, which results in a purple color. As the myogl obin is exposed to oxygen, it is converted into oxymyoglobin. Oxymyoglobin results in the typical bright cherry red color that the consumer

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102 desires. As meat ages, oxymyoglobin experien ces oxidation and the formation of metmyoglobin occurs (Aberle et al., 2001). Concurrently, lipid oxidation occu rs in the intramuscular fat, membrane phospholipids, and intermuscular fat. Th is oxidative system results in the formation of metmyoglobin in the meat and correlates to the amount of discolorat ion observed (Faustman and Cassens, 1990). In the retail display case, consumers visually evaluate numerous factors when considering which product to purchase (MacKinney et al., 1966). These factors may include portion size, leanness, ease of preparation, or color. A ccording to Hedrick et al. (1994) and Kropf (1980), color is the single most importa nt visual component th at determines if a consumer will purchase a meat product. Th erefore, the effect of RAC on metmyoglobin accumulation on the surface of steaks requires attention. Regardless of treatment, muscles observed in the study have different reducing potential, or ability to maintain this potential, during display. The differences observed in reducing potential translated into differe nces in a* values and (K/S)572/(K/S)525 ratios. From day 0 of the study, the LL and SM displayed the largest pe rcent NOM reducing ability, while the RF exhibited the least. The LL ma intained the greatest NOM redu cing ability throughout the five day display period. The VL and GRA began display with similar NOM reducing abilities, and the VL was able to maintain the second highest percentage of NOM reduced by the end of the study. The ADD and RF had the worst NOM reduc ing abilities. The ADD did not possess the capability to maintain NOM reducing ability throughout the 5 day study. Initial percent NOM reduced from this muscle was similar to th e GRA or VL, and the ADD ultimately possessed the lowest percentage of any muscle. Spectral data used to calculate ratios of the accumulation of metmyoglobin and oxymyoglobin indicate that muscle s followed patterns similar to those reported in the NOM

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103 reducing data. At the beginning of the displa y period, all muscles exhibited similar ratios for oxymyoglobin and metmyoglobin accumulation. By the end of the display period, the LL and VL had the least amount of metmyoglobin and subsequently the highest amount of oxymyoglobin. In contrast, the ADD and RF had th e lowest ratios and accumulated the highest concentration of metmyoglobin a nd lowest concentration of oxy myoglobin. The rapid increase in ADD and RF metmyoglobin was apparent by the s econd day of retail display; similar results were observed with a* values. The LL and SM ma intained larger a* values throughout the five day study indicating that muscles were redder in appearance. These findings agree with McKenna et al. ( 2005) who found that the LL and VL have the highest and the ADD has the worst NOM reducing ability. However, these researchers found that the RF had greater reduci ng ability than the SM. (K/S)572/(K/S)525 ratios indicated that the LL, VL, and SM were the most resistant to metmyoglobin accumulation, and the RF and ADD accumulated metmyoglobin rapidly. Instrument a* values indicated that the LL was the reddest during display. The SM, VL, and RF maintained similar redness values, and the adductor rapidly lost its red hue. Our re sults, in agreement with McKenna et al. (2005), demonstrate that muscles degrade in color at diffe rent rates and should be marketed accordingly to reduce product losses or devaluation. Dietzel (1990) reported that supplementation of RAC produced steaks with a brighter cherry red lean color after four days of display when compared to untreated steaks. Additionally, steaks from ractopamine fed steers maintained thei r overall appearance longer than steaks from non-treated steers. In the cu rrent study, spectral and absolu te data indicates that RAC supplementation did not affect the color stability or metmyoglobin accumulation in steaks from the six muscles analyzed. More importantly, metmyoglobin accumulation, as measured by

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104 (K/S)572/(K/S)525 ratio, was unaffected by RAC supplementa tion. However, visual panel data indicates that RAC did have a detrimental effect on the beef lean color and surface discoloration scores of several muscles. Ractopamine steaks from the VL were darker on days 3 and 5 of display. In addition, CON steaks from the VL app eared to reach a stable lean color score during the last three days of display, while the RAC steaks continued to become darker. Ractopamine treated VL steaks had more surface discoloratio n during the last three days of display. Ractopamine steaks from the GRA, RF, and SM al so demonstrated greater discoloration on day 5 of display. Therefore, wh ile objective measures of color were unable to detect treatment differences, visual panelists observed differences that a consumer ma y also detect. In agreement with our findings, Neill et al. (2008) found that steaks originati ng from the knuckle of implanted cull cows fed zilpaterol-HCl were darker and ha d more discoloration on day 5 of display than cows that were grass fed, concentrate fed, and con centrate fed with the incl usion of zilpaterol. The decrease in shelf-life witnessed in this study may be attributed to a physiological effect commonly observed when feeding beta-agonists, a shifting of MyHC isoforms. In swine, RAC increases the percentage of type IIB fibers at the expense of type IIA and IIX fibers (Depreux et al., 2002; Guanawan et al., 2007). In older fema le cattle, RAC included in a high concentrate feeding program can shift MyHC is oforms from type I to type IIA and from type IIA to type I by as much as 30% depending on the muscle ev aluated (Gonzalez et al., 2007, 2008). In the steers evaluated in the current study, all the muscles observed had RAC induced MyHC fiber type shifts except the SM. The lack of fiber shifting in the SM is surprising since the SM had one of the largest MyHC isoform shifts in RA C supplemented cull cows (Gonzalez et al., 2008). In the current study, the VL and GRA had the largest percent shift with 21% and 11% of their type I fibers shifting to type II fi bers, respectively. This isoform shift in the VL is in line with

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105 previous report (Gonzalez et al 2008). The ADD and LD demons trated 7% and 6% type I to type II shift, respectively. In contrast, the RF had 14% increase in type I fibers at the expense of type II fibers. The shifting of type II fibers to type I fibers seen in the RF was also seen in the infraspinatus of cull cows (Gonzal ez et al., 2008), which further exemplifies that muscles of beef cattle have differential responses to RAC. Muscle type is a major factor in determining the rate of meat discoloration (Hood, 1980). The fiber type composition and biochemical pro cesses with which the fibers of the muscle utilizes, affects the visual color of the meat. Fa ustman and Cassens (1990) reported that the fiber type composition of steaks under aerobic display a ffects the rate of discoloration through accumulation of metmyoglobin by altering the rate of oxygen diffusion and consumption, autoxidation of myoglobin in the presence of oxygen, and rate of metmyoglobin reducing activity ( MRA ). Of these factors, McKenna et al (2005) and Bekhit and Faustman (2005) identified MRA as the basis for meat disc oloration by reducing metmyoglobin accumulation. The MRA of muscle is strongly affected by the fiber composition of muscle due to the type of metabolism utilized within the fiber. Type I fibers rely more heavily on oxidative metabolism than type II fibers, which utili ze glycolytic metabolism (Aberle et al., 2001). Because of these differences in metabolism, type I fibers contain more mitochondria than type II fibers. Giddings (1974) hypothesized and others confirmed (Arihara et al., 1996) that mitochondria contribute to metmyoglobin reduction by supplyi ng NADH as a reducing cofactor. Kim et al. (2008) confirmed muscle fiber type has an effect on NADH presence by demons trating that porcine muscle with a high percentage of oxidative fibers contains more NADH. Based on these findings and the fiber type shift toward glycol ytic in the current study, one would expect RAC treated muscles to exhibit less MRA. The fiber ty pe percentage shift needed to affect the MRA

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106 of muscles remains unknown, but the current data s uggests that it must be larger than 21%. Steaks which exhibited muscle fiber type shifts due to RAC supplementa tion did not exhibit the expected changes in MRA as measured by NOM reducing ability. The inability to detect changes in metmyoglobi n reduction may be related to the assay of choice as the role of the MRA sy stem and tests used to measure it remain controversial (Bekhit and Faustman, 2005). Beef MRA can be measured by total reducing activity, aerobic reducing ability, metmyoglobin reductase activity, and NOM reducing assays (Sammel et al., 2002; McKenna et al., 2005). Previous reports indicate that NOM is intermediately correlated (r = 0.61) with metmyoglobin accumulation (McKenna et al., 2005) and only correlated well with metmyoglobin accumulation when discoloration differences were the largest (Sammel et al., 2002). However, Ledward (1972) re ported that aerobic reducing ab ility is highly correlated (r = -0.94) to metmyoglobin concentration, and Sammel et al. (2002) found this assay to be the best method for measuring reducing ability. Theref ore, employment of additional methods to measure MRA than the one utilized in this study may help explain the surface discoloration data reported on day 5 of the study and resolve the discontinuity found between the NOM reducing data and the fiber type shift. Conclusion Supplementing beef steers with 200 mghd-1d-1 of RAC for the final 28 days of feeding induced the MyHC fiber shift in all muscles obser ved except the SM. The shift observed was not consistent between muscles, which confirm th at RAC differentially affects muscles in young beef cattle. The expected effects of the RA C induced shift on NOM reducing ability, L*a*b* values, and (K/S)572/(K/S)525 ratio was not seen. Therefore, a gr eater shift toward type II fibers may be required to affect these shelf-life char acteristics. However, visual panel surface discoloration scores indicated th at by day five of the study RAC had a detrimental effect on color

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107 stability. The inconsistencies between the MRA results and visual panel surface discoloration may be due to the test used to meas ure MRA, but requires further research.

