An Examination of the Temporal and Spatial Expression Pattern of Muscle Proteins in the Early Bovine Embryo

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An Examination of the Temporal and Spatial Expression Pattern of Muscle Proteins in the Early Bovine Embryo
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Waits, Christy M
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Master's ( M.S.)
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University of Florida
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Animal Sciences
Committee Chair:
Johnson, Sally
Committee Members:
Ealy, Alan
Yelich, Joel V

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bovine -- embryogenesis -- immunohistochemistry -- myogenesis
Animal Sciences -- Dissertations, Academic -- UF
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Animal Sciences thesis, M.S.
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Bos taurus or Bos indicus cattle are used in crossbreeding operations to take advantage of heterosis to obtain an animal capable of handling Florida’s subtropical climate and produce a better meat product.  This study used ultrasonography to examine fetal development of Bos taurus (Angus) and Bos taurus x Bos indicus (Brangus) in early gestation.  Repeated measures ANOVA showed that Brangus fetuses tended (P = 0.06) to be larger than Angus fetuses on d 33 of gestation.  At d 55 of gestation, Angus fetuses were larger (P
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by Christy M Waits.
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Thesis (M.S.)--University of Florida, 2012.
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Adviser: Johnson, Sally.
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1 AN EXAMINATION OF TH E TEMPORAL AND SPATI AL EXPRESSION PATTER N OF MUSCLE PROTEINS IN T HE EARLY BOVINE EMBR YO By CHRISTY MARIE WAITS THIS THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFI LLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 CHRISTY WAITS

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3 To Mom and Dad thanks for all the love and support and to Elizabeth and Katelynn t he world is what you make of it, k isses on your f aces

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4 ACKNOWLEDGMENTS First ly I would like to thank my mentor, Dr. Sally Johnson for her guidance, patience, and intellectual support throughout my graduate program and willingness to take a chance on a returning student seven years out of practice and with very little experience in the molecular world. I would also like to thank the members of my supervisory committee, Drs. Alan Ealy and Joel Yelich, for their insight and contributions to my growth as a scientist. Special thanks are extended to th e members of m y lab, Dr. John Michael Gonzalez Marni Lapin, and Renan Di Giovanni Isola and also a special thanks to adopted members of the lab Marisa White and Dr. Regina Esterman. Their time, assistance, and willingness to help with these projects were invaluable. My lab partners have made my graduate experience more enjoyable. I would also like to thank the staff of the Santa Fe Beef Research Unit and the Dairy Unit as well as the Meat Processing Lab for their cooperation and assistance during the ex periments. Finally, I would like to thank my friends and family for all of their love and support. I thank God for each and every one of my friends you brought so much to me through this time, thanks for the laughter, the shoulders to lean on, and the wiping away of my tears when things got tough. I really could not have done this without all of you, thank you.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUC TION ................................ ................................ ................................ .... 10 2 LITERATURE REVIEW ................................ ................................ .......................... 13 Breeds of Cattle ................................ ................................ ................................ ...... 13 Overview ................................ ................................ ................................ .......... 13 Bos taurus ................................ ................................ ................................ ........ 13 Bos indicus ................................ ................................ ................................ ....... 14 Advantages of using Bos indicus in S outhern United States ............................ 15 Disadvantages of using Bos indicus in Southern United States ....................... 16 Crossbreeding ................................ ................................ ................................ .. 16 Muscle Growth and Development ................................ ................................ ........... 17 Overview ................................ ................................ ................................ .......... 17 Bovine Gestational Development ................................ ................................ ..... 17 The Muscle Fiber ................................ ................................ .............................. 21 Muscle Growth and Regeneration ................................ ................................ .... 23 Skeletal Muscle D evelopment ................................ ................................ .......... 23 Tongue Development ................................ ................................ ....................... 25 Satellite Cells and Stem Cells ................................ ................................ .......... 26 Myogenic Regulatory Factors ................................ ................................ ........... 28 Myf 5 ................................ ................................ ................................ ................ 29 Pax3 and Pax7 ................................ ................................ ................................ 30 3 DIFFERENCES MEASURED BY ULTRASONOGRAPHY I N BOVINE FETAL GROWTH IN EARLY GEST ATION BETWEEN ANGUS AND BRANGUS CATTLE ................................ ................................ ................................ .................. 34 Background ................................ ................................ ................................ ............. 34 Materials and Methods ................................ ................................ ............................ 35 Results and Discussion ................................ ................................ ........................... 37 Implications ................................ ................................ ................................ ............. 39

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6 4 SPATIAL AND TEMPORAL EXPRESSION OF MYOGEN IC PROTEINS IN THE EARLY BOVINE EMBRYO ................................ ................................ ..................... 41 Background ................................ ................................ ................................ ............. 41 Materials and Methods ................................ ................................ ............................ 45 Embryo Collection ................................ ................................ ............................ 45 Whole Mount Myosin Immunostaining ................................ .............................. 46 Im munohistochemistry ................................ ................................ ...................... 46 Results and Discussion ................................ ................................ ........................... 47 Morphological Features of Bovine Embryos at d 28 Gestation ......................... 47 Whole Mount Myosin Immunostaining Indicates Presence of Skeletal Muscle in Myotome ................................ ................................ ....................... 47 Immunofluorescence Staining Reveals Myogenic Cells in d 28 E mbryos ........ 48 Morphological Features of Bovine Embryos at d 45 Gestation ......................... 50 Myogenesis in the d45 Limb and Intercostal Muscles ................................ ...... 51 Primary Muscle Development in Tongue of d 45 Embryo ................................ 53 Pax7 Expression in the Olfactory System ................................ ......................... 56 Implications ................................ ................................ ................................ ............. 57 5 CONCLUSIONS ................................ ................................ ................................ ..... 76 LIST OF REFERENCES ................................ ................................ ............................... 77 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 88

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7 LIST OF TABLES Table page 4 1 Body Measurements and Means of D28 Embryos ................................ .................. 59 4 2 D45 Embryo Measurements ................................ ................................ ................... 65

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8 LIST OF FIGURES Figure page 3 1 Bovine fetal measurments and growth during early gestation on days 33 40, 47, and 55 of gestation ................................ ................................ ...................... 40 4 1 Gross morphological features of bovine embryos at d28 of gestation. ............... 58 4 2 Skeletal muscle is present within the myotome compartment. ......................... 60 4 3 Skeletal muscle is present within the myotome compartment and Myogenic Regulatory Factor Myf 5 is migrating into the limb bud in d28 embryo. .............. 61 4 4 Localization of Myf5 immunoreactiviy in the presumptive forelimb bud. ............. 62 4 5 Skeletal muscle is present within the myotome compartment an terior to the limb bud ................................ ................................ ................................ ............. 63 4 6 Day 45 Embryos. ................................ ................................ ................................ 64 4 7 Primary muscle fibers are present in the f orelimb upon completion of embryogenesis in d45 embryo. ................................ ................................ ........... 66 4 8 Primary skeletal muscle fiber formation in the developing limb showing Myf 5 and Pax7 positive cells throughout primary skeletal m uscle. .............................. 67 4 9 Satellite cells positive for Pax7 migrating throughout primary skeletal muscle in the developing limb and intercostal muscles. ................................ ................ 68 4 10 Satellite cells positive for Pax7 migrating throughout primary skeletal muscle in the developing limb and intercostal muscles. ................................ .................. 69 4 11 Primary muscle fibers in the tongue i n d45 embryo head. ................................ 70 4 12 Primary muscle fibers in the tongue in d45 embryo head. ................................ .. 71 4 13 Primary muscle fibers in the tongue i n d45 embryo head. ................................ .. 72 4 14 Pax7 positive cells in forming muscle fibers of the tongue in d45 embryo head. 73 4 15 Tongue muscle formation in d45 embryo marked with anti myosin heaving chain (MyHC) and Pax 7. ................................ ................................ .................... 74 4 16 Pax7 positive cells in forming olfactory epithelial cells of the nasal cavity in d45 embryo head. ................................ ................................ ............................. 75

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AN EXAMINATION OF TH E TEMPORAL AND SPATI AL EXPRESSION PATTER N OF MUSCLE PROTEINS IN T HE EARLY BOVINE EMBR YO By Christy M. Waits August 2012 Chair: Sally Johnson Major: Animal Science Bos taurus or Bos indicus cattle are used in crossbreeding operations to take advantage of heterosis to obtain an animal capable of ha climate and produce a better meat product. T his study used ultrasonography to examine fetal development of Bos taurus (Angus) and Bos taurus x Bos indicus (Brangus) in early gestation R epeated measures ANOVA show ed that Bran gus fetuses tended ( P = 0.06) to be larger than Angus fetuses on d 33 of gestation A t d 55 of gestation, Angus fetuses were larger ( P < 0.05) than Brangus fetuses. To define early bovine myogenesis immuno histochemistry was performed for myosin heavy cha in (MyHC), Myf5, and Pax7 in d 28 and d 45 embryos Results indicate in d 28 embryos myoblasts are present in the myotome and the somatic mesoderm and migratory myoblasts are present In the developing limb bud a large number of nuclear Myf5 is present i n the somatic mesoderm. Satellite cells are intermixed with primary muscle fibers in d 45 embryos in intercostal, tongue limb muscle as well as the lateral nasal mesenchyme and the olfactory epithelium

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10 CHAPTER 1 INTRODUCTION Florida is a subtropical region known for high temperatures and humidity. C attle that are raised and live in Florida face increased exposure to parasites and increase risk of infestation by parasites. Additionally cattle in Florida often consume pasture forages that are low er in nutritional quality than forages in the rest of the country (Koger, 1963; Rechav, 1987; Hunter and Siebert, 1985 ) In the beef industry there is high demand for fast maturing cattle that yield a meat product that is both tender and flavorful. Bos tauru s breeds are preferred and selected for superior performance and meat quality (Marshall, 1994) T hese breeds perform well in temperate climates but are poorly adapted to the sub tropical climate of Florida therefore their performance decreases Bos indic us cattle are more tolerant to warmer, sub tropical climates as well as resistant to parasi te s and disease ; however their meat is lower in quality due to decreased tenderness and they do not marble as well making the end product less desirable among consu mers (Koger, 1963; Hansen, 1990; Elzo et al., 2012; Whipple et al., 1990) To counter the negative effects of both breeds and build on the positive aspects Bos taurus x Bos indicus crossbred cattle. Through crossbreedin g producers select for an animal capable of maintaining good performance in a sub tropical climate but maintain the preferred meat characteristics if tenderness and marbling. calf producers are l ocated in the southern United States, Bos indicus cattle and their crosses dominate these areas (Morrison, 2005). Brangus is one example of crossbred cattle commonly used, as with all cross breeds this animal falls in between the two

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11 purebred breeds for t he preferred characteristics of cattle (Reynolds et al., 1980; Elzo et al., 2012). The ultimate goal of the cow calf producer is to produce a viable calf crop from year to year that will be sold to feeders and growers and upon reaching finishing weight wil l go to slaughter and produce quality meat products to be bought by consumers. A considerable amount of research has been place d on animal growth particularly muscle growth. There are numerous studies evaluating how to produce a better meat product, thr ough enhancing feed efficiency growth implants, and feed additives which has been mostly focused at post natal muscle growth (Elzo et al., 2012; Whipple et al., 1990) To gain a better understanding of how muscle growth occurs we also need to evaluate p re natal growth particularly when comparing cattle of different breed types. S tudies have shown th at different breeds hav e different birth weights as well as dam and sire breed effec ts on birth weight (Casas et al., 2010; Reynolds et al., 1980) The va riation in birth weights between breeds of cattle draws focus to what is occurring developmentally in gestation. In the early stages of gestation cells are dividing and proliferating, development occurs through a series of signaling factors that make deve loping and proliferating cells undergo differentiation (Guillomot, 1995) As pregnancy proceeds, different migrating signals are released and act upon cells determining what type of cell it will become, such as endothelial, bone, and muscle; these cells t oo are capable of migrating to further growth and development (Cossu and Biressi, 2005) Muscle is formed through myoge nesis, via the myogenic cascade, in the vertebrate embryo somites are where skeletal muscle originates (Arnold and Braun,

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12 1993). Progen itor cells respond to signals and activate basic helix loop helix transcription factors which commit cells to myogenesis (Cossu and Biressi, 2005). The goals of these experiments are to determine if in early gestational development differences in size of t he fetuses exist between Bos taurus (Angus) and Bos taurus x Bos indicus (Brangus) and when these differences occur and to define early bovine myogenesis. Based on previous, unpublished work in by Gonzalez, muscle fiber morphometrics indicate fiber muscle sizes are different at birth between Angus and Brangus calves this leads to the hypothesi s that differences in fiber sizes and numbers at birth between Angus and Brangus are due to differences in myogenesis in utero.

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13 CHAPTER 2 LITERATURE REVIEW Breeds o f Cattle Overview In order to optimize cattle production, diverse biological types of cattle that differ with respect to mature size, milk production, growth and maturation rates, and adaptability to regional differences in feed resources and climatic cond itions are needed. In the Southern United States Bos taurus and Bos indicus cattle are two subspecies of cattle that are commonly used in the livestock industry for beef production. Beef cattle are selected and bred based on their reproductive capacity a nd postnatal growth. Beef animals are sold based on weight and the quality of meat product they produce, therefore understanding muscle growth and development is important in creating a better meat animal. Through evaluation of embryonic muscle growth an d examination of its differences between breeds of cattle, further insight is gained to aid in production of an animal that will yield a better meat product. Bos taurus Bos taurus cattle are broken down into four functional groups based upon their uses and performance, these groups are British cattle, Continental cattle, Dual purpose cattle, and Dairy cattle. In the beef industry the most commonly used of these functional groups are British and Continental breeds. British breeds such as Angus or Herefo rd are most frequently utilized for their maternal abilities and have a long standing reputation for producing high quality meat products (Marshall, 1994). Continental breeds, such as Charolais, are characterized by large size and known to be late maturin g with higher muscle and protein retention capacity and better food

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14 efficiency (Geay and Robelin, 1978). Bos taurus breeds are characterized by fast rate at which they reach reproductive maturity (Warnick et al., 1956; Temple et al., 1961; Reynolds et al. 1967; Casas et al., 2011), approximately a 21 day estrous cycle (Hansel et al., 1973), shorter gestation lengths, the average Angus gestation length is 282 days (Burris and Blunn, 1952), are more tender and have higher marbling scores than Bos indicus ca ttle (Elzo et al., 2012; Whipple et al., 1990). Bos indicus Bos indicus cattle are of Indian origin characterized by large ears and a hump and are best known for their tolerance to heat and humidity (Koger, 1963), which is aided by a light colored, sle ek, shiny hair coat that reflects a proportion of solar radiation (Hansen, 1990). In addition to thermotolerance, they also have resistance to parasite infection and ticks, and possess the ability to digest poor quality forages (Koger, 1963; Rechav, 1987; Hunter and Siebert, 1985; Turner, 1980). Bos indicus cattle reach reproductive maturity at older ages than their English counterparts (Plasse et al., 1968a; Warnick et al., 1956; Temple et al., 1961; Reynolds, 1967) the a verage age at puberty for Brahman heifers is 19 months (Plasse et al., 1968a), and have shorter estrus durations, approximately 21 days (Plasse et al., 1970; Randel, 1984; Pinheiro et al., 1998; Richardson et al., 2002; Schams et al., 1977; Hansel et al., 1973). For the Brahman breed the average estrous cycle length averages 29 days and duration of estrus averages to 7 hours (Plasse et al., 1970). The most common Bos indicus breed in the U.S. that is utilized in the southern region is the Brahman these cows have an average gestation len gth of 293 days (Plasse et al., 1968b). Brahman calves are reported to be heavier at birth when compared to Angus calves (Paschal et al., 1991). Purebred Brahman cattle are plagued

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15 with a reputation for poor neonatal performance and low calf survival rate s (Cartwright, 1980), poor nursing instinct (Olcott et al., 1987), and low milk yield and lactation persistency (reviewed in Hansen, 2004). Additionally, Brahman meat is less desirable when compared to Bos taurus meat because it is less tender and has mor e connective tissue (Elzo et al., 2012; Whipple et al., 1990). Advantages of using Bos indicus in Southern U nited S tates Florida and the Gulf Coast region are characterized by subtropical temperatures, with mild winters and hot summers (West, 2003). Bos t aurus particularly Angus cattle, are poorly adapted to warmer climates their dark, thick coats and their ability to accumulate more fat, makes them more prone to heat stress (Hansen, 1990) brought on the extended period of intense radiant energy, and the presence of high relative humidity (West, 2003). Heat stress or heat production and accumulation, coupled with compromised cooling capability because of environmental conditions, causes heat load in the cow to increase to the point that body temperature r ises, intake declines and causes a decline production of both milk and meat (West, 2003). Bos indicus cattle are equipped with special adaptations which include light colored, sleek, shiny coats, larger sweat glands, and a lower basal metabolic rate than Bos taurus which when exposed to heat stress allows Bos indicus breeds to experience less severe alterations in feed intake, growth rate, milk yield, and reproduction than cattle of Bos taurus breeds (Hansen, 1990; Pan, 1963) In addition to thermotoleran ce Bos indicus cattle are tick resistant and efficient at digesting poor quality forages both of which conditions are prevalent in the Southern United States (Rechav, 1987; Hunter and Siebert, 1985).

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16 Disadvantages of using Bos indicus in Southern U nited S t ates While Bos indicus particularly Brahman cattle are well suited to the climate of the Southern U. S. these cattle do have some disadvantages that limit their use and value ustry requires cattle enter the reproductive cycle at 1 year of age and to undergo first parturition by 2 years of age, Brahman heifers are reported to reach reproductive maturity at 19 months of age and have longer gestational rates than Bos taurus cattle (Plasse et al., 1968a; Casas et al., 2010. Also, Brahman cattle have low calf viability, and reports of lower numbers of survival of calves from birth to weaning than in progeny of any other breeds (Casas et al., 2010). Riley et al. (2007) and Prayaga ( 2004) found that calves derived from a cross with Brahman sires had higher perinatal mortality. Additionally Brahman cattle have a reputation for inadequate tenderness, meat from Brahman cattle is less tender and scores lower for marbling than meat from B os taurus cattle (DeRouen et al., 1992; Koch et al., 1982; Crouse et al., 1989; Elzo et al., 2012). Crossbreeding The variation that exists in biological traits important for beef production is vast; crossbreeding is used to exploit heterosis in cattle h erds (Cundiff et al., 1986). located in the southern United States; an estimated 30% of cattle in the United States contain some percentage of Bos indicus genetics (Morrison, 2 005; Chase et al., 2005). In subtropical climates, Bos indicus breeds are crossed with Bos taurus breeds to provide advantages for heat and disease resistance, lower occurrence of dystocia, improve calf viability and survival, and improve feed efficiency (Crockett et al., 1979; Wythe, 1970; Turner, 1980; Casas et al., 2010; Riley et al., 2007; Prayaga, 2004; Smith

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17 et al., 2007; Elzo et al, 2009). However, the advantages are tempered by older age at reproductive maturity, slightly longer gestation rate whe n compared to purebred Bos taurus and reduced meat tenderness as the proportion of Bos indicus increases (Crouse et al., 1989; Reynolds et al., 1980; Koch et al., 1988; Riley et al., 2005). One common cross found in Florida is the Brangus which is 5/8 Ang us and 3/8 Brahman, this breed reaches reproductive maturity earlier than and a shorter gestation length than Brahman cattle, as well as increased tenderness and marbling when compared Brahman cattle (Reynolds et al., 1980; Elzo et al., 2012). Muscle Growt h and Development Overview Extensive research has been conducted studying muscle growth and development and the regulatory factors in chicken, mice, and zebra fish There is minimal research in the area of bovine muscle development most immunohistochemist ry work is from day 9 21 of gestation and then from gestation day 80 and later ( Alexopoulos et al., 2008 ; Maddox Hyttel et al., 2003 ; Martyn et al., 2004 ) Ultrasound is becoming a tool utilized more frequently as a means of measuring fetal size during early embryonic growth (Curran et al., 1986; Riding et al, 2008). Understanding the means of growth, structure and function of skeletal muscle cells provides the basis for understanding embryological muscle growth and development. Bovine Gestational Deve lopment In bovine species, blastocyst formation occurs ~7 days after fertilization, followed by placentation initiation with apposition to the uterus around two weeks later (Guillomot, 1995). The vast majority of embryonic loss occurs between day 8 and 1 6 as seen through IVP (in vitro production) and SCNT (somatic cell nuclear transfer); most

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18 embryonic loss has already occurred by day 14 gestation (Alexopoulos et al., 2008). The stages of bovine post hatching embryonic development from days 9 21 have b een examined and documented. Maddox Hyttel et al. (2003) saw on day 9 of gestation, embryos are devoid of zona p e llucida and present a well defined inner cell the Ra underlying ICM; the hypoblast, visible as a thin confluent cell layer is separated from the ICM and the trophoblast by intercellular matrix (Maddox Hyttel et al., 2003). The epibla st represents the final embryonic founder cell population with the potential for giving rise to all cell types of the adult body; in day 12 embryos, two cell populations of the epiblast are identified: one constituting distinctive basal layer apposing the hypoblast, and one arranged inside or above the former layer, including cells apposing the Rauber layer (Vejlsted et al., 2005). Day 14 embryos are ovoid to tubular and display a confluent hypoblast, the epiblast is inserted into the trophoblast epitheliu m and tight junctions and desmosomes are present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells (Maddox Hyttel et al., 2003). On day 21, Maddox Hyttel et al. (2003) had much variation in embryo sizes and in wh at was developing; the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites, and allantois development with a gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate, and lateral plate mesoderm.