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108 Table 5-1. Least squares means of mu scle fiber myosin heavy chain isoform distribution, cross-sectional area and diameter from six muscles from steers fed with and without ractopamine-HCl Type I Fiber Type II Fiber Muscle Percentage Area ( m2)Percentage Area ( m2) Adductor Control 32.7a1,644.3 67.3a2,957.0 Ractopamine 30.7 b 1,774.0 69.3 b 2,865.6 Gracilis Control 37.9a1,312.1 62.1a1,786.3 Ractopamine 33.6 b 1,396.1 66.4 b 1,727.7 Longissimus lumborum Control 32.5a2,244.6 67.5a3,333.8 Ractopamine 30.5 b 2,323.6 69.5 b 3,318.2 Rectus femoris Control 13.8a1,179.0 86.3a2,032.3 Ractopamine 15.8 b 1,356.5 84.2 b 2,245.1 Semimembranosus Control 27.9 1,893.8 72.2 3,267.0 Ractopamine 27.5 2,001.6 72.5 3,616.6 Vastus lateralis Control 22.4a1,704.5 77.6a2,581.2 Ractopamine 17.6 b 1,523.8 82.4 b 2,369.1 SEM 187.0 187.0 a, b Means within a muscle and column with different letters are significantly different (P < 0.05).

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109Table 5-2. Percent nitric oxide metmyoglobin reduced and ratios of oxyand metmyogl obin accumulation from steaks originating from six muscles of steers fed with a nd without ractopamine-HCl displayed under simulated retail display conditions for 5 days Muscle Adductor Gracilis Longissimus lumborum Rectus femoris Semimembranosus Vastus lateralis Item1 Day CON RAC CON RAC CON RA C CON RAC CON RAC CON RAC NOM Reduced2 0 66.8a 81.7 b 62.8 61.8 70.6 75.6 28.8 32.7 82.0 79.9 61.5 68.2 1 44.0a 62.5 b 54.7 47.3 84.4 76.4 28.3 20.6 63.0 64.9 57.7 62.3 2 36.7 38.7 48.6 49.4 57.9a74.9 b 20.3 14.6 49.6 56.8 52.0 59.8 3 31.1 27.8 49.3 48.2 61.5 71.8 20.4 15.9 43.2 34.3 50.4 47.0 4 15.3 18.4 40.2 39.7 50.7 62.4 19.5 14.3 31.1 26.5 50.0 52.6 5 15.4 11.3 23.8 26.5 44.9 36.8 24.8 18.2 28.6 24.6 40.7 43.4 SEM 5.5 5.5 5.5 5.5 5.5 5.5 (K/S)572/(K/S)525 3 0 1.27 1.28 1.34 1.28 1.33 1.34 1.34 1.29 1.33 1.30 1.34 1.35 1 1.13 1.14 1.31 1.31 1.36 1.34 1.21 1.20 1.24 1.26 1.29 1.34 2 1.06 1.05 1.27 1.22 1.30 1.28 1.13 1.13 1.13 1.16 1.30 1.24 3 0.99 0.97 1.23 1.22 1.27 1.26 1.08 1.10 1.12 1.11 1.21 1.17 4 0.95 0.92 1.19 1.20 1.28 1.27 1.04 1.04 1.10 1.01 1.15 1.14 5 0.90 0.84 1.06 1.11 1.23 1.24 1.01 0.98 1.06 0.99 1.14 1.07 SEM 0.03 0.03 0.03 0.03 0.03 0.03 (K/S)610/(K/S)525 4 0 0.17 0.17 0.21 0.22 0.16 0.17 0.18 0.20 0.16 0.17 0.17 0.17 1 0.20 0.22 0.18 0.19 0.12 0.14 0.25 0.21 0.15 0.15 0.13 0.14 2 0.23 0.25 0.19 0.19 0.14 0.14 0.21 0.22 0.15 0.15 0.15 0.17 3 0.27 0.28 0.20 0.21 0.14 0.14 0.22 0.23 0.14 0.17 0.16 0.18 4 0.29 0.30 0.22 0.22 0.14 0.15 0.22 0.25 0.17 0.18 0.16 0.19 5 0.28 0.31 0.26 0.26 0.13 0.14 0.23 0.25 0.18 0.19 0.16 0.19 SEM 0.02 0.02 0.02 0.02 0.02 0.02 ab Means within a row without common superscript significantly differ ( P < 0.05). 1CON = steers supplemented 0 mghd-1d-1 of ractopamine; RAC = steers supplemented 200 mghd-1d-1 of ractopamine. 2Percent nitric oxide metmyoglobin reduced. 3 Ratios derived from Kubelka -Munk equations calculated from spectrophotometer measurement s taken at 572 nm and 525 nm used to estimate the percentage of Metmyoglobin accumulation. Smaller values indicate an increase in Metmyoglobin. 4 Ratios derived from Kubelka -Munk equations calculated from spectrophotometer measurement s taken at 610 nm and 525 nm used to estimate the percentage of Oxymyoglobin accumulation. Larger values indicate a decrease in Oxymyoglobin.

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110 Table 5-3. Least squares means of HunterLab MiniScan XE L*, a*, and b* values from st eaks originating from six muscles of stee rs fed with and without ractopamine-HCl displayed under simulated retail display conditions for 5 days Muscle Adductor Gracilis Longissimus lumborum Rectus femoris Semimembranosus Vastus lateralis Item1 Day CON RAC CON RAC CON RA C CON RAC CON RAC CON RAC L*2 0 49.1 49.3 45.7 44.9 43.6 41.9 47.9 47.7 43.5 42.7 44.4 44.5 1 46.3 46.1 44.9 43.5 42.4 42.3 45.7 46.9 42.8 40.8 42.1 41.8 2 45.4 45.2 44.3 43.3 42.5 41.0 43.8 44.4 42.0 41.5 41.7 41.8 3 45.5 44.7 44.4 42.2 41.4 40.1 43.7 44.4 41.5 40.4 41.2 41.8 4 43.4 44.2 44.1 42.7 40.8 39.7 42.5 42.4 41.3 40.1 41.1 40.8 5 44.9 43.5 43.5 41.8 40.3 39.2 43.6 43.6 41.3 40.0 41.0 41.0 SEM 1.6 1.6 1.6 1.6 1.6 1.6 a*3 0 32.8 32.3 30.7 29.7 33.2 33.3 32.4 30.4 34.7 34.0 34.7 33.7 1 28.3 27.1 30.8 32.2 36.5 35.2 30.9 28.6 33.6 34.4 35.9 35.6 2 24.9 24.2 30.0 30.0 34.2 34.6 28.6 26.7 32.0 31.7 33.4 31.4 3 22.7 22.1 28.3 28.8 33.9 34.5 27.1 25.1 30.9 30.4 32.2 29.3 4 22.3 21.1 27.3 27.6 33.8 33.8 25.9 25.0 29.9 29.1 31.4 29.4 5 20.8 19.5 23.6 24.4 33.3 32.9 24.8 22.8 28.5 27.6 30.0 27.7 SEM 1.4 1.4 1.4 1.4 1.4 b*4 0 30.6 30.1 26.4 25.5 30.4 30.4 29.2 27.3 32.5 31.9 31.5 30.5 1 28.6 27.6 27.0 28.5 34.2 32.8 29.5 27.2 32.2 33.3 33.6 33.1 2 26.5 26.2 26.2 26.6 31.9 32.1 28.2 26.3 31.3 31.1 31.5 29.7 3 25.6 25.5 25.4 26.0 31.9 32.1 27.3 25.5 30.6 30.7 30.8 28.4 4 25.5 24.9 24.7 24.8 31.7 31.7 27.1 25.7 29.8 29.8 30.1 28.5 5 25.0 24.6 23.1 23.4 31.4 31.0 25.9 24.6 29.1 29.0 29.3 27.6 SEM 1.1 1.1 1.1 1.1 1.1 1.1 1CON = steers supplemented 0 mghd-1d-1 of ractopamine; RAC = steers supplemented 200 mghd-1d-1 of ractopamine. 2 Lightness: 100 = White; 0 = Black. 3 Redness: 60 = Red; -60 = Green. 4 Blueness: 60 = Yellow; -60 = Blue.