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19 Research conducted using immunohistochemistry of fetuses in the second trimester of pregnancy provides an interesting look at muscle development using muscles in t he limb. Martyn et al. (2004) determined beginning around day 120 muscle fiber types are being discerned into Type I or Type II fibers. Presumptive primary and secondary fibers of day 120 bovine M. vastus lateralis muscle fibers are positive for embryoni c myosin heavy chain (MHC) at day 160 there is a similar pattern however some fibers are negative for embryonic myosin heavy chain (Martyn et al., 2004). The average sizes of primary myotubes initially decrease from 120 to 160 days of gestation as they de veloped into mature myofibers (Strickland, 1978; Martyn et al., 2004). The disappearance of fetal myosin heavy chain indicates muscle contractile differentiation from day 180 onwards (Cassar Malek et al., 2007). The time period during which a difference develops in the average area of Type I fibers is from 160 to 210 days of gestation, a t 210 days, presumptive secondary fibers Type I fibers vary in positive staining for embryonic MHC and slow MYC; at 260 days, all fibers are negative for embryonic MHC (M artyn et al., 2004). Skeletal muscle matures during late gestation in cattle at approximately day 210 of gestation (Greenwood et al., 1999). Ultrasonography provides the opportunity to improve the methods of evaluation of ovarian function and diagnoses of pregnancy in beef cattle and provides another way of examining fetal growth in vivo In early development embryonic vessels are first detected in bovine heifers at 11.7 days of gestation, detection of embryo proper at 20 days of gestation and heartbeat h as been detected on the first day of detection of the embryo proper or the following day (Curran et al., 1986). From days 20 60 of gestation the growth curve of the embryo is quadratic, with an increasing growth rate

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20 after approximately day 50; detectio n of various structures occur as follows: allantois, 23.2 days, spinal cord 29.1 days, forelimbs 29.1 days, amnion 29.5 days, optic area 30.2 days, hind limbs 31.2 days, placentomes 35.3 days, optic lens, 40.0 days, split hooves 44.6 days, fetal movements 44.8 days, and ribs 52.8 days (Curran et al., 1986). Determination of the sex of a fetus early in pregnancy (d 55 to 85) and verification of embryo viability by monitoring fetal heartbeat are unique methods involving ultrasound scanning (Beal et al., 1992 ). Morphological differences between Bos taurus and Bos indicus breeds are evident by Day 100 of gestation (Lyne, 1960). However; the time during gestation at which fetal weights diverge due to breed is not clearly defined. Some suspect this event likely occurs during the first trimester, birth weight differences among purebred cattle breeds reflect the combined genotypic effects of sire and dam and are relatively dramatic, generally being greater than differences observed within breeds (Lyne, 1960; Holla nd and Odde, 1992). Others suspect maternal ability in late gestation, Ferrell (1991a) suggests maternal ability may play a role in size regulation during late gestation, at day 232 of gestation, no difference in weight was reported between fetuses of Bra hman or Charolais cattle regardless of sire or dam effect. However, by day 274 of gestation Brahman fetuses in Charolais cows were heavier than Brahman fetuses in Brahman cows, and Charolais fetuses in Brahman cows were lighter than Charolais fetuses in C harolais cows (Ferrell, 1991a). Maternal ability may be defined as the physiological capacity of the dam to nurture the developing fetus, independent of the contribution to fetal genotype (Holland and Odde, 1992). Ferrell (1991b) also proposed that birth weight suppression was due to limitations of uteroplacental function and uterine blood

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21 flow. Correlation between birth weight and gestation length is considered to be positive and is low to moderate in magnitude (Burfening et al., 1978). Fetal growth ra te near term varies between 100 and 250g/day (Prior and Laster, 1979). Thus while extended gestation periods result in additional fetal weight gain, the actual magnitude of the increase in birth weight is slight (Holland et al., 1990). The Muscle Fiber On e of the most highly organized cells in the animal body is the muscle cell, which performs a diverse array of mechanical functions: locomotion and maintenance of balance and coordination. Skeletal muscle cells are long, multinucleated cells that through g rouping and bundling form the muscle ((McComas, 1996; Lieber, 2002). The organization, structure and metabolism of the muscle determine its function and aid in the maintenance of its integrity during muscle contraction (Lonergan et al., 2010). Organizati on of muscle begins with the basic unit of the muscle is, the sarcomere joins with other sarcomeres making strings of sarcomeres to form the myofibril; these arrange side by side to compose the muscle fiber, muscle is composed of bundles of muscle fibers c alled fascicles (McComas, 1996; Lieber, 2002). Skeletal muscle is striated due to the structure of the sarcomere in the myofibrils which are punctuated with light and dark bands forming the striations (Goll et al., 1984; Schroeter et al., 1996). Striated muscle is characterized by its ability to contract and generate tension and then return to its original length and form after contraction or stretching ceases (Vigoreaux, 1994; Knupp et al., 2002). The skeletal muscle contractile system is formed from an array of thick filaments, thin filaments and Z lines (Bloom and Fawcett, 1968). These thick and thin filaments are contractile filaments that are large polymers of myosin and actin proteins that compose the sarcomere which are

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22 found between two successiv e Z lines (Gordon et al., 1966a; Gordon et al., 1966b; Lieber, 2002). The Z line act as acting anchoring structures for the thin filaments and interdigitate the thick filament, repeating units of these structures are in each myofibril (Bloom and Fawcett, 1968; McComas, 1996; Lieber 2002; Goll et al., 1984; Schroeter et al., 1996). In muscle contraction the thick filament, the myosin containing filament, generates tension and the thin filament, the actin containing filament, regulates the tension generated (Gordon et al., 1966a; Gordon et al., 1966b; Lieber, 2002). Movement occurs as the result of the interaction of the thick and thin filaments driven by adenosine triphosphate (ATP) and ATPase and regulatory proteins troponin and tropomyosin (Goll et al., 1984; Lieber, 2002). These filaments interact via the tail and globular head region of myosin which extends from the thick filament to interact with actin in the thin filament forming an actomyosin complex upon contraction (Goll et al., 1984). The globul ar head of myosin has enzymatic activity and is capable of hydrolyzing ATP to liberate energy, ATPase acts on myosin providing energy for myosin bound to actin to swivel and pull the thin filaments toward the center of the sarcomere thus stimulating contra ction, dissociation of myosin and actin occurs when a new molecule of ATP is bound to the myosin head triggering relaxation (Goll et al., 1984). There are two main regulatory proteins involved in stimulating muscle contraction; troponin and tropomyosin re ly on ATP for attachment and release to stimulate contraction and relaxation (Lieber, 2002). Troponins are responsible for turning on contractions and tropomyosin is a long, rigid and insoluble rod shaped molecule that stretches along in close contact wit h the strands of the thin filament (Lieber, 2002).

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23 Muscle Growth and Regeneration Muscle growth occurs in two manners, hyperplasia and hypertrophy which are responsible for growth at different time points in life. Hyperplasia is muscle growth by which the number of muscle fibers increases and occurs in prenatal development and continues for the first few months of life (Aberle et al., 2001). Growth by hypertrophy is growth by enlargement of individual muscle fibers through increase in cross sectional area or diameter; therefore, the number of fibers present in a muscle is set at birth, and postnatal muscle growth is accomplished through muscle fiber hypertrophy (Pas et al., 2004). Muscle regeneration is needed in the postnatal period and throughout life when for both growth and muscle repair it is currently considered as a recapitulation of muscle development (Cossu and Biressi, 2005). An important difference between embryonic myogenesis and muscle regeneration is commitment to the myogenic fate is induc ed by Borello, 1999; Tajbakhsh, 2003). During post natal muscle regeneration, a quiescent but already myogenically committed cell is activated by signals emanating from infiltrating cells and damaged muscle (Charge and Rudnic ki, 2004). Skeletal Muscle Development Skeletal muscle development is initiated during the embryonic stage of development; multipotential cells become progressively committed to follow a defined differentiation pathway (Cossu and Borello, 1999). Skeleta l muscle in the vertebrate embryo is derived from somites that initially appear as spherical structures and are composed primarily of epithelial like mesoderm cells that differentiate into several cell

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24 types (Arnold and Braun, 1993). Embryonic myogenesis begins in newly formed somites where progenitors located in the dorso medial and in the ventro lateral lips of the dermomyotome respond to signals, such as Wingless and Int (Wnt) and Sonic hedgehog (Shh) which emanate from the adjacent neural tube, notocho rd and ectoderm, and activate basic helix loop helix transcription factors (Myf5 and MyoD) that commit cells to myogenesis (Cossu and Biressi, 2005). During embryogenesis, skeletal muscle arises from three different locations: segmented somite paraxial mes oderm, unsegmented paraxial head mesoderm, and prechordal mesoderm; the trunk and limb muscles originate from the somite epithelial dermomyotome (reviewed in Kalcheim and Ben Yair, 2005). Body muscles are derived from condensation of the paraxial mesoderm into the somites, which form along the rostro caudal axis of the embryo and are organized into dorso ventral compartments (Braun et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995). As development progresses somites differentiate into two regio ns the dermomyotome and the sclerotome; the sclerotome is formed from the ventromedial portion, making it the most vent r al part of the somite which will give rise to the axial skeleton, and the dermomyotome is formed from the dorso lateral portion and will give rise to the dermis and muscle progenitor cells (MPC; Braun et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995). The dorso medial, or epaxial, part of the dermomyotome and myotome gives rise to the back muscles, while the ventro lateral or hypaxial somite generates the trunk and limb muscles (Parker et al., 2003). Epaxial body wall muscles are formed from the dorso medial lip of the dermomyotome and hypaxial muscle, limbs, tongue, diaphragm,

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25 and ventral wall musculature are formed from the dorso lateral lip (Chevallier et al., 1977; Beddington and Martin, 1989; Ordahl and LeDouarin, 1992; Wilting et al., 1995; Kardon et al., 2002). The dermomyotome undergoes further change in which the borders make a transition from epithelial to mesenchym al cells to form the third somatic compartment the myotome, where the first differentiated myofibers are seen (reviewed in Bismuth and Relaix, 2010). In limb bud development progenitor cells respond to molecular signals from the adjacent lateral plate mes oderm and delaminate and migrate distally into the developing limb bud (Chevallier et al., 1977; Christ et al., 1977; Solursh et al., 1987; Hayashi and Ozawa, 1995). In mice, the myotome forms on embryonic day 9 (E9) in the mouse, followed by fusion of my oblasts to form primary fibers, at approximately day E 11 12 (reviewed by Cossu and Biressi, 2005). In bovine embryonic development the majority of muscle fibers form in the fetal stage between 2 months and 7 or 8 months of gestation; on day 21 of gestati on somites are visible (Russell and Oteruelo, 1981; Maddox Hyttel et al. 2003). Terminal differentiation of skeletal myoblasts involves alignment of the mononucleated cells, fusion into multinucleated syncitia, and transcription of muscle specific genes. A second wave of fiber formation in the mouse occurs at E 15 17, giving rise to secondary (fetal) fibers that are originally smaller and surround primary fibers (Zhang and McLennan, 1998). In the bovine fiber types are being discerned beginning around da y 120 at day 160 some fibers are negative for embryonic myosin heavy chain indicating secondary fiber formation (Martyn et al., 2004). Tongue Development The tongue is a unique muscle as it develops differently from other muscles. It is a complex arra y of muscles with many traits, the tongue is coated with sensors on the

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26 dorsal surface for taste, temperature, pain, and tactile information and performs the function of mixing, controlling, and propelling consumed food toward the throat and clear the mout h of food debris (Gilroy et al., 2008; Moore and Persaud, 2008). The mammalian tongue is composed of eight muscles, receives its blood supply from the lingual artery and is constituted by striated muscle, cranial neural crest cell (CNCC) derived mesenchym e, and stratified, squamous, non keratinized epithelium (Gilroy et al., 2008). The tongue is derived from all of the branchial arches (BAs) and development begins with the formation of a medial elevation on the floor of the pharynx (Huang et al., 1999). It is not clear whether the myogenic progenitors that migrate from the boundary of the trunk and head mesoderm to form the tongue share more characteristics with the trunk or head mesoderm, but the tongue muscles originate from the somites (Noden, 1983; Hu ang et al., 1999). Satellite Cells and Stem Cells Satellite cells are a heterogeneous population of myogenic precursors responsible for muscle growth and repair in mammals. Satellite cells are classically defined as quiescent mononucleated cells, locate d between the sarcolemma and the basal lamina of adult skeletal muscle (Bischoff, 1994). In 1961, Alexander Mauro discovered mononucleated cells existing between the plasma membrane and the basal lamina, and r position relative to the muscle fiber. Mauro (1961) proposed the cells were remnants of multinucleated muscle cells from embryonic development. These cells were theorized to be dormant myoblasts that did not fuse into the muscle cell but were lying in a quiescent state until stimulated by signals to repair the damaged muscle cell (Mauro, 1961).

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27 At its discovery, the satellite cell w as credited with the function of the cell responsible for growth and maintenance of skeletal muscle, yet it was not propo sed to be a stem cell (reviewed by Pault et al., 2007). Satellite cells are normally mitotically quiescent, but are activated and enter the cell cycle in response to stress induced by weight bearing or by trauma su ch as injury (Bischoff, 1994). B ecause these cells become activated following muscle damage triggering a number of cell divisions producing fusion competent cells and other cells that return to quiescence to maintain the progenitor pool, they represent a type of stem cell (Miller et al., 1999; Zammit and Beauchamp, 2001). During peri and post natal development, satellite cells divide at a slow rate and part of the progeny fuse with the adjacent fiber to contribute new nuclei and to increase to size of muscle fibers whose nuclei cannot divide, a number of studies have confirmed that the satellite cell is the principle source of muscle regeneration in the adult mouse (reviewed by Cossu and Biressi, 2005; reviewed by Pault et al., 2007). There are a number of molecular markers that have been de scribed that allow for identification of the majority of satellite cells these markers include Myf5, Pax7, M cadherin, CD34, vascular cell adhesion molecule 1 (VCAM 1), c met (receptor for hepatocytes growth factor), neural cell adhesion molecule 1 (CD56), Foxk1, and syndecans 3 and 4 (reviewed by Pault et al., 2007). LEK1 may serve as a useful marker for satellite cells that are preparing to fuse into adjacent fibers as well as an indicator of recently added myonuclei (Ouellette et al., 2009). Prolifera ting cell nuclear antigen (PCNA) expression in satellite cells can be used as a marker to follow entry of satellite cells into the cell cycle in primary mass cultures. (Johnson and Allen, 1993).

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28 Differences in marker expression indicates heterogeneity in the satellite cell population, however, all satellite cells express Pax7 (Seale et al., 2000). The descendants of activated satellite cells, called myogenic precursor cells, or myoblasts, undergo multiple rounds of division before fusion and terminal diff erentiation (Tajbakhsh, 2009). M yogenic R egulatory F actors Multiple pathways drive the embryonic process of cellular differentiation for formation of skeletal muscle. The Wnt family of secreted glycoproteins acts through autocrine or paracrine mechanis ms to influence the development of many cell types (Johnson and Rajamanan, 2006). Wingless and Int signaling causes cell proliferation, differentiation, or maintenance of precursor cells, and is crucial for myogenesis in fetal muscle (Du et al., 2010). W ingless and Int and Shh regulate paired box (Pax) 3 and Pax7 which regulates the action of myogenic regulatory factors (MRFs) on undifferentiated muscle cells are called myoblasts (Kassar Duchossoy et al., 2005). Myogenic regulatory factors (MRFs) mediat e the process of myogenic determination and muscle specific gene expression enabling multipotent, mesodermal cells to give rise to mononucleated myoblasts, withdraw from the cell cycle and differentiate into multinucleated muscle fibers, which are the fram ework of whole muscle (Stockdale et al., 1999). Skeletal muscle differentiation is dependent on four basic helix loop helix (bHLH) transcription factors, Myf5, MyoD, Myogenin, and Mrf4 (Myf6; to regulate myogenesis, forming a mature muscle fiber (Du et al., 2010). Myogenic factor (Myf) 5 and MyoD are considered to be the primary MRFs required for determination of skeletal myoblasts. Myf5 is the first gene expressed in all muscle progenitors (Ott et al., 1991). Originally it was believed mice lacking Myf5 die

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29 shortly after birth due to lack of ribs and rib cage and that muscle developed normally because of MyoD or mice with null mutations for both MyoD and Myf5 genes died shortly after birth from rib cage formation failure and complete lack of myoblasts and skeletal myofibers (Rudnicki et al., 1993). Further experiments revealed that the original knockout experiments actually were a triple knockout instead of a double knockout silencing or re moving Myf6 along with Myf5 and MyoD (Braun et al., 1992). Originally, Myf6 (also known as MRF4) was thought to act later as a differentiation factor (reviewed by Rudnicki and Jaenisch, 1995) however later research showed Myf6 acts as a determining factor in the absence of Myf5 and MyoD (Kassar Duchossoy et al., 2004). Myf 5 Expression of the muscle determination factor Myf5 is associated with proliferating myoblasts and tightly regulated by the cell cycle (Lindon, 1998). Myf5 functions as a myogenic fact or that is important for specification of muscle cells (Chen et al., 2007). Cell culture and i mmunocytochemistry of bovine satellite cells (BSC) reveals the majority of Pax7 expressing BSC also express Myf5 with a minor population failing to exhibit Myf5 immunoreactivity (Li et al., 2011). It is the first gene expressed in all muscle progenitors, beginning in the dorso medial lip of the dermomyotome, which rapidly generates the epaxial myotome (Ott et al., 1991; Buchberger et al., 2003 ). Myogenic progen itor cells become restricted to the epithelium of the dermomyotome when somites mature. The onset of myogenesis is defined by stable activation of the Myf5 promoter in the epaxial dermomyotome, which is followed by differentiation of Myf5 expression cells that have migrated under the dermomyotome (Buckingham, 2006). Myf5 is first activated in the dorsomedial edge in the epithelial and

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30 ball shaped somites beginning in the cranial region of the mouse embryo 8 days post conception (dpc; Ott et al., 1991). M yf 5 transcripts are still present in differentiated myotomal cells and start to appear in the pre muscle masses of the limb buds in mice around 10.5 dpc (Ott et al., 1991; Buchberger et al., 2003; Arnold and Braun, 1993). However, in mice beginning aroun d 11.5 days post conception Myf5 expression decreases rapidly throughout the embryo; Pax3 and Pax7 W ingless and I nt and Shh regulate expression of Pax3 and Pax7, which then initiate expression of myogenic regulatory factors (MRFs; Munsterberg et al., 1995 ; Stern et al., 1995; Petropoulos and Skerjanc, 2002). P aired box (Pax ) 7 and Pax3 genes arose by duplication from a unique ancestral Pax3/7 gene, and similarities in their protein sequence and expression pattern reflect this common origin (Relaix et al., 2004). The paired box (Pax) family of transcription factors has important functions in the regulation of development and differentiation of diverse cell lineages during embryogenesis (Mansouri et al., 1999). Pax3 and Pax7 are together essential for the myogenic potential, survival, and proliferation of myogenic progenitors (Relaix et al., 2005). After the initial formation of the myotome subsequent embryonic myogenesis depends on the expression of Pax3 and Pax7, making these factors key upstream regula tors of the myogenic process (Relaix et al., 2005). During gestation, Pax3 and Pax7 expression patterns diverge (Horst et al., 2006). Pax 3 is essential for skeletal myogenesis and acts upstream of MyoD during skeletal muscle development (Ridgeway and Ske rjanc, 2001; Gustafsson et al., 2002). Pax7 is most commonly known for its role postnatally in which it is required for the presence of satellite cells;

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31 however in culture of primary muscle fibers Pax7 is expressed in proliferating primary myoblasts and i s down regulated after myogenic differentiation and expressed strictly in myogenic cells (Seale et al., 2000). Pax3 plays an essential role in regulating the developmental program of MyoD dependent migratory myoblasts during emb ryogenesis (Maroto et al., 1997 ). Pax3 is initially expressed in the presomitic mesoderm (Fan and Tessier Lavigne, 1994) and soon after somite formation Pax3 expression becomes restricted to the dermomyotome, with higher expression levels at the lateral lip. Pax3+/Pax7+ cells at the central just beneath the dermomyotome and abutting the Myf5+ and MyoD+ myotome this is most likely the source for embryonic muscle expansion (Relaix et al., 2005). Pa x3 + /Pax7 + progenitors originating in the embryonic somite are thought to be the precursors of satellite cells in adult muscle (Kassar Duchossoy et al., 2005; Relaix et al. 2005). Splotch (Sp) mice, lacking a functional Pax3 gene, do not survive to term an d fail to form limb muscles owing to impaired migration of Pax3 expressing cells originating from the somite (Daston et al., 1996). Compound mutant Sp/Myf5 / mice do not express MyoD in their somites, suggesting that Myf5 and Pax3 function upstream of My oD in myogenic determination (Tajbakhsh et al. 1997). Committed myogenic progenitors (that express Pax3 but not Myf5 or MyoD) migrate from somites to the limb (Buckingham et al., 2003). Pax7 can efficiently substitute for Pax3 during somite segmentation and in the development and maintenance of the dermomyotome ; myogenesis proceeds normally in the trunk in the absence of Pax3 (Buckingham, 2001) Expression of Pax7 starts in

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32 the dermomyotome of more mature somites (Fan and Tessier Lavigne, 1994), and is evenly distributed in the medial and central portions of the dermomyotome. It is required for maintenance and function of many satellite stem cells (Relaix et al., 2004, 2005; Seale et al., 2000). Pax7 expression is detected in mono nucleated cells assoc iated with trunk and limb muscles throughout embryogenesis and the cells are associated with the sublaminal space of myofibers (Relaix et al., 2005). Lepper and Fan (2010) determined in mice that between E9.5 and E10.5, Pax7 expressing cells are multi pot ent and only become restricted to the myogenic lineage after E12.5; limb muscles and their satellite cells do not come from dermomyotomal cells that express Pax7 at E9.5. During the embryonic stage, a portion of cells in the mesoderm first express Pax3 a nd Pax7, and then these cells express the MRF myogenic factor 5 (Myf5) and myogenic differentiation 1 (MyoD; Buckingham, 2001). In culture of BSC the majority of cells that express Pax7 also express Myf5. In adult muscle, most satellite cells express Myf 5 with the exception of a small subpopulation of Pax7+ satellite cells that have never expressed Myf5. In cultures of BSC from young animals rapid rate of fiber formation is apparent as the percentage of Pax7:Myf5 decreases at a faster rate than adult BSC Pax7 also plays a role in the development of the olfactory epithelium (OE; Davis and Reed, 1996; LaMantia et al., 2000; Murdoch et al., 2010). Pax7 is more widely expressed in the developing mouse embryo at E9.5 in the hindbrain, forebrain, and front onasal mesenchyme (Murdoch et al., 2010), and it is present in the nose at E10 to E14.5 (Jostes et al., 1991). By E11.5, the mesenchyme of the lateral nasal process expresses Pax7, which in the OE showed a ventral to dorsomedial gradient (Murdoch et

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33 al., 2010). In muscle, Pax7+ satellite cells contribute to growth and regeneration (Charge and Rudnicki, 2004); most likely, Pax7+ olfactory precursors also represent a hierarchical heterogeneous progenitor cell population with mostly committed progenitors (Mu rdoch et al., 2010). Growth and development has been well documented in the mouse, extensive research has been conducted examining the myogenic cascade, the origin and determination of proliferating myoblasts as well as muscle fiber development. Some exa mination of bovine development has been conducted however there is a void in this research as the majority of the work focuses on early gestation up to day 21 and development shortly before the second trimester around day 85 of gestation. Ultrasonography allows for examination of fetal growth throughout gestation. Monitoring rate of fetal growth and defining early bovine myogenesis through immunohistochemistry during the time point of gestation where there is a void in the literature will provide better i nsight to bovine fetal development.