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111 Table 5-4. Least squares means of visual panel scores for steaks from six muscles di splayed under simulated retail display con ditions for 5 days from cattle fed w ith and without ractopamine-HCl Muscle Adductor Gracilis Longissimus lumborum Rectus femoris Semimembr anosus Vastus lateralis Item1 Day CON RAC CON RAC CON RA C CON RAC CON RAC CON RAC Beef Lean Color2 0 5.5 5.4 5.7 5.9 5.5 5.3 5.8 5.6 5.4 5.4 5.6 5.3 1 4.5 4.3 5.3 4.9 5.4 5.3 5.7 5.2 5.0 4.7 5.1 5.0 2 4.1 3.9 5.1 4.9 5.4 5.2 5.6 5.4 5.0 4.6 4.9 4.9 3 3.9 3.7 4.9 4.4 5.3 4.9 5.6 5.3 4.7 4.4 5.0 4.6 4 3.6 3.3 4.4 4.2 5.0 4.9 5.3 5.3 4.5 4.2 4.9 4.7 5 3.4 3.2 4.3 4.3 5.1 4.7 5.5 5.3 4.5 4.1 5.0 4.5 SEM 0.3 0.3 0.3 0.3 0.3 0.3 Fat Color3 0 1.9 1.9 2.0 2.0 2.0 2.0 1.8 1.8 1.9 1.9 1.9 1.8 1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.1 2.0 2.0 2 2.0 2.1 2.2 2.1 2.1 2.1 2.0 2.1 2.1 2.1 2.1 2.0 3 2.0 2.0 2.1 2.1 2.0 2.0 2.1 2.0 2.0 2.1 2.0 2.0 4 2.4 2.5 2.4 2.3 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.2 5 2.7 2.6 2.6 2.7 2.4 2.4 2.3 2.4 2.3 2.4 2.2 2.4 SEM 0.1 0.1 0.1 0.1 0.1 0.1 Surface Discolor4 0 1.2 1.1 1.0 1.0 1.1 1.0 1.1 1.1 1.0 1.1 1.1 1.0 1 2.5 2.6 1.1 1.1 1.0 1.0 1.7 1.8 1.3 1.5 1.3 1.4 2 3.8a 4.3 b 1.2 1.0 1.1 1.0 2.0 2.2 1.7 1.9 1.6 1.8 3 4.5 4.8 1.3 1.5 1.0 1.1 2.5 2.7 2.1 2.4 2.1 2.5 4 4.9 5.1 1.6 1.9 1.3 1.3 2.8 3.0 2.5 2.8 2.5a3.0 b 5 5.1 5.3 2.5 2.8 1.6 1.6 2.9a 3.6 b 2.7a3.2 b 3.0x3.4 y SEM 0.2 0.2 0.2 0.2 0.2 0.2 ab Means within a row without common superscript significantly differ ( P < 0.05). x-yMeans within a row without common superscript tend to differ ( P < 0.10). 1 CON = steers supplemented 0 mghd-1d-1 of ractopamine; RAC = steers supplemented 200 mghd-1d-1 of ractopamine. 28 = Extremely bright cherry-red; 7 = Bright cherry-red; 6 = Moderately bright cherry-red; 5 = Slightly bright cherry-red; 4 = S lightly dark cherryred; 3 = Moderately dark red; 2 = Dark red; 1 = Extremely dark red. 35 = Yellow; 4 = Moderately yellow; 3 = Slightly yellow; 2 = Creamy white; 1 = White. 47 = Total discoloration (100%); 6 = Extensive discoloration (80-99%); 5 = Moderate discoloration (60-78%); 4 = Modest discolora tion (40-59%); 3 = Small discoloration (20-39%); 2 = Slight discoloration (1-19%); 1 = No discoloration (0%).

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112 CHAPTER 6 EFFECT OF RACTOPAMINE-HCl ON LIVE AND CAR CASS CHARACTERISTICS WHEN FED TO STEERS DURING THE FINAL 28 DAYS OF FEEDING Introduction The supplem ent ractopamine-HCl ( RAC ) belongs to a class of compounds called betaadrenergic agonists ( BAA). The Food and Drug Administra tion approves the use of RAC in both swine (Paylean in 1999) and cattle (Optaflexx in 2003). Ractopamine is approved for cattle fed in confinement during the last 24 to 42 days of feeding before slaughter. Ractopamine supplementation during the final days of f eeding commonly improves live performance by increasing average daily gain and gain to feed ratio in steers (Gruber et al., 2007) and heifers (Quinn et al., 2008). In steers, RAC supplementation increased gain to feed ratio and ADG by 17.2% and 15.3%, respectively (Grube r et al., 2007). Schroeder et al. (2005a) reported that RAC supplementation can improve average daily gain a nd gain to feed ratio by as much as 26%. Through its repartitioning abilit y, ractopamine elevates skelet al muscle protein deposition at the expense of fat deposition. Numerous studies report that ractopamine increases hot carcass weight and ribeye area, decreases fat, and incr eases dressing percentage by as much as 3.6% (Schroeder et al., 2005b; Winterholler et al., 2007 ). While the majority of published data on ractopamine documents its effects on whole carcass parameters, little data exists on yields from muscles throughout the carcass of beef cattle. In pigs, RAC in creases wholesale cut yields, trimmed wholesale cut yields, and boneless, wholes ale cut yields (Crome et al., 1996). Stites et al. (1991) found that RAC increased trimmed loin a nd ham yields, but did not affect the yields of the boston butt and picnic shoulder. Plascencia et al. (1999) reported that the BAA, zilpaterolHCl, increased the percentages of the knuckle, skir t, neck, and inside roun d in cattle. In addition, RAC supplemented to cull cows differentially increased muscle fiber hypertrophy of various

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113 muscles originating from the chuck and round (G onzalez et al., 2008). Therefore, RAC also may have the ability to increase yields from individual musc les of supplemented carcasses. In todays market condition with of high co rn/feed prices, the us e of RAC to improve average daily gain, gain to feed ratio, and carca ss characteristics becomes an attractive option for producers to lower the cost of b eef production. The objective of th e study was to investigate the effect of RAC on live performance and carcass characteristics, while also evaluating its effect on whole muscle yields from the carcass. Materials and Methods Animals and Pre-Harvest Diets This exper iment was approved by the University of Florida Institutional Animal Care and Use Committee. Thirty-four crossbred steers house d at the University of Florida Beef Teaching Unit were selected for the study. Steers were sepa rated into four harvest groups (three groups of eight and one group of ten) and followed the same implantation program consisting of a Ralgro implant (36 mg Zeranol) followed by a RevalorS implant (120 mg trenbolone acetate and 24 mg estradiol). Once a harvest group wa s established, steers were separa ted into two pens so that breed type, initial pen weight, and visual back fat thickness were similar. This separation occurred approximately two weeks before the beginning of each RAC su pplementation period to allow the steers time to establish a new pen dynamic. At 6 p.m. daily, steers were fed a basal concen trate diet (Table 1) in concrete bunks that provided 76.2 cm per head of bunk space. To allow the steers time to adjust to the top dress, both the control and treatment pens were suppl emented with a hand mixed blank top dress (0 mghd-1d-1 of RAC; Lakeland Animal Nutrition, Lakeland, FL ) at a rate of 0.45 kg per head per day for two weeks before the beginning of th e 28 day RAC supplementation period. Once the supplementation period began, the control pen co ntinued to receive th e hand mixed blank top