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34 CHAPTER 3 DIFFERENCES MEASURED BY ULTRASONOGRAPHY I N BOVINE FETAL GROWT H IN EARLY GESTATION B ETWEEN ANGUS AND BRA NGUS CATTLE Background The variation that exists in biological traits important for beef production is va st; crossbreeding is used to exploit heterosis in cattle herds (Cundiff et al., 1986). An estimated 30% of cattle in the United States contain some percentage of Bos indicus genetics (Morrison, 2005; Chase et al., 2005). Brahman calves are reported to be heavier at birth when compared to Angus calves (Paschal et al., 1991). One common cross found in Florida is the Brangus which is 5/8 Angus and 3/8 Brahman, this breed reaches reproductive maturity earlier than and a shorter gestation length than Brahman cattle, as well as increased tenderness and marbling when compared Brahman cattle (Reynolds et al., 1980; Elzo et al., 2012). Ultrasound is becoming a tool utilized more frequently as a means of measuring fetal size in vivo during early embryonic growth ( Curran et al., 1986; Riding et al, 2008). Ultrasonography provides the opportunity to improve the methods of evaluation of ovarian function and diagnoses of pregnancy in beef cattle and provides a way of examining fetal growth in vivo Embryonic vessels are first detected in bovine heifers at 11.7 days of gestation, detection of embryo proper at 20 days of gestation and heartbeat has been detected on the first day of detection of the embryo proper or the following day (Curran et al., 1986). The growth c urve of the embryo is quadratic from days 20 60 of gestation, with an increasing growth rate after approximately day 50; detection of various structures can be detected as development progresses: allantois, 23.2 days, forelimbs 29.1 days, hind limbs 31.2 days, placentomes 35.3 days, optic

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35 lens, 40.0 days, split hooves 44.6 days, fetal movements 44.8 days, and ribs 52.8 days (Curran et al., 1986). At birth weights of calves among breeds vary, Angus calves at birth weigh the least when compared to Brahman and Brangus (Reynolds et al., 1980; Casas et al., 2010). The time during gestation at which fetal weights diverge due to breed is not clearly defined, morphological differences between Bos taurus and Bos indicus breeds are evident by Day 100 of gestation (Lyne, 1960). There is some speculation this event likely occurs during the first trimester (Lyne, 1960; Holland and Odde, 1992) while others suspect maternal ability in late gestation plays a role in differences of weights between breeds (Ferrell, 1991a ). Correlation between birth weight and gestation length is considered to be positive and is low to moderate in magnitude, fetal growth rate near term varies between 100 and 2 50g/day (Burfening et al., 1978; Prior and Laster, 1979). Thus while extended ge station periods result in additional fetal weight gain, the actual magnitude of the increase in birth weight is slight (Holland et al., 1990). In this experiment, ultrasonography was used to measure fetal sizes (crown rump length) at weekly intervals over a four week period in the first trimester of pregnancy beginning on day 33 of gestation and ending on day 55 of gestation. This experiment set out provide an initial insight to understanding the potential causes in birth weight variations between breeds b y determining when variation in growth rates occurs between developing Angus and Brangus fetuses. Materials and Methods Santa Fe River Ranch Unit were artificially inseminated to pre assigned multiple sires within their respective breeds. The average age for Angus cows was 5 3 years (range

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36 2 11 yrs) and Brangus cows was 5 2 years (range 2 9 yrs). Body condition scores (1 = severely emaciated; 5 = moderate; 9 = very obese; Wagner et al., 1988) were taken monthly by two individuals. A t the time of insemination body condition scores ranged from 3.0 to 6.5 (x = 4.7 0.7) for Angus and 4.0 to 6.5 (x = 5.2 0.7) f or Brangus Prior to the start of AI cows were on one of two nutrition plans. They were either 1) limit grazing on rye ryegrass pasture for 2 hrs daily or 2) supplemented 3 days/week with whole cottonseed (WCS) at a rate of 0.5% of the pen mean body weight per day (3.3kg DM/day). All cows had ad libitum access to Coastal be rmudagrass ( Cynodon dactylon L.) hay throughout the time period before AI. After breeding all cows were on pasture and did not receive any additional supplement or feed, thus during the time period cow were pregnant, they were just on pasture. All cow s over 30 d postpartum were randomly assigned to either a 5 day Select Synch CIDR and Timed AI (5D) or a Modified 7 day Select Synch CIDR and Timed AI (7D) synchronization program. On day 0, cows received a CIDR an 25 mg Prostaglandin F2 (Lutalyse Sterile Solution, Pfizer). On day 2, 5D cows received a CIDR (Eazi Breed CIDR, Pfizer) and all cows in 5D and 7D treatments received a shot of gonadotropin releasing hormone (Cystorelin, Merial, GnRH). On day 7, CIDRs were removed from all c ows in both treatment groups; all cows received 25 mg FGF at CIDR removal and 25 mg PGF ap proximately 6 8 hrs later. For 72 hrs cows were observed for estrus, those in estrus were bred via artificial insemination (AI) 8 12 hrs after the onset of estrus All cows that did not exhibit estrus by 72hrs after CIDR removal received GnRH and were timed AI. Pregnancy was detected approximately 30 days after AI with ultrasonography.

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37 Beginning on d 33, each pregnancy was evaluated by trans rectal ultrasonog raphy by a single ultrasound technician, using an Aloka 500V machine equipped with a 5.0 MHz transducer and fetal crown rump length (CRL) was measured. CRL was measured at d33/34, d40/41 and d47/48. At d54/55, crown to nose was measured and converted to CRL (Riding et al., 2008). y = 0.3203x + 3.3978, R2=0.9899 The fetal measurements from the ultrasound data were analyzed with mixed procedures, repeated measures ANOVA in SAS (SAS Inst. Inc.) using a 4 x 2 factorial design looking at length on da y of gestation by breed effects, with P<0.05 considered significant. In Figure 4 1b percent growth was calculated by taking the difference between fetal lengths on two consecutive days of measurement divided by total growth for the whole period of measure ment. Results and Discussion Early gestation growth patterns differ for Angus and Brangus fetuses. Angus and Brangus cows impregnated through a timed artificial insemination program were examined weekly and fetal size was measured by ultrasonography. Cro wn rump length was measured at d33/34, d40/41 and d47/48 of gestation. At d54/55, crown to nose was measured and converted to CRL (Riding et al., 2008). Brangus fetuses (n=30) tended ( P = 0.06) to be larger than Angus fetuses (n=44) on d 33 of gestation (1.38 0.03 cm, and 1.30 0.02 cm, respectively; Figure 3 1A). Fetal CRL on d 40 and d 47 did not differ between the breeds. At d 55 of gestation, measurements of crown snout lengths (CSL) indicate Angus fetuses were larger ( P < 0.05) than Brangus fetu ses with CSLs of 2.08 0.03 cm and 1.95 0.03 cm, respectively. Calculation of CRL of d

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38 55 fetuses maintains that Angus fetuses were larger ( P < 0.05) than Brangus fetuses with CRLs of 5.44 0.08 cm and 3.65 0.09 cm, respectively. Angus gestation len gth is shorter compared to most breeds of cattle, in the Reynolds et al. (1980) study straightbred Angus calves average 280 day gestation where Brahman averaged 291.1 days and Brangus averaged 286 days. Shorter period of time in gestation means Angus fetu ses must develop at a faster rate than other breeds of cattle. The rapid increase in CRL demonstrated by the Angus calves suggests divergent patterns of growth between the two breeds during the late embryo and early fetal period. Weekly growth as a perce nt of total growth for the period was calculated (Figure 3 1B). Angus and Brangus fetuses exhibited similar relative growth at wk 5 (15.51 1.01 and 12.78 1.38, respectively) and wk 6 of gestation (28.69 1.04 and 30.46 1.77, respectively). At wk 7 of gestation, Angus fetuses demonstrated a robust increase ( P < 0.05) in relative growth rate by comparison to Brangus contemporaries. For the observation period, Angus fetuses accomplished 55.79 1.10 % of their total growth during wk 7 of gestation, a time synonymous with the beginning of fetogenesis. Angus fetuses (4.14 0.08 cm) had greater ( P < 0.05) growth rates than the Brangus fetuses (3.65 0.09 cm). Additional studies of crossbred cattle have demonstrated that animals with Angus inheritan ce have the shortest gestation period averaging 284 days and those with Brahman inheritance are among the longest gestation lengths averaging 294 days (Browning et al., 1995; Casas et al., 2010). In addition to length of gestation affecting fetal growth, the breed of sire is also a factor for consideration. Research has shown there is a breed of sire effect on birth weight;

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39 Brahman sires increase birth weights compared with Bos taurus sire breeds when bred to Bos taurus cows (Roberson et al., 1986; Comerf ord et al., 1987; Paschal et al., 1991). Implications Trajectory growth indicates Angus fetuses grow more rapidly than Brangus fetuses at the entry of the fetal period at d 55 of gestation. This knowledge is beneficial to those in the beef industry as it provides better insight into when variation in size of breeds occurs gestationally which manifests itself by variation in birth weights between different breeds of cattle. Additionally, this ultrasonographic examination provides a starting point of when i n gestational development of the fetus to begin tracing the myogenic lineage to examine muscle growth and development.

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40 A. B. Figure 3 1 Bovine fetal measurements and growth during early gestation on days 33, 40, 47, and 55 of gestation. Brangus fetuses tended (P = 0.06) to be larger than Angus fetuses on day 33, fetuses were similar (P > 0.05) in size on days 40 and 47, by day 55 of gestation, Angus fetuses were (P < 0.05) larger than Brangus fetuses (A). Angus and Brangus fetuses had similar (P > 0.05) growth percentages at week 5 and week 6, at week 7 (d 47 54) Angus fetuses had a significantly higher percent growth than Brangus fetuses (P < 0.05) (B). 0 1 2 3 4 5 6 33 40 47 55 Crown rump length, cm Gestation, days Angus Brangus # 0 10 20 30 40 50 60 5 6 7 Percent growth Gestation, wks Angus Brangus

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41 CHAPTER 4 SPATIAL AND TEMPORAL EXPRESSION OF MYOGEN IC PROTEINS IN THE E ARLY BOVINE EMBRYO Background Muscle growth and development and its regulatory factors are well studied in chicken, mice, and zebra fish however minimal research in the area of bovine muscle development has been conducted. Most of the research examines very early ge station or later gestational development from day 9 21 of gestation and then from gestation day 85 and later ( Alexopoulos et al., 2008 ; Maddox Hyttel et al., 2003 ; Martyn et al., 2004 ) On day 21, Maddox Hyttel et al. (2003) had much variation in embr yo sizes and in w hat was developing; the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites, and allantois development with a gradual formation of the endo derm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate, and lateral plate mesoderm. B eginning around day 120 muscle fiber types are being discerned into Type I or Type II fibers ( Martyn et al. 2004). The average sizes of primar y myotubes initially decrease from 120 to 160 days of gestation as they developed into mature myofibers (Strickland 1978; Martyn et al ., 2004). The disappearance of fetal myosin heavy chain indicates muscle contractile diffe rentiation from day 180 onward s (Cassar Malek et al., 2007). Skeletal muscle matures during late gestation in cattle at approximately day 210 of gestation (Greenwood et al., 1999). Skeletal muscle development is initiated during the embryonic stage of development; multipotential cells become progressively committed to follow a defined differentiation pathway (Cossu and Borello, 1999). Embryonic myogenesis begins in newly formed somites where progenitors located in the dorso medial and in the ventro

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42 lateral lips of the dermomyotome res pond to signals which emanate from the adjacent neural tube, notochord and ectoderm, and activate basic helix loop helix transcription factors that commit cells to myogenesis (Cossu and Biressi, 2005). Skeletal muscle arises from three different locations : segmented somite paraxial mesoderm, unsegmented paraxial head mesoderm, and prechordal mesoderm; the trunk and limb muscles originate from the somite epithelial dermomyotome; body muscles are derived from condensation of the paraxial mesoderm into the so mites, which form along the rostro caudal axis of the embryo and are organized into dorso ventral compartments (Braun et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995). Somites differentiate into two regions the dermomyotome and the sclerotom e; the sclerotome will give rise to the axial skeleton, and the dermomyotome is formed from the dorso lateral portion and will give rise to the dermis and muscle progenitor cells (MPC; Braun et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995). In limb bud development progenitor cells respond to molecular signals from the adjacent lateral plate mesoderm and delaminate and migrate distally into the developing limb bud (Chevallier et al., 1977; Christ et al., 1977; Solursh et al., 1987; Hayashi and O zawa, 1995). In bovine embryonic development the majority of muscle fibers form in the fetal stage between 2 months and 7 or 8 months of gestation; on day 21 of gestation somites are visible (Russell and Oteruelo, 1981; Maddox Hyttel et al. 2003). Termin al differentiation of skeletal myoblasts involves alignment of the mononucleated cells, fusion into multinucleated syncitia, and transcription of muscle specific genes. A second wave of fiber formation in the mouse occurs at E 15 17, giving rise to second ary