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114 dress at a rate of 0.91 kg per head per day. The treatment pens received 0.91 kg per head per day of hand mixed top dress designed to provide 200 mghd-1d-1 of RAC (Elanco Animal Health, Greenfield, IN) for 28 days prior to harvest. Harvesting and Carcass Data Collection Following day 28 of supplementation, final steer body weights ( BW ) were collected and steers were transported to the University of Florida Meats Laboratory for harvesting. Steers were harvested under Federal Inspection followi ng the Humane Methods of Slaughter Act of 1978. No carcasses from any harvest group were el ectrically stimulated during the harvesting process. During harvest, weights of inedible offal items were collected, including head, feet, pluck, empty rumen, and hide weights. Following harvest, hot carcass weights (HCW ) were recorded and carcasses were plac ed in a cooler at 0C. A pproximately thirty-six hours postmortem, carcasses were ribbed at the 12th-13th rib juncture and allowed to bloom for 30 minutes prior to carcass data collection. An experienced unive rsity employee collected carcass data including lean and bone maturi ty, marbling score, color score, texture score, firmness score, 12th rib fat, 12th rib ribeye area, kidney heart and pelvic fat ( KPH ), and average preliminary yield grade. These measurements were used to calculate dressing percent, USDA Yield grade and Quality grade (USDA, 1997). Whole Muscle Extraction and Measurement Seventy-two hours postmortem subprimals incl uding the bone-in strip loin, knuckle, and top round were excised from the right side of eac h carcass. From the subprimals, whole muscles were removed. The Longissimus lumborum ( LL ) was removed from the bone-in strip loin, and the Adductor ( ADD ), Gracilis ( GRA ), and Semimembranosus ( SM ) were removed from the top round. Similarly, the Rectus femoris ( RF ) and Vastus lateralis ( VL) were removed from the knuckle.

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115 After separation, each muscle was trimmed to 0.635 cm of fat and the commodity weight was taken. Muscles were then trimmed of all visible fat and epimysium connective tissue, and reweighed for a denuded weight measurement. Finally, muscle lengt h, width, maximum depth, and minimum depth measurements were taken on each muscle with a measuring tape. Prior to vacuum packaging and aging, muscle L*a*b* values were measured using a HunterLab MiniScan XE (HunterLab, Reston, VA). Whole mu scles were placed in heat shrink vacuum bags (B2570; Cryovac, Duncan, SC), vacuum pack aged using a Multivac C500 (Multivac, Inc., Kansas City, MO), and wet aged for 13 days postmortem at 2 3C. Warner-Bratzler Shear Force Analysis After aging, muscles were rem oved from thei r vacuum bags, weighed for purge loss, and cut into six 1.27 cm steaks and one 2.54 cm steak. Steaks were cut from the same end of each muscle, perpendicular to the orientation of the musc le fibers. The first si x steaks were used for shelf-life analysis (data presen ted elsewhere). The seventh 2.54 cm steak was vacuum packaged and stored at -40C for Warner-Bratzler shear force analysis. Twenty-four hours prior to cooking, steaks were thawed at 4 2C. Steaks were cooked on preheated Hamilton Beach Indoor/Outdoor open top grills (Hamilton Beach Brands, Washington, NC) following the guidelines of the American Meat Science Asso ciation. Thermocouples (Omega Engineering, Inc., Stamford, CT) were placed in the geometric center of each steak and internal temperatures were constantly monitored and recorded using 1100 Labtech Notebook for Windows 1998 (Computer Boards, Inc., Middleboro, MA). Steaks were cooked to an internal temperature of 71C, turned once at 35C (AMSA, 1995). Cooked steaks were chilled at 4 2C for 24 hours. Once chilled, six 1.27 cm cores were obtained from each steak, parallel to the orientation of the muscle fibers. Each core was sheared once thr ough the center of the core and perpendicular to

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116 the orientation of the muscle fiber using an Instron Universal Testing Machine (Instron Corporation, Canton, MA) with a Warner-Bratzler head (crosshead speed of 200 mm/min). Statistics Live performance and carcass data was anal yzed as a randomized complete block design with harvest group as the blocking factor and trea tment as the fixed effect. Pen was considered the experimental unit fo r live performance and carcass data. Muscle and Warner-Bratzler shear force data were analyzed as a split-plot design with repeated measures. Kill group and treatment was considered the whole plot and muscle was considered the sub-plot Kill group treatment was utilized as the random error for the w hole-plot, and kill group the treatment/muscle interaction was considered the random error for the sub-plot. All measured variables were analyzed with the PROC MIXED procedure of SAS (SAS Inst. Inc., Carry, NC, 2002). Pairwise comparisons between the least square means of the factor levels were computed using the PDIFF option of the LSMEANS statement. Differ ences were considered significant at an alpha = 0.05 and tendencies at an alpha = 0.15. Results Live perform ance for both treatment groups was recorded during th e final 28 days of feeding (Table 6-2). At the beginning of the 28 day supplementation period, both treatment groups body weights were not significantly different ( P = 0.55). During the supplementation period, both treatment groups had similar ( P > 0.05) dry matter intake, ADG, and G:F ratio. At the end of the trial period, body weights for both treatm ent groups were not significantly different (P = 0.70) due to both groups having similar ( P = 0.34) gains during the final 28 days of feeding. During the harvesting process, inedible offa l weights were collected, and 36 hours later, postmortem carcass data was collected (Table 63). The weights and percentage of total body

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117 weight of the head, pluck, viscera, f eet, and hide were not significantly ( P > 0.05) affected by the inclusion of RAC in the diet. Important carca ss measurements that indi cate increased muscling, including hot carcass weight, longissimus muscle area, and longissimus muscle area per 100 pounds of HCW were unaffected ( P > 0.05) by RAC supplementation. However, RAC supplementation tended ( P = 0.15) to increase dressing percentage. Carcass lean quality parameters including color and texture scores were unaffected ( P > 0.05) by RAC supplementation. Lean maturity of RAC supplemented animals appeared ( P = 0.02) older, and RAC also tended ( P < 0.15) to soften the firmness of th e lean of supplemented animals. Marbling score was significantly ( P < 0.0001) decreased by RAC supplementation. However, quality grade was unaffected ( P = 0.64) by RAC supplementation. Other carcass fat measurements, namely 12th rib fat thickness and KP H were not affected ( P > 0.05) by RAC supplementation. Therefore, this resulted in both treatment groups having similar ( P = 0.94) yield grades. Commodity weights, denuded weights, musc le dimensions, and Warner-Bratzler shear force values are presented in Table 6-4. Muscle weights and dimensions were measured with the hope of demonstrating the ability of RAC to improve muscling of steers. However, RAC did not significantly (P > 0.05) affect the muscle weights or dimensions of most of the muscles analyzed. Ractopamine did significantly ( P = 0.02) increase the widt h of the GRA and tended ( P = 0.08) to increase the width of the LL. Ractopamine supplementation also tended ( P = 0.12) to increase the minimum depth of the GRA. The effect of RAC on whole muscle color and tenderness was also analyzed (Table 6-4). For all six muscles observed except the RF RAC supplementation did not affect ( P > 0.05) L*a*b* values. Ractopamine supplementation tended ( P < 0.11) to cause a darker appearance

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118 in the RF. Warner-Bratzler shear force analysis indicated that the tendern ess of steaks from all six muscles were unaffected by RAC supplementation (P > 0.05). Discussion Since RAC was approved for use in beef cat tle in 2003, numerous studies report that the inclusion of RAC in the diet during the final da ys of feeding prior to harvest improves both live performance and carcass characteristics. Therefor e, producers have incentive to utilize this feed additive in their feeding program as a means to lower costs of gain. Commonly, ADG and G:F ratio are the live performance characteristics improved by supplementation. However, in the current study, all live perfor mance characteristics measured were unaffected by RAC supplementation over the final 28 days of feeding. Winterholler et al. ( 2007) reported that the same feeding protocol increas ed ADG and G:F ratio by 4.6 and 3.8%, respectively. In addition, Winterholler et al. (2007) f ound that RAC supplementation increased DMI by 3.5%, which contrasts the findings of this study and most other published studi es. Schroeder et al. (2005a) and Laudert et al. (2005a) both found that RAC supplemented at 200 mghd-1d-1increased both ADG and G:F ratio by as much as 20%. In st eers of differing biological type, RAC increased ADG and G:F ratio without having a biological type interaction (Gruber et al., 2007). This finding suggests that the lack of RA C effect in the current study was not due to the utilization of steers with differing genetic backgrounds. The numerical live performance values from this study were similar to significant values published in other studies. In the current st udy ADG and G:F ratio were increased numerically by 10 and 7%, respectively. These values are in the range of signifi cant values published by Wineterholler et al. (2007) and Gr uber et al. (2007). Overall, ga in data is the most encouraging data from the live performance data. Publishe d studies indicate that RAC can increase gain significantly by 6% (Winterholle r et al., 2007). In the curren t study, RAC increased gain 9%