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43 (fetal) fibers that are originally smaller and surround primary fibers (Zhang and McLennan, 1998). In the bovine fiber types are being discerned beginning around day 120 at day 160 some fibers are negative for embryonic myosin heavy chain indicating s econdary fiber formation (Martyn et al., 2004). The tongue is a unique muscle as it develops differently from other muscles. It is a complex array of muscles with many traits, the tongue is coated with sensors on the dorsal surface for taste, temperat ure, pain, and tactile information and performs the function of mixing, controlling, and propelling consumed food toward the throat and clear the mouth of food debris (Gilroy et al., 2008; Moore and Persaud, 2008). The tongue is derived from all of the br anchial arches (BAs) and development begins with the formation of a medial elevation on the floor of the pharynx (Huang et al., 1999). It is not clear whether the myogenic progenitors that migrate from the boundary of the trunk and head mesoderm to form t he tongue share more characteristics with the trunk or head mesoderm, but the tongue muscles originate from the somites (Noden, 1983; Huang et al., 1999). During peri and post natal development, satellite cells divide at a slow rate and part of the prog eny fuse with the adjacent fiber to contribute new nuclei and to increase to size of muscle fibers whose nuclei cannot divide, a number of studies have confirmed that the satellite cell is the principle source of muscle regeneration in the adult mouse (rev iewed by Cossu and Biressi, 2005; reviewed by Pault et al., 2007). There are a number of molecular markers that have been described that allow for identification of the majority of satellite cells these markers include Myf5, Pax7, M cadherin, CD34, vascu lar cell adhesion molecule 1 (VCAM 1), c met (receptor for hepatocytes growth

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44 factor), neural cell adhesion molecule 1 (CD56), Foxk1, and syndecans 3 and 4 (reviewed by Pault et al., 2007). LEK1 may serve as a useful marker for satellite cells that are p reparing to fuse into adjacent fibers as well as an indicator of recently added myonuclei (Ouellette et al., 2009). Proliferating cell nuclear antigen (PCNA) expression in satellite cells can be used as a marker to follow entry of satellite cells into the cell cycle in primary mass cultures. (Johnson and Allen, 1993). Differences in marker expression indicates heterogeneity in the satellite cell population, however, all satellite cells express Pax7 (Seale et al., 2000). Multiple pathways drive the embry onic process of cellular differentiation for formation of skeletal muscle. Wingless and Int and Shh regulate paired box (Pax) 3 and Pax7 which regulates the action of myogenic regulatory factors (MRFs) on undifferentiated muscle cells are called myoblasts (Kassar Duchossoy et al., 2005). Myogenic regulatory factors (MRFs) mediate the process of myogenic determination and muscle specific gene expression enabling multipotent, mesodermal cells to give rise to mononucleated myoblasts, withdraw from the cell cycle and differentiate into multinucleated muscle fibers, which are the framework of whole muscle (Stockdale et al., 1999). Skeletal muscle differentiation is dependent on four basic helix loop helix (bHLH) transcription factors, Myf5, MyoD, Myogenin, a nd Mrf4 (Myf6; Weintraub et al., mature muscle fiber (Du et al., 2010). Myogenic factor (Myf) 5 and MyoD are considered to be the primary MRFs required for determination of ske letal myoblasts. Myf5 is the first gene expressed in all muscle progenitors (Ott et al., 1991).

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45 Limited literature exists in bovine development during gestation, past research shows somites are forming at d 21, fetogenesis begins on d 45, and secondary myogenesis begins by d 84, however little is known about development between d 21 and d 84. Through immunohistochemistry this experiment defines early bovine myogenesis at d 28 and d 45 of gestation, revealing some similarities to mouse development but al so some differences from mouse development. Materials and Methods Embryo Collection Non lactating Holstein cows (n=20 (Cystorelin; Merial) and fitted with a vaginal progesterone insert (CIDR; Pfizer). After 7 six hours post PGF2 treatment; the cows receiv inseminated with Holstein semen 18 hr later. Time of insemination was denoted as d0, pregnancy was determined by ultrasonography for each insemination, 4 5 cows were diagnosed as pregnant Cows were killed by cap tive bolt stunning and exsanguination at d28 or d45 after insemination and embryos were collected from the uterus. Embryos were dissected free of placental fluid and structures and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 60 m inutes on ice. Subsequently, the tissues were rinsed with PBS and infiltrated with 30% sucrose in PBS overnight at 4 C. Embryos were embedded in Optimal Cutting Temperature (OCT) compound and frozen in super cooled isopentane, the heads were removed from the bodies of d 45 embryos prior to embedding and frozen separately. Processed embryos were stored at 80 C.

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46 Whole Mount Myosin Immunostaining E mbryos (d28) were fixed sequentially in 4% paraformaldehyde for 60 min and 4% paraformaldehyde containing h ydrogen peroxide (5% final concentration) for 20 minutes. Fixed embryos were incubated overnight at 4 C with PBS containing 1% bovine serum albumin and 0.5% Triton X100 (PBS.5T) to permeabilize membranes and block nonspecific antigen binding sites. Embry os were incubated sequentially with anti myosin heavy chain (MyHC) hybridoma supernatant (MF20; Developmental Hybridoma Studies Bank (DSHB), Iowa City, IA) at 4 C overnight followed by extensive washing with PBS.5T. Following and overnight incubation with goat anti mouse peroxidase (1:1000; Invitrogen) at 4 C, immune complexes were visualized colorimetrically with 3,3 diaminobenzidine and nickel chloride. Representative photomicrographs were captured with NIS Elements using a DXM1200F CCD camera attached to a stereomicroscope (SMZ1500; Nikon Instruments) Immunohistochemistry All embryos were sectioned into ten micron cryosections were collected onto glass slides (SuperFrost, Fisher) and allowed to air dry for 60 min at room temperature. Cryosections were incubated with PBS containing 2% bovine serum albumin and 0.1% TritonX 100 (PBS.1T) for 20 min at room temperature to remove nonspecific antigen sites. For all embryos primary antibodies and conditions include anti MyHC (DSHB) and anti desmin (DSHB) hybri doma culture supernatant, 60 min at room temperature, anti Pax7 (DSHB) hybridoma culture supernantant, overnight, 4 C and anti Myf5 (Santa Cruz Biotechnology), 1:100 in PBS.1T, overnight at 4 C. Following incubation with the appropriate secondary antibody (donkey anti rabbit AlexaFluor 568; goat anti mouse AlexaFluor 488), immunocomplexes were visualized on a Nikon TE2000U equipped

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47 with epifluorescence, a digital camera (CoolSnap EF, Photometris) and NIS Elements software. Results and Discussion Morphol ogical F eatures of B ovine E mbryos at d 28 G estation Embryos were harvested on d 28 of gestation from Holstein cows at slaughter. After removal of placental structures and fixation in paraformaldehyde, the embryos were imaged and measurements were recorde d. Gross morphological features of bovine embryos at d 28 of gestation are shown in Figure 4 1 Panel A is a representative embryo and panel B demonstrates the areas used for body measurements and head lengths were measured s omite pairs were enumerated ( C and D ), and panel E shows t he initial stages of forelimb development. In the mouse embryo, somites form from about 8 days post coitum (dpc) in an anteroposterior gradient by segmentation of the paraxial mesoderm (Rugh, 1990). In addition the developing heart is visible in panel F the formation of the h indbrain and isthmus formation at rhomboid 2 4 in panel G and there are t hree visible pharyngeal arches panel H Morphometric for the embryos are recorded in Table 4 1. The body length of the d 28 embr yos averaged 6.0 0.5 mm and 40 2 somite pairs were noted. Whole M ount M yosin I mmunostaining I ndicates P resence of S keletal M uscle in M yotome Muscle represents the largest tissue in the body. The extent of myogenesis was examined in early bovine embryo s. In brief, intact e mbryos (d28) were incubated with anti myosin heavy chain (MyHC) and immune complexes were visualized colorimetrically with 3,3 diaminobenzidine and nickel chloride. Whole mount immunocytochemistry revealed the presence of skeletal mu scle within the myotome

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48 compartment of the somite (Figure 4 2 ). E xtensive myosin immunoreactivity is found in the somites and heart. Rostral somites exhibit more robust myosin immunostaining than caudal somites (Figure 4 2 A). Myo t omes adjacent to the pr esumptive limb bud (LB) contain 5 10 primary muscle fibers (Figure 4 2 B and C). Transverse cryosections were collected at the level of the indicated somite in Panel A and incubated with a fluorescent tagged second antibody. Representative images demonstr ate that the myotome muscle fibers are positioned immediately below the dermamyotome (Figure 4 2 D). Multiple fibers are evident that are grouped together and not found as a single cell layer of tissue. The arrangement is analogous to that found in mammal s and birds. Immunofluorescence S taining R eveals M yogenic C ells in d 28 E mbryos In mammals myogenesis occurs in two phases, the first phase begins before embryonic day (E) 12.5 in the mouse where the primary muscle fibers that form will constitute approxim ately 20% of the muscle in the newborn (Kablar and Belliveau, (2005). After delamination of cells from the dermomyotomal layer, myogenesis starts by stable expression of the myogenic regulatory factor (MRF) Myf5 followed by expression of MyoD myogenin and finally MRF4 (Neuhaus and Braun, 2002). A d 28 bovine embryo is closely similar in development to a 10 10.5 dpc mouse embryo Cryosections of d 28 embryos were collected in the transverse plane through the mid point of the presumptive limb bud (Fi gure 4 3 A). The tissue sections were immunostained with anti MyHC, anti Myf5 and the appropriate fluorescent secondary antibody (Figure 4 3 B). Myoblasts, as defined by Myf5 immunoreactivity, represent a discrete cell population that does not overlap wit h the primary myofibers. In the mouse, Myf5 occurs in the rostral somites around 8 days post coitum (dpc) and is down regulated after 14 dpc with maximal accumulation of M yf 5 transcripts visible between

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49 10.5 and 11 days of develo pment in the mouse (Ott et al., 1991). In d 28 bovine embryos co staining for MyHC and Myf5 reveal m yoblasts positioned near dorsal lip of dermomyotome with primary muscle fibers in medial and ventral regions below somite structure this is c onsistent with new myoblasts forming f rom the dorsal lip of the dermomyotome. Closer examination of the myotome compartment demonstrates that the myoblasts are positioned near the dorsal lip of the dermamyotome with primary fibers located in the medial and ventral regions below the distinctiv e somite structure (Figure 4 3 C). This immunostaining pattern is consistent with new myoblasts forming from the dorsal lip of the dermamyotome. Limb muscles are formed by cells that delaminate from the hypaxial dermomyotome of somites facing the limbs a nd then migrate into the limb bud; muscle progenitor cells initiate migration around the 20 somite stage (E9.5) to the forelimb and hypoglossal chord (Relaix et al. 2004). Myf 5 and MyoD are not expressed until after the Pax 3 expressing migratory cells ha ve arrived in the limb bud (Tajbakhsh and Buckingham, 1994). A noticeable feature of the Myf5 localization pattern was the unexpected immunoreactivity found in the presumptive forelimb bud (Figure 4 4 ). Immunofluorescence is observed throughout the epit helial barrier (surface ectoderm) surrounding the embryo with localization to the cytoplasm or extracellular membrane. The ring of antigenic activity is regarded as a non specific ridge effect. Within the limb bud, anti Myf5 denotes mesoderm derived cel ls with localization of the protein in the nucleus and perinuclear compartment (Figure 4 4 ). The intensity of the limb mesoderm field immunoreactivity is stronger than that found in the myoblasts within the myotome (Figure 4 3 C). The heightened fluoresc ent intensity also is noted in the lateral plate

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50 mesoderm located anterior to the limb bud (Figure 4 5 ). Second antibody only controls are void of immunofluorescence. These results argue that anti Myf5 recognizes a specific epitope within myoblasts as we ll as within cells of the somatapleure. In the mouse limb bud, Myf5 is expressed transiently between days 10 and 12 (Ott et al., 1991). Transgenic mouse lines and numerous transient transgenic embryos for 58/ 56 Myf5 enhancer conferred robust muscle s pecific expression in myotome and in muscle progenitor cells in limbs of mouse embryo (Buchberger et al., 2007). Yvernogeau et al., (2012) established a timetable for mouse forelimb colonization by endothelial and myogenic precursors looking at expression of Pax3 positive cells, onset of delamination of somite derived Pax3+ myogenic cells towards the forelimb takes place at 9 dpc, when endothelial cells have already invaded the limb bud and formed a vascular plexus. Staining of d 28 bovine limb mesoderm w ith anti Myf5 exhibits stronger immunoreactivity than the myoblasts in the myotome. Myf5 detection in developing embryos begins to decrease from 11.5 days and onward (Ott et al., 1991) less intense staining in the myotome is expected as Myf5 expression de creases throughout the embryo and limb mesoderm myoblasts are in the beginning phases of muscle formation. Mouse into chicken chimeras reveals mouse presomitic mesoderm first emits endothelial cells, followed by myogenic cells delaminating from the somite (Yvernogeau et al., 2012). Morphological F eatures of B ovine E mbryos at d 45 G estation Embryos (n=4) were harvested from non lactating Holstein cows (n=5; 3 0.4 yr) at slaughter on d 45 of gestation. After removal of placental structures and fixation in paraformaldehyde, the embryos were imaged and measurements were recorded. Gross morphological features and measures of d45 embryos are shown in Figure 4 6

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51 Embryos were approximately 28 mm in length and 10 mm in width at the thoracic level with little inter animal variation (Table 4 2). The forelimb was slightly larger than hind limb, a reflection of the anterior to posterior developmental progression. Cloven hooves were noted placing the embryo within the ungulate order of mammals but no other easil y distinguishable features characteristic of bovidae was evident. Myogenesis in the d45 L imb and I ntercostal M uscles The extent of skeletal myogenesis during the late embryonic and early fetal period was examined through immunolocalization of myosin, Myf5 and Pax7. Cryosections were incubated with the respective muscle antibodies coupled with fluorescent detection of antigens. As expected large multinucleated myosin expressing fibers are present within the developing forelimb (Figure 4 7 A, B). It does no t appear that the individual fibers span from origin to insertion indicating that myoblast addition to the growing tips is delayed by comparison to limb elongation. Numerous myoblasts are interspersed throughout the muscle bed as noted by large numbers of Myf5 expressing cells (Figure 4 7 C). Similar to the observation for myotome muscle development, Myf5 nuclei do not appear within the fiber boundaries. The identity of the presumptive myoblasts was further explored by co localization of Myf5 and Pax7. S ubstantial numbers of Pax7 immunopositive cells are found within the forelimb muscle bed (Figure 4 8 ). Many of these cells also express the committed myoblast marker, Myf5. However, distinct Pax7 only cells are located within the muscle. These results i ndicate that satellite cells, as denoted by Pax7 expression, infiltrate the limb structures and contribute to the myoblast pool responsible for initial myofiber formation. In the mouse, between E 9.5 and E 10.0, dermomyotomal cells from the ventrolateral edge involute and form the hypaxial myotome and the somatic bud,

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52 contributing progenitors to the intercostal and ventral body wall muscles (reviewed in Buchberger et al., 2003). E mbryonic limb Pax7 transcripts are first detected in the mouse at E11.5 in t he proximal limb and then by E12.5 in more distal limb muscles as well (Relaix et al. 2004). Immunostaining of d 45 bovine forelimb suggests that primary muscle fibers are present in the forelimb upon completion of embryogenesis at d 45, which is equivale nt of a 14.5 dpc mouse embryo. In the bovine embryo at d 45 Pax7 is expressed in the developing limb structures and intercostal muscles. There are similar findings in the mouse where expression of Pax7 reveals multinucleated muscle fibers in the trunk an d limbs of E 13.5 to E 15.5 mice (Relaix et al., 2005). Pax7 is most commonly known for its role postnatally in which it is required for the presence of satellite cells; however in culture of primary muscle fibers Pax7 is expressed in proliferating primar y myoblasts and is down regulated after myogenic differentiation and expressed strictly in myogenic cells (Seale et al., 2000). Migratory myoblasts that populate the limb structures originate from the dorsal medial lip of the dermamyotome (Chevallier et a l., 1977). Intercostal muscles are formed from myogenic cells that originate from the ventral lateral aspects of the myotome (Parker et al., 2003). Saggital cryosections collected from a d45 embryo were incubated with phalloidin AlexFluor568 and anti Pa x7 (Figure 4 9 ). Again, large numbers of Pax7 expressing cells are evident within the forelimb muscle and lie adjacent to the presumptive fibers. A similar localization pattern is observed for the intercostal muscles. These results suggest that Pax7 exp ressing cells are present throughout all aspects of the dermamyotome and migrate to all muscle forming regions of the bovine embryo. Indeed, Pax7 is expressed throughout the early somite compartment at d28 of embryogenesis this supports the

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53 argument for migration of committed myoblasts. Phalloidin intercalation into actin fibers provides a stark outline of the somite structures in transverse cryosections collected from a d28 embryo (Figure 4 1 0 ). Central to the dermatome and myotome is a population of Pax7 immunopositive cells. The stripe of cells extends from the dorsal aspects of the somite compartment to the ventral lip portion of the dermamyotome. The expression of Pax7 in mice is seen in the dermomyotome, however, this expression is mainly restri cted to the central region and is excluded from the epaxial and hypaxial extremities (Relaix et al., 2004; Tajbakshs et al., 1997). The presence of these cells argues that myogenic cells in the bovine embryo migrate as committed myoblasts to the muscle fo rming regions, similar to that of avian and mouse embryos. Marcelle et al., (1995) found the chicken differs, where Pax7 transcripts are not detected in muscle progenitor cells migrating to the limb. Primary M uscle Development in Tongue of d 45 E mbryo The tongue was chosen to look at muscle development in a non traditional muscle as development of the tongue is different from other muscles. Muscles of the tongue and face form by migration of precursor cells from the somites in concert with cells that come from the neural crest (for review see Parada et al., 2012). Myogenic precursors delaminate from the lateral lip of the dermamyotome of the occipital somites and migrate through the hypoglossal cord into the pharyngeal arch. The first pharyngeal arc h gives rise to the tongue. The tongue contains an oral component that is freely mobile and represents nearly 2/3 of the entire structure with the remaining posterior portion acting as a fixed anchoring muscle (Wedeen et al., 2001). Myosin immunocytochem istry reveals that the anterior tip of the tongue contains at least two distinct muscle groups with fiber orientation primarily in the horizontal anterior posterior

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54 axis (Figure 4 1 1 ). Sporadic MyHC positive fibers that appear to align horizontal to the t ransverse plane of the tongue are found in the central core region of the tongue. By contrast, the posterior tongue contains muscle fibers oriented in several trajectory planes (Figure 4 1 2 ). The cellular origins of the tongue are a hybrid, it is derive d from all of the branchial arches, and most of the tongue muscles originate from myoblasts that have migrated from the occipital somites (Parade et al., 2012; Noden, 1983; Noden and Frances West, 2006). Fibers emanating upwards in a rostral dorsal manner from the base of the tongue are noted with additional fibers traversing the dorsal ventral and medial lateral planes of the tongue. Closer examination of the posterior tongue demonstrates possible mononucleate differentiated myocytes at the ventral end o f fibers running perpendicular to the anterior posterior axis (Figure 4 1 2 C). The differentiation status of the unique myocytes was further examined using anti desmin, a marker protein of committed myoblasts destined for fusion (Allen et al., 1991). Cry osections of the posterior tongue were incubated with anti desmin followed by fluorescent antigen detection. Weak desmin immunoreactivity is evident on the interior of the muscle fiber with intense signal located on the ends of the structures (Figure 4 1 3 ). These results suggest that myoblasts are being added to the growing tips of the dorsal ventral oriented fibers. Interestingly, comparison of the desmin and MyHC localization patterns within the fibers projecting upwards from the base of the tongue s how no apparent differences. Desmin is present throughout the fiber with no obvious inconsistencies in spatial intensity.

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55 Pax7 and Pax3 denote migratory cells from the somite that populate the tongue. To confirm the presence of a precursor population in the tongue, cryosections were immunostained with anti Pax7. U nlike Pax3, Pax7 is expressed in the branchial arches and later in facial muscles, some of which derive from muscle precursor cells in the arches in the mouse (Relaix et al., 2004 ). Horst et al. (2006) discovered a reverse pattern of Pax7 expression during myogenesis in the head, where in contrast to somites the myogenic regulatory factors are expressed before Pax7. Migratory cells positive for Pax7 in the tongue most likely are migratory ce lls from the somite; the pattern of dispersion of these cells suggest these fibers are neofibers that elongate by addition of nuclei at the tips. Pax7 expression is seen in proliferating primary myoblasts and becomes down regulated after myogenic differen tiation in the mouse and appears to be specific to the satellite cell myogenic lineage (Seale et al., 2000). As shown in Figure 4 1 4 Pax7 expression is located in all regions of the tongue. In the anterior tip, the Pax7 cells are found as a cluster w ithin the central core area. The more developed muscles of the posterior base of the tongue contain the myogenic precursors throughout the examined area. Due to the divergent regional immunostaining patterns, serial cryosections were immunostained for M yHC or Pax7 and incubated with Hoechst 33245 to detect nuclei (Figure 4 1 5 ). Serial sections were aligned based upon the nuclei staining pattern. Pax7 is expressed predominantly in the central core of the tip interior to the muscle fibers. By contrast, the posterior region of the tongue contains a higher density of Pax7 cells per unit area with the cells found in many regions devoid of fibers. However, a large number of Pax7 precursors are located within the dorsal ventral oriented fibers. The presenc e of these cells further

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56 supports the contention that these are neofibers elongating by addition of nuclei at the tips. Pax7 Expression in the Olfactory Sy stem Pax7 is expressed in multiple tissues of the developing mouse embryo including the nasal cavity (Murdoch et al., 2010). Faithful expression of the transcription factor was examined in the d45 olfactory system of the bovine nasal passages. In brief, cryosections of the fetal nose were incubated with anti Pax7 followed by epifluorescent detection of antigens. Pax7 protein expression is detected in mouse embryos at E7.5 E8.5 in the lateral region of the cephalic neural folds, in the caudal neural plate neuroepithelium and in the cephalic mesenchyme and is more widely expressed in the E9.5 developing hindbrain, forebrain, and frontonasal mesenchyme (Murdoch et al., 2010). Similar to the mouse, strong Pax7 expression is observed in the lateral nasal mesenchyme (Figure 4 1 6 ). Regionalized expression of the transcription factor is also found in the olf actory epithelium. Murdoch (2010) found Pax7 expression in the nose at stages E10 E14.5 specifically in the frontonasal mesenchyme and the lateral margin of the nasal pit, and by E11.5 the mesenchyme of the lateral nasal process robustly expressed Pax7 in the olfactory epithelium. The Pax7 cells located within the nasal cavity likely represent invading and differentiating neurons. These results duplicate mouse olfaction developmental patterns and provide evidence that the sensory neurons form during t he early fetal period in the bovine embry o. In d 45 bovine embryos the regionalized expression of Pax7 cells found in the olfactory epithelium most likely represent invading and differentiating sensory neurons. This is in congruence with Murdoch et al. ( 2010) which demonstrated that Pax7 derivatives contribute to various cell types in the lamina

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57 propria, which include olfactory ensheathing glia which are a special type of glial cell that supports the growth of olfactory axons from the peripheral olfactory epithelium to their central nervous system target, the olfactory bulb, where they form the nerve fiber layer. Implications Previous research in bovine embryonic development has left a void in knowledge of early bovine myogenesis from d 21 to d 85 of gesta tion. This work has taken the initial steps for tracing myogenic lineage in cattle during this time of development for which information is lacking. Immunostaining d 28 and d 45 embryos yields results similar in mouse and avian models with a few differen ces; myogenesis begins in the somites and myoblasts delaminate from the dermomyotome and migrate to form limb and body muscles. Future work needs to be done in comparing myogenic development of different breeds of cattle to see if there is variation among breeds in the onset of various myogenic regulatory factors as well as examine other factors involved in myogenesis such as desmin expression, is there overlapping expression of Pax3 and Pax7, and neural stains in the head.