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119 over the final 28 days of feeding. The glar ing difference between the present study and published studies is the large difference in numbe r of animals used. The lack of significance found in this study may be due to the low number of animals used and the variation in individual animal response when compared to other studies. Insignificant live performance data also may be due to the RAC dosage level and duration employed. We used the regimen suggest ed by the manufacturer of 200 mghd-1d-1 for the final 28 days of feeding. In the registration trials used to gain FDA approval, RAC supplemented at 100, 200, and 300 mghd-1d-1 improved ADG linearly by 17.1, 19.6, and 25.7%, respectively (Schroeder et al., 2004). Abney et al. (2007) investigated the effects of supplementing 100 and 200 mghd-1d-1 of RAC for 28, 35, and 42 days. These researchers found that RAC supplementation at 100 mghd-1d-1 yielded a linear increase in growth performance characteristics as time increased, with a maximum benefit at day 42. However, RAC supplemented at 200 mghd-1d-1 produced a quadratic increase in the same parameters and maximum benefit was reached at day 35. The researchers concluded that RAC fed at 200 mghd1d-1 for 35 days provided optimal live performance. These findings suggest that modifying our regimen to include RAC s upplementation at 200 mghd-1d-1 for 35 days or 100 mghd-1d-1 for 42 days may yield a greater live performance response. Ractopamine supplementation produces mixed carcass characteristic results. Effects commonly include modest increases in HCW, dressi ng percentage, and ribeye area in steers, with no changes in 12th rib fat thickness or other carcass char acteristics (Schroed er et al., 2004; Schroeder et al., 2005b; Laudert et al., 2005b). Walker et al. (2006) reported that RAC increased HCW by 6.9 kg, and tended to incr ease ribeye area by 1.74 cm in he ifers. Gruber et al. (2007) demonstrated that RAC administration to cattle differing in biological type increased HCW and

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120 ribeye area by 2 and 4%, respectively. In the current study, RAC supplementation did not increase HCW, longissimus muscle area, or l ongissimus muscle area per 100 pounds of HCW. In agreement, Quinn et al. (2008) reported that a variety of RAC supplementation regimens, including the protocol utilized in this study, had no effect on any carcass characteristics. Numerical increases in ribeye ar ea in the current study are similar to the significant increases in recent published studies, indicating that the lack of significance could be a result of the lower number of animals used. When feeding RAC to cattle, producers are of ten concerned about is its effect on fat deposition. In the current study, RA C supplementation did not affect 12th rib fat thickness and KPH, which contributed to both treatment groups having similar yi eld grades. Schroeder et al. (2004) reported that RAC supplemented at 200 mghd-1d-1 did not alter 12th rib fat thickness or KPH, but tended to decrease the yield grade of steers. In contrast, Sissom et al. (2007) found that RAC supplementation at the same dosage and time interval decreased 12th rib fat thickness and yield grades. Winterholler et al. (2008) found that when 200 mghd-1d-1 of RAC was supplemented for 37 days 12th rib fat thickness was unaffected, but yield grade was decreased. However, when the supplementation period was reduced to 28 days, there was no effect on 12th rib fat thickness or yield grade. In general, most studies report that RAC supplemented at 200 mghd-1d-1 does not affect 12th rib fat thickness or yield grade (Laudert et al., 2005b; Walker et al., 2006; Abney et al., 2007; Qui nn et al., 2008). In the current study marbling score was decreased by supplementation. However, concer n about this effect should be minimal since RAC supplemented steers achieved in the same Slight category of marbling as the non supplemented steers and quality grade was unaffect ed. Gruber et al. (2007) also found that RAC supplementation tended to lower marbling, but not e nough to affect quality grad e. Therefore, the

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121 literature and the current study indicate that RAC supplemented at 200 mghd-1d-1 for 28 days prior to harvest has little to no detrimental effect on fat measurements or yield and quality grades. Ractopamine supplementation tended to improve dressing percentage. Since RAC supplementation did not affect the weights or percentage of tota l body weight of the head, pluck, empty rumen, feet, or hide, differe nces between treatment groups inedible offal item weights did not contribute to this improveme nt. Early published studies from Schroeder et al. (2005b) and Laudert et al. (2005b) reported that RAC supplementation imp roves dressing percentage. However, recent studies published by Gruber et al. (2007), Quinn et al. (2008), and Winterholler et al. (2007, 2008) found that RAC supplementation was unable to in crease dressing percentage. The increase in dressing percenta ge without large decreases in fat measurements may indicate that RAC supplementation increased muscling. To date, RAC-induced increases in ribeye area is the only indicator of increased muscling. As discussed earlier, this result is mixed across studies and is not pronounced. However, commodity weights, denuded weights, and muscle dimensions of the six muscles analyzed in the current study suggest that RAC supplemented at 200 mghd-1d-1 for the final 28 days of feeding has no ability to globally increase muscling. In agreement, Dijkhuis (2007) reported that RAC administered in a similar manner did not affect the same measurements of the same six muscles from cull cows. Fiber morphometric data from the LL and SM of these cull cows found that RAC supplemented at 200 mghd-1d-1 did not increase muscle fiber hypertrophy in type I or IIA fibers of the LL, and only increased type IIA fibers by 34% in the SM. The 34% increase in type IIA fibers of the SM was not sufficient to cau se an overall increase in muscle weight or dimensions (Gonzalez et al., 2008). In the current study, fiber mo rphometric data (Gonzalez,

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122 Chapter 5) indicated that RAC did not stimulate hypertrophy in either muscle fiber isoform from any of the six muscles examined. Thus, the inability of RAC supplem entation to stimulate muscle fiber hypertrophy results in a lack of whole muscle growth. Reduced muscle protein degradation plays an active role in muscle protein accretion due to BAA supplementation (Koohmaraie et al., 1991). Specifically, BAAs may increase activity of the postmortem proteolytic enzyme inhibitor, ca lpastatin, by as much as 348% (Koohmaraie et al., 1992, Garssen et al., 1995). While not officially identified as the main culprit of decreased meat tenderness, increased calpastatin activity ma y contribute to decreases in tenderness. When RAC is supplemented at high levels, such as 300 mghd-1d-1, steak tenderness is decreased (Schroeder et al., 2004; Avendano-Reyes et al ., 2006). At a lower dosage level (200 mghd-1d1), Quinn et al. (2008) reported that RAC supplementation did not affect steak tenderness. However, RAC supplemented at 200 mghd-1d-1 for 28 days decreased the tenderness of steaks from cattle of three different biological backgrounds when measured by Warner-Bratzler shear force and trained sensory pane l (Gruber et al., 2008). All of these studies examined the tenderness of steaks originating from the longissimus dorsi in steers. However, the effect of RAC supplementation on other muscles of the carcass is unknown. In cull cows, RAC supplemented at 200 mghd-1d-1 did not affect tenderness in the same six muscles when compared to controls (Dijkhuis et al., 2008). In the current study, RAC supplemented at the same dosage level also di d not affect the tenderness, as measured by Warner-Bratzler shear force, of any of the muscles observed. The ADD, GRA, LL, and RF were all similar in tenderness. The SM and VL were similar in tenderness to one another, but were less tender than the other muscles. However, steaks from all muscles except the VL, regardless

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123 of treatment group, were considered acceptably/intermediately tende r (Miller et al., 1995; Miller et al., 2001) when analyzed by Wa rner-Bratzler shear force. Quinn et al. (2008) stated th at since neither HCW nor ribe ye area were increased by RAC supplementation, muscling was not increased by s upplementation and it would be expected for RAC to yield no effect on meat tenderness. In addition, Gruber et al. (2008) hypothesized that because RAC is a 1-agonist, decreases in tenderness were due to increased protein synthesis and muscle fiber hypertrophy. However, RAC did not increase the cross-secti onal area or diameter of the muscle fibers in any of the muscles examined (Gonzalez, Ch apter 5). Therefore, the lack of a RAC effect on muscle tenderness in the current study is not surp rising, given that RAC supplementation did not affect measurements of muscling or musc le fiber hypertrophy. Conclusion Data indicates that feeding RAC at 200 mghd-1d-1 for the final 28 days before slaughter has little or no effect on live, ca rcass, and individual muscle char acteristics. This study provides valuable data documenting the effects of 200 mghd-1d-1 of RAC on muscle dimension, weights, and tenderness of numerous muscles of the beef carcass. While ADG, G:F ratio, and ribeye area numerical differences were comparable to signi ficant published values, more animals may be needed to detect significant di fferences. Employing a feeding st rategy (greater RAC dosage or feeding time) different than the one followed in the current study may be n ecessary to elicit more beneficial effects on both live perfor mance and carcass characteristics.

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124 Table 6-1. Composition of basal diet1 and carrier for top dress2Item Amount, percent of DM Basal Diet Whole corn 85.0 Cottonseed hulls 7.5 Protein pellet 7.5 Trace minerals 0.05 Carrier Dehydrated alfalfa meal 50.8 Corn meal 33.3 Calcium carbonate3 12.5 Soybean oil 1.7 Mineral oil 1.7 1 Diet provided 75.1% TDN, 11.9% crude protein, 3.3% crude fat, 6.5% crude fiber, 9.9% acid detergent fiber, 16.1% neutral detergent fiber, 0.17 % calcium, and 0.28% phosphorus. 2Carrier provided 91.4% DM, 11.8% crude prot ein, 5.6% crude fat, 16.9% crude fiber, and 62.1% TDN. 3Calcium carbonate was reduced to 7.5% wh en ractopamine-HCl was added to the carrier. All nutrient values remained the same.

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125 Table 6-2. Least squares means of live performance characteristics of steers supplemented with and without ractopamine-HCl1 Item Control Ractopamine, 200 mghd-1d-1 SEM Initial BW, kg 531.58 522.78 10.32 Final BW, kg 564.32 558.45 10.73 DMI, kg/d/pen 39.14 39.69 2.48 Gain, kg 32.68 35.62 2.30 ADG, kg 1.20 1.31 0.09 G:F 0.13 0.14 0.01 1Live performance characteristics measured during the final 28 days of feeding.

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126 Table 6-3. Least squares means of offal we ights and carcass characteristics of steers supplemented with and without ractopamine-HCl Item Control Ractopamine, 200 mghd-1d-1 SEM Offal Weights Head, kg 13.98 14.15 0.44 Head Percentage1 2.49 2.53 0.06 Feet, kg 10.34 10.38 0.26 Feet Percentage1 1.84 1.86 0.04 Pluck, kg 7.59 7.42 0.26 Pluck Percentage1 1.35 1.33 0.04 Empty Rumen, kg 56.27 55.05 2.72 Empty Rumen Percentage1 9.94 9.85 0.43 Hide, kg 41.81 41.84 1.58 Hide Percentage1 7.42 7.52 0.28 Carcass Performance HCW, kg 344.09 345.45 7.57 Dressing Percent2 60.92x 61.86y0.98 Lean Maturity3 145.29a156.47 b 3.96 Bone Maturity4 150.79 148.44 5.93 Marbling Score5 335.88a324.12 b 4.40 Color Score6 3.20 3.48 0.25 Texture Score7 3.36 3.53 0.23 Firmness Score8 2.06x 2.47y0.20 12t h rib fat, cm 0.92 0.93 0.14 LM area, cm2 85.04 87.36 3.29 LM/100 kg HCW9 24.78 25.38 0.71 KPH 2.17 2.28 0.10 Yield Grade 2.61 2.60 0.19 Quality Grade10 16.35 16.11 0.35 ab Means within a row without common superscript significantly differ ( P < 0.05). x-yMeans within a row without common superscript tend to differ ( P < 0.15). 1Percentage of live weight. 2Dressing Percentage = (HCW/ Final BW)* 100. 3100 = A; 200 = B; 300 = C; 400 = D; 500 = E. 4100 = A; 200 = B; 300 = C; 400 = D; 500 = E. 5100 = Practically Devoid; 200 = Tr aces; 300 = Slight; 400 = Small. 61 = Bright Cherry Red; 8 = Extremely Dark Red. 71 = Very Fine; 7 = Extremely Course. 81 = Very Firm; 7 = Extremely Soft. 9Longissimus muscle area per 100 kg of hot carcass weight. 1013-15 = Standard; 16-18 = Select; 19-21 = Choice; 22-24 = Prime.

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127Table 6-4. Least squares means of muscle weights, dimensions, color m easurements, and Warner-Bratzler shear force values from six muscles of the round from cattle fed with and without ractopamine-HCl Adductor Gracilis Longissimus lumborum Rectus femoris Semimembranosus Vastus lateralis SEM Item1 CON RAC CON RAC CON RAC CO N RAC CON RAC CON RAC Commodity, kg 1.76 1.67 2.15 2.26 5.93 5.79 1.78 1.80 5.55 5.53 2.02 2.11 0.19 Percent HCW2 0.51 0.49 0.62 0.65 1.73 1.68 0.52 0.52 1.61 1.60 0.59 0.61 0.04 Denuded, kg 1.57 1.50 1.06 1.07 3.94 4.12 1.60 1.61 4.98 4.97 1.71 1.80 0.12 Percent HCW2 0.46 0.44 0.31 0.31 1.14 1.19 0.47 0.47 1.44 1.43 0.50 0.52 0.02 Length, cm 21.26 20.82 32.20 31.34 39.89 39.31 23.71 25.27 34.08 34.42 25.56 26.09 0.79 Width, cm 14.85 14.50 18.13a19.54 b 16.02x17.10y13.06 12.94 19.95 19.28 18.39 19.07 0.45 Minimum Depth, cm 2.13 1.98 1.27x 1.57y 3.04 3.12 2.21 2.43 2.02 2.16 1.89 1.81 0.43 Maximum Depth, cm 9.18 9.02 2.95 3.00 6.26 6.41 8.40 8.35 11.65 11.78 7.45 7.37 0.19 L* 33.61 33.32 33.50 34.03 37.98 36.72 50.79x 48.30y36.77 35.39 41.69 40.25 1.08 a* 26.41 26.25 22.82 23.64 27.43 28.22 27.98 27.58 27.70 27.48 28.56 28.49 2.80 b* 18.22 18.75 12.73 13.96 20.45 20.98 21.83 20.46 20.09 19.52 20.91 20.58 4.73 WBS3, kg 3.25 3.50 3.78 3.31 3.49 3.21 3.65 3.52 4.14 3.84 5.11 4.67 0.25 ab Means within a row without common superscript significantly differ ( P < 0.05). x-yMeans within a row without common superscript tend to differ ( P < 0.15). 1 CON = steers supplemented 0 mghd-1d-1 of ractopamine; RAC = steers supplemented 200 mghd-1d-1 of ractopamine. 2Muscle weight percentage of hot carcass weight. 3Warner-Bratzler shear force values.

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128 CHAPTER 7 OVERALL CONCLUSIONS AND IMPLICATIONS Supplem enting cull cows with 200 mghd-1d-1 of RAC for the final 28 days of a 92 day feeding period increased Longissimus dorsi type I muscle fiber CSA by 31% when compared to controls. Addition of an impl ant to RAC fed cull cows increased type I fiber CSA by an additional 13% when compared to non-implan ted RAC supplemented cows. Neither RAC supplementation nor employment of an implant st rategy affected the CSA of type IIA muscle fibers in the Longissimus dorsi When RAC was combined with an implant, muscle fiber isoform distribution altered, with type I fibers shifting to t ype IIA fibers. Ractopamine supplementation did not affect the number of satellite cells or fiber associated nuclei counted when identified with immunohistoc hemical techniques. The lack of an effect on both fiber associated nuclei and satellite ce ll numbers indicates that the hypert rophy of type I muscle fibers stimulated by RAC supplementation could be due to alteration in protein synthesis/degradation rates. Because older animals have a limited abil ity to synthesize protein, it is hypothesized that this caused the limited increases in muscle fi ber hypertrophy e xperienced when feeding RAC to younger animals. Various concentration levels (100, 200, and 300 mghd-1d-1) of RAC supplemented to cull cows during the final 28 days of feeding differe ntially affected the mo rphometrics of various muscles throughout the carcass. In creasing the dosage level of RAC in the diet did not linearly increase the CSA of type I or type IIA fibers of muscles orig inating from the chuck, loin, or round. The inclusion of RAC in the diet at any level did not affect the CSA of type I fibers in the Longissimus dorsi as was seen when RAC was supplemented for 35 days. When RAC supplementation affected muscle fiber CSA, impr ovements in CSA were minimal. Interestingly,

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129 the lower concentration of RAC only increased fiber size in type I and II muscle fibers of the INF, a muscle characterized by a higher proportion of red fibers. Ractopamine addition at any of the three suppl ementation levels caused a biological effect on muscle fiber isoform distribut ion. Muscle fibers of the Longissimus dorsi Semimembranosus and Vastus lateralis demonstrated shifts from type I to IIA, while fibers of the Infraspinatus showed shifts from type IIA to I. Si nce no cows were implanted in this trial, this indicates that an implant is not needed to cause a shift in myosin heavy chain isoform distribution. Ractopamine supplementation at all levels decreased the number of fiber associated nuclei counted in multiple muscles. This provides evidence that fiber associated nuclei incorporation into the muscle fiber is not needed for RAC induced muscle fiber hypertrophy. This finding further substantiates the hypothesis that RAC stimulated hypertrophy is most likely accomplished by altering protein synthesis/degradation rates. When RAC was supplemente d to steers at 200 mghd-1d-1 during the final 28 days of feeding, results indicated that RAC has little to no effect on live, carcass, or individual muscle characteristics. While RAC did not significantly improve live performance, including average daily gain and gain to feed ratio, numerical di fferences were similar to significant published values. Ribeye area also demonstrated numeri cal increases in cross-sectional area with RAC supplementation similar to published values. It is hypothesized that usi ng more animals in the study could have added signi ficance to these values. The literature has failed to define the effect s of RAC supplementation on other muscles of the beef carcass. When RAC was s upplemented to steers at 200 mghd-1d-1 for the final 28 days before slaughter, muscle dimensions, weights, and tenderness of muscle s of the loin and round were unaffected. The lack of increased muscling can be attributed to the inability of RAC to

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130 increase the CSA of either type I and IIA muscle fibers in all muscles evaluated. However, RAC did stimulate the muscle fiber isoform shift seen in cull cows. The shift in isoforms was not consistent among all the muscles ev aluated; further indicating that RAC has a differential effect on muscles. While RAC supplementation did not affect metmyoglobin reducing ability or objective measures of color stability during retail di splay, trained panelist s did find that RAC supplemented steaks had a higher percentage of su rface discoloration toward the end of the five day display period. This could indicate the RAC supplementation detrimentally affects meat color stability, but this warrants further researc h. If RAC affects shelflife through a reduction in metmyoglobin reducing ability, fu rther research using different tests to measure metmyoglobin reducing ability are needed. The literature published over the last four years concerning th e supplementation of RAC to beef cattle reveals two trends Firstly, in the live animal, RAC supplementation improves average daily gain and gain to feed ratio with little to no effect on dry matter intake. Second, RAC supplementation modestly increases ribeye ar ea, can occasionally decrease yield grade, and rarely affects carcass fat measurements. While RAC does increase ribeye area, it seems that its ability to increase muscling is limited. The goal of this dissertation was to describe the cellular events responsible for the lack of a gross increase in muscling in multiple muscles of old and young beef cattle. Results indicate that RA C supplementation was unable to significantly stimulate muscle fiber hypertrophy in type I and type IIA muscle fibers in muscles originating from the chuck, loin, or round. Immunohist ochemical analysis indicated that RAC supplementation did not increase the numbers of satelli te cells or fiber associated nuclei. Therefore, RAC may stimulate muscle hypertrop hy by altering protein synthesis/degradation

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131 rates. Because older animals have a limited cap acity to synthesize protein, it was hypothesized that RAC may not be able to stimulate muscle fiber hypertrophy in cull cows. However, results that RAC also has a limited ability to stimulate hypertrophy of muscle fi bers from young steers, which resulted in an inability to increase muscle weights or dimensions. Therefore, the results from cull cows may not be due to an inability of RAC to stimulate increases in synthesized protein, but possibly the inability of RAC to decrease protein degradation. In the literature, re searchers report that most BAAs stim ulate muscle hypertrophy by altering protein degradation rate s through increased calpastatin ac tivity. As a consequence of this increased activity, th e studies report decreases in tender ness. However, in the current body of work, the tenderness of various muscles was unaffected by RAC supplementation. The lack of growth observed may be attributed to a failure of RAC to stimulate increased calpastatin activity and reduce protein degradation in the mu scle fiber. Recently, the FDA approved the use of a new BAA in beef cattle, labeled as a 2-receptor agonist. Results from a few studies indicate that this product has a greater capacity to incr ease muscling. Steaks from cattle fed this product become less tender, which may indicate that grow th is occurring through increased calpastatin activity. Muscles of beef cattle contain more 2-receptors than 1-receptors. Ractopamine is labeled as a 1-agonist. Lower levels of 1-receptors in the muscles of beef cattle may limit the ability of RAC to increase calpastatin ac tivity, which lowers pr otein degradation and consequently yields greater muscle mass. To date, the effect of RAC on protein degradation rates, specifically its effect on calpastatin ac tivity, has not been examined and requires further research. Producers must consider what type of bene fits they hope to reap from employing RAC supplementation in their feeding program. While published data and the current body of work

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132 indicate that RAC can positively affect live performance, the effects at the carcass level are minimal. Therefore, producers must decide if th e improvement in the live performance of their beef cattle outweighs the cost of feeding the supplement. They also must be aware that there will be little benefit to the carcass from feeding RAC due to its inability to stimulate muscle fiber hypertrophy. The beef industry must consider that whole muscle cuts from RAC supplemented cattle can have decreased shelf-life due to shifts in muscle fiber isofor ms, and evaluate if the decrease in shelf-life is worth the potential loss es at retail. Finally, because RAC has a limited ability to increase muscling in beef carcass, researchers and producers may consider feeding RAC at different dosages or increase the length of the supplementation period to elicit greater effects.

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133 APPENDIX A REPRESENTATIVE PHOTOMICROGRAPHS OF INFRASPINATUS MUSCLE IMMUNOS TAINED FOR DETECTION OF SATELLITE CELLS Methodology for staining can be found in the immunohistochemistry section of the materials and methods section of chapters 3 and 4. Satellite cells were identified as Hoechst dye and -Pax7 positive nuclei located outside the -dystrophin barrier. Figure A-1. Photomicrographs of immunohistochemical stains used to identify satellite cells. A) Hoechst Dye; B) -Pax7; C) -Dystrophin; D) Combined stain photomicrographs. A B C D

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134 APPENDIX B REPRESENTATIVE PHOTOMICROGRAPH OF LONGISSIMUS DORSI MUSCLE IMMUNOS TAINED FOR DETECTION OF FIBER ASSOCIATED NUCLEI Methodology for staining can be found in the immunohistochemistry section of the materials and methods section of Chapters 3 and 4. Fiber associated nuclei were identified as propidium iodide stained objects within the -dystrophin barrier. Figure B-1. Photomicrograph of Longissimus dorsi muscle immunostained for fiber associated nuclei detection. -Dystrophin identified as green st ain and propidium iodide stain (red stain) identified nuclei.

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135 LIST OF REFERENCES Aalhus, J. L., A. L. Schaefer, A. C. Murray, an d D. M. Jones. 1992. The effect of ractopam ine on myofibre distribution and morphology and their relation to meat quality in swine. Meat Sci. 31:397-409. Aberle, E. D., J. C. Forrest, D. E. Gerrard, a nd E. W. Mills. 2001. Principles of Meat Science. 4th ed. Kendall/Hunt Publishing Co. Dubuque, IA. Abney, C. S., J. T. Vasconcelos, J. P. McMeniman, S. A. Keyser, K. R. Wilson, G. J. Vogel, and M. L. Galyean. 2007. Effects of ractopamine hydrochloride on performance, rat and variation in feed intake, and acid-base balance in feedlot cattle. J. Anim. Sci. 85:30903098. Adeola, Olayiwola, Evans Asare Dar ko, Ping He, and Leslie Graham Young. 1990. Manipulation of porcine carca ss composition by ractopamine. J. Anim. Sci. 68:3633-3541. AMSA (American Meat Science Association). 1995. Research Guidelines for Cookery, Sensory Evaluation, and Instrumental Tenderness Meas urements of Fresh Meat. Am. Meat Sci. Assoc., Chicago, IL. AMSA (American Meat Science Association). 2003. Meat colo r evaluation guidelines. Am. Meat Sci. Assoc., Savoy, IL. Anderson, D. B., E. L. Veenhuizen, J. F. Wagne r, M. I. Wray, and D. H. Mowery. 1989. The effect of ractopamine hydrochloride on nitrogen retention, growth performance and carcass composition of beef cattle. J. Anim Sci. 67(Suppl. 1):222(Abstr.). Apple, J. K., C. V. Maxwell, B. R. Kutz, L. K. Rakes, J. T. Sawyer, Z. B. Johnson, T. A. Armstrong, S. N. Carr, and P. D. Matzat. 2008. Interactive effect of ractopamine and dietary fat source on pork quality characteristics of fresh pork chops during simulated retail display. J. Anim. Sci.jas.2007-0327v1-20070327. Arihara, K., M. Itoh, and Y. Kondo. 1996. Contribution of the glycolytic pathway to enzymatic metmyoglobin reduction in myocytes. Biochem. Mol. Biol. Inter. 38:325-331. Armstrong, T. A., D. J. Ivers, J. R. Wagner, D. B. Anderson, W. C. Weldon, and E. P. Berg. 2004. The effect of dietary ractopamine concen tration and duration of feeding on growth performance, carcass characteristics, a nd meat quality. J. Anim. Sci. 82:3245-3253. Avendano-Reyes, L., V. Torres-Rodriguez, F. J. Meraz-Murillo, C. Pere z-Linares, F. FiguroaSaaverdra, and P. H. Robinson. 2006. Effects of two -adrenergic agonists on finishing performance, carcass characteristics, and meat quality of feedlot steers. J. Anim. Sci. 84:3259-3265. Bardsley, R. G., S. M. J. Allcock, J. M. Dawson, N. W. Dumelow, J. A. Higgins, Y. V. Lasslett, A. K. Lockley, T. Parr, and P. J. Buttery. 1992. Effect of -agonists on expression of calpain and calpastatin activity in skeletal muscle. Biochimie 74:267-273.

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144 Plascencia, A., N. Torrentera, a nd R. A. Zinn. 1999. Influence of the -agonist, zilpaterol, on growth performance and carcass characteristic s of feedlot steers. Proceedings, Western Section, American Society of Animal Science 50:331-334. Polla, B., V. Cappelli, F. Morello, M. A. Pellegrino, F. Boschi, O. Pastoris, and C. Reggiani. 2001. Effects of the beta(2)-agon ist clenbuterol on respir atory and limb muscles of weaning rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280: R862-869. Prince, T. J., D. L. Huffman, and P. M. Br own. 1987. Effects of ractopamine on growth and carcass composition of finishing swine. J. Anim. Sci. 65(Suppl. 1):309(Abstr.). Prince, J. C., J. T. Yean, and W. G. Pond. 1983. Ga strointestinal, carcass and performance traits of obese versus lean genotype swine: Eff ect of dietary fiber. Nutr. Rep. Int. 27:259. Quinn, M. J., C. D. Reinhardt, E. R. Loe, B. E. Depenbusch, M. E. Corrigan, M. L. May, and J. S. Drouillard. 2008. The effects of ractopa mine-hydrogen chloride (optaflexx) on performance, carcass characteristics, and meat quality of finishing feedlot heifers. J. Anim. Sci. 86: 902-908. Rajab, P., J. Fox, S. Riaz, D. Tomlinson, D. Ba ll, and P. L. Greenhaff. 2000. Skeletal muscle myosin heavy chain isoforms and energy metabolism after clenbuterol treatment in the rat. Am. J. Physiol. Regulatory Interg rative Comp. Physiol. 279:R1076-R1081. Reeds, P. J., S. M. Hay, P. M. Dorwood, and R. M. Palmer. 1986. Stimulation of muscle growth by clenbuterol: Lack of effect on muscle protein biosynthesi s. Br. J. Nutr. 56: 249-258. Relaix, F., D. Montarras, S. Zaffran, B. Gayraud-Morel, D. Rocancourt, S. Tajbakhsh, A. Mansouri, A. Cumano, and M. Buckingham. 2006. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J. Cell Biol. 172:91-102. Ricks, C. A., P. K. Baker, and R. H. Dalrymple. 1984a. Use of repartitioning agents to improve performance and body composition of meat animals. Pages 5-11 in Proc. 37th Recip. Meat Conf., Lubbock, TX. Ricks, C. A., R. H. Dalrymple, P. K. Baker, and D. L. Ingle. 1984b. Use of a -agonist to alter fat and muscle deposition in steers. J. Anim. Sci. 59:1247-1255. Ruffolo, R. R. 1991. Chirality in and -adrenoceptor agonists and antagonists. Tetrahedron 47:9953-9980. Sainz, R. D., Y. S. Kim, F. R. Dunshea, and R. G. Campbell. 1993. Temporal changes in growth enhancement by ractopamine in pigs: performa nce aspects. Aust. J. Agric. Res. 44:14491455. Sajko, S., L. Kubinova, E. Cvetko, M. Kreft, A. Wernig, and I. Erze n. 2004. Frequency of Mcadherin-stained satellite cells declines in human muscles during aging. J. Histochem. Cytochem. 52:179-185.

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BIOGRAPHICAL SKETCH John Michael Gonzalez was born in San Antonio, Texas in 1978 to Joe L uis Gonzalez and Linda Martha Gonzalez. He was the youngest of three children born in th e Gonzalez family. His older sister, Rebecca Lynn Gon zales is a school teacher in Sa n Antonio, Texas; and his older brother Joe Luis Gonzalez, Jr. is a loan officer living in Austin, Texas. John Michael began his formal education attending Charles C. Ball Elem entary School from grades kindergarten through five. John Michael was then given the great opportunity to attend Sa int Marys Hall School where he attended both middle and high school. While attending Saint Marys Hall, John Michael was exposed to agriculture by visiting ranches belonging to his friends families and by helping his best fr iend rope steers. Upon graduation from Saint Marys Hall, J ohn Michael attended Texas A&M University in College Station, Texas. When deciding wh ich major to purse in college, John Michael decided that he enjoyed all asp ects of production agriculture and d ecided to major in agricultural economics (with an emphasis in farm and ranch management); and also to major in poultry science. While attending A&M, John Michael received his first agriculture-relat ed employment opportunity when he was hired for a job in the Te xas A&M University Department of Veterinary Physiology and Pharmacology, managi ng a herd of miniatur e Sinclair swine. At this time, John Michael swore to his boss that he would never pu rsue any form of graduate education. Toward the end of his education at Texas A&M, John Michael gained experience managing a 10,000 acre cow-calf cattle operation located in Enicnal, Texas. At the Triple-Bar ranch, John Michael assisted in the daily operations associated w ith maintaining the cow-calf herd, while also managing the nutrition program for the ranchs trophy whitetail deer operation. During this experience, John Michael realized that he woul d have to win the mega-lottery to have an operation of this size and scope and woul d need to make other life plans.

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Upon graduation from Texas A&M in May of 2002, John Michael quickly broke his promise to his boss at the Texas A&M Veterina ry School and decided to pursue his Masters degree at Sul Ross State University in Alpine, Texa s. While attending this little school located in the most beautiful country of Texas, John Mich ael decided to pursue his Masters in meat science under Paul A. Will. Because of the large population of meat goats located in West Texas, John Michael decided to study the effect s of preharvest supplementation methods on goat meat quality. John Michaels thesis was titled The Effects of Vitamin D3 on Goat Meat Tenderness and Color Stability In between his studies and re search, John Michael spent time helping the West Cattle Company and working at a restaurant at the Hotel Paisano in Marfa, Texas. At Sul Ross, John Michael coordina ted and taught all the General Animal Science classes, and also assisted in al l meat science related courses. For his efforts, John Michael was awarded Outstanding Graduate Student in Anim al Sciences and Graduate Student Teaching Award of Merit by the North American Colleges and Teachers of Agriculture and Sul Ross State University in 2003. After many trial and tribulat ions (and no meat lab), John Michael received his Master of Science degree in May of 2002. Upon completion of his Masters degree, John Michael moved to Gainesville, Florida to work on his Doctorate degree in meat science un der the supervision of D. Dwain Johnson in the Department of Animal Sciences at the University of Florida. For his Doctorate program, John Michael worked in collaboration with Sally Johnson, examining the effects of ractopamine supplementation on the muscle biol ogy of beef animals. During hi s time at the University of Florida, John Michael extensively aided the Depart ment of Animal Sciences to fulfill its mission of teaching and extension by serving as a graduate assistant in several undergraduate courses and state extension programs. As a testament to his hard work and dedication during his four years at

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the University of Florida, John Michael has publi shed two peer reviewed articles and received the 2008 Animal Science Graduate Student Associ ation Ph.D. Student of the Year Award. Upon receiving his doctorate, John Michael hopes to en ter the NASA Astronaut Candidate Program to conduct research for the United States space prog ram. John Michael is also considering a position in the meats industry or academia. His ma in goal in life is to ha ve a career that is adventurous, exciting, that will make him and his family happy.