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58 Figure 4 1 Gross morphological features of bovine embryos at d28 of gestation. Representative d28 embryo shown in panel A, areas measured shown in panel B. Somite pairs were enumerated (C and D). The initial stages of forelimb development were observed (E). The develo ping heart is visible (F). Hindbrain and isthmus formation was apparent at rhomboid 2 4 (G). Three visible pharyngeal arches are present (H). Scale bar equals 1 mm. A. B. C. E. G. D. F. H.

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59 Table 4 1 Body Measurements and Means of D28 Embryos Embryo Body l ength 1 mm Head l e ngth 2 mm Number of s omite pairs 3 1 6.3 1.7 40 2 5.5 1.5 42 3 6.7 2.0 41 4 5.6 1.7 39 5 5.8 1.9 37 Mean 6.0 0.5 1.8 0.2 40 2 1 Body lengths were taken at the longest spread of the embryo. 2 Head length was measured from the crown of the head t o the tip of the snout 3 Number of somites on one side of the body were counted based on segmentation

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60 Figure 4 2 Skeletal muscle is present within the myotome compartment. Embyros (d28) were incubated in anti myosin heavy chain (MyHC) for the detection of myosin. Extensive myosin immunoreativity is found in the somites (S) and heart (H). Rostral somites exhibit more robust myosin immunostaining than caudal somites (A). Myotomes adjacent to the presumptive limb bud (LB) contain 5 10 primary muscle fibers (B and C). Cross section demonstrates myotome muscle fibers are positioned immediately below dermamyotome (D). Scale bar A, B, and C equals 0.5 mm in D equals 50 m. S H LB B. C. A. D.

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61 A. MyHC Myf5 Merge B. C. Figure 4 3 Skeletal muscle is present within the myotome compartment and Myogenic Regulatory Factor Myf 5 is migrating into the limb bud in d28 embryo. Embryos were co stained with immunofluorescence using anti myosin heavy chain (MyHC) and anti Myf5. Panel A shown for reference of location of section from embryo. Myosin (green) and Myf5 (red) immunoreactivity is seen in the myotome of the somite, myoblasts are positioned near the dorsal lip of the dermamyotome (B, C). Nuclei wer e stained using Hoechst 33342 (blue). Secondary antibody only positive control yielded not staining for MyHC or Myf5. Scale bar in row A equals 100 m, and in rows B and C equals 50 m.

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62 Figure 4 4 Localization of Myf5 immunoreactiviy in the presumptive forelimb bud. Epithelial barrier staining is regarded as a non specific ridge effect, within the limb bud anti Myf5 denotes mesoderm derived cells with localization of the protein in the nucleus and perinuclear compartment. Nuclei were stained using Hoechst 33342 (blue). Secondary antibody only positive control yielded no staining for MyHC or Myf5. Scale bar equals 50 m. MyHC Hoechst 33342 Merge Myf 5

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63 Figure 4 5 Skeletal muscle is present within the myotome compartment anterior to the limb bud. Embryos (d28 ) were co stained with immunofluorescence using anti myosin heavy chain (MyHC) and anti Myf5. Panel A shown for reference of location of section from embryo. Myosin (green) immunoreactivity is seen in the myotome of the somite (B, C) and Myf5 (red) immuno reactivity is seen not as pronounced in the somite but exhibits heightened intensity in the lateral plate mesoderm (B, C). Nuclei were stained using Hoechst 33342 (blue). Secondary antibody only positive control yielded no staining for MyHC or Myf5. Sca le bar in whole embryo equals 100 m, and in rows B and C equals 50 m. Hoechst 33342 MyHC Myf 5 Merge A. B. C.

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64 A. B. C. D. E. Figure 4 6 Day 45 Embryos. A) D45 embryos lined up to show similar size. B)D45 embryo picture from G box with ruler for size measurements. C E depicted with ruler indicating where measurements were take on t he embryo A Crown rump length, B Crown snout le ngth, C Forelimb, D Hind limb E Body width, F Longest rib, G Shortes t rib, H Tail.

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65 Table 4 2 D45 Embryo Measurements Embryo Crown rump length 1 mm Crown snout length 2 mm Forelimb 3 mm Hindlimb 4 mm Body width 5 mm 5th rib 6 mm 13th rib 7 mm 1 28 13 10 9 10 4 3 2 28 13 8 7 10 6 4 3 29 13 8 8 10 5 3 4 29 14 8 9 10 5 2 Mean 28.5 0.6 13.3 0.5 8.5 1 8.3 1 10.0 0 5.0 0.8 3.0 0.8 1 Crown rump length full body measurement from top of head to end of the rump 2 Crown snout length measurement of head from top of head to end of snout 3 Forelimb measureme nt from end of hoof to base of leg where it attaches to the body 4 Hind limb measurement from end of hoof to base of leg where it attaches to the body 5 Body width measurement taken from flat of back to line of body above the rib cage 6 5th rib measurement taken from longest visible rib to back 7 13th rib measurement taken from shortest visible rib to the back

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66 Figure 4 7 Primary muscle fibers are present in the forelimb upon completion of embryogenesis in d45 embryo. Ten micron cryosection were imm unostained for myosin (MyHC) and Myf5. Nuclei were visualized with Hoechst 33342. Multiple primary fibers are present in the forelimb (B). Myoblasts are interspersed throughout the region but distinct from the fibers (C). Trans saggital cryosection image shown in (A). Red box denotes area of interest in series B and C. Secondary antibody only positive control yielded no staining for MyHC or Myf5. Scale bar in A equals 1 mm in B and C equals 50 m. B. C. 1 mm Hoechst 33342 Myf 5 Merge Hoechst 33342 MyHC Merge A.

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67 Figure 4 8 Primary skeletal muscle fiber formation i n the developing limb showing Myf 5 and Pax7 positive cells throughout primary skeletal muscle. Embryos (d45) were stained for immunofluorescence using anti Myf 5 and anti Pax7 to detect Myf 5 and Pax7 positive satellite cells. Hoechst 33342 was used as nu clear stain and whole image for location reference. Red box is shown in the panels to the right of large image. Myf 5 (red) associated with nuclei indicate growing and merging fibers and Pax7 (green) indicates presence of primary muscle fiber formation. Secondary antibody only positive control yielded no staining for Pax7 or Myf5. Scale bar in large image equals 1 mm and in the four smaller panels equals 50 m. Hoechst 33342 Pax 7 Myf 5 Merge

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68 Figure 4 9 Satellite cells positive for Pax7 migrating throughout primary skeleta l muscle in the developing limb and intercostal muscles. In d 45 embryos Pax7 satellite cell activity is around muscle fibers in the developing limb (B) and intercostal muscles (C). Panel A shows nuclear stains for location reference. Secondary antibody only positive control yielded no staining for Pax7. Scale bar in A equals 1 mm in B and C equals 50 m. B. C. A. Phalloidin Pax 7 Merge

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69 Figure 4 10 Satellite cells positive for Pax7 migrating throughout primary skeletal muscle in the developing limb and intercostal muscles. Embry os (d28) were stained with immunofluorescence using anti Pax7 to detect Pax 7 positive satellite cells and phalloidin to stain for actin. In d28 embryos Pax7 is present in the myotome (B). Panel A is a nuclear stain to show reference for location. Second ary antibody only positive control yielded no staining for Pax7. Scale bar in A equals 0.5 mm and in B equals 50 m. Phalloidin Pax 7 Merge A. B.

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70 Figure 4 11 Primary muscle fibers in the tongue in d45 embryo head. Twelve micron cryosections we re immunostatined for myosin (MyHC) nuclei were visualized with Hoechst 33342. Primary muscle fibers are present in the tip of the tongue (B). Panel C is increased magnification of MyHC staining. Box in nuclear stained cryosection of head in longitude ( A) for position reference. Secondary antibody only positive control yielded no staining for MyHC. Scale bar in A equals 0.5 mm and in B and C scale bar equals 50 m. A. B. C. Hoechst 33342 MyHC Merge

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71 Figure 4 12 Primary muscle fibers in the tongue in d45 embryo head Twelve micron cryosections were immunostatined for myosin (MyHC) nuclei were visualized with Hoechst 33342. Primary muscle fibers are present in the back of the tongue (B). Panel C is increased magnification of MyHC staining. Box in nuclear stained c ryosection of head in longitude (A) for position reference. Secondary antibody only positive control yielded no staining for MyHC. Scale bar in A equals 0.5 mm and in B and C scale bar equals 50 m. A. B. C. Hoechst 33342 MyHC Merge

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72 Fi gure 4 13 Primary muscle fibers in the tongue in d45 embryo head. Twelve micron cryosections were immunostatined for Desmin, nuclei were visualized with Hoechst 33342. Desmin positive muscle staining present in the back of the tongue (B). Panel C is increased magnification of Desmin staining. Box in nuclear stained cryosection of head in longitude (A) for position reference. Secondary antibody only positive control yielded no staining for Desmin. Scale bar in A equals 0.5 mm and in B and C scale bar equals 50 m. A. B. C. Hoechst 33342 Desmin Merge

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73 Figure 4 14 Pax7 positiv e cells in forming muscle fibers of the tongue in d45 embryo head. Twelve micron cryosections were immunostatined for Pax7 nuclei were visualized with Hoechst 33342. Pax7 cells present in developing muscle fibers in the anterior tip of the tongue (B). P ax7 cells present in developing muscle fibers in the posterior of the tongue (C). Box in nuclear stained cryosection of head in longitude (A) for position reference. Secondary antibody only positive control yielded no staining for Pax7. Scale bar in A e quals 0.5 mm and in B and C scale bar equals 50 m. 10x A. B. Hoechst 33342 Pax7 Merge C.

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74 Figure 4 15 Tongue muscle formation in d45 embryo marked with anti myosin heaving chain (MyHC) and Pax 7. In the tip of the tongue (top panels) MyHC staining shows greater immunoreactivity toward t he outer edge of the tongue and Pax 7 is prevalent toward the center of the tongue, suggesting that the muscle cells toward the edge of the tongue older. At the back of the tongue (lower panels) development of the different muscles seen through MyHC stain ing, Pax 7 cells immunoreactive cells are present around the fibers. Hoechst 33342 shows nuclear staining for reference in the section. Secondary antibody only positive control yielded no staining for MyHC or Pax7. Scale bar in reference section equals 0.5 mm, in remaining panels represent 50 m. Hoechst 33342 MyHC Pax 7 Merge

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75 Figure 4 16 Pax7 positive cells in forming olfactory epithelial cells of the nasal cavity in d45 embryo head. Twelve micron cryosections were immunostatined for Pax7 nuclei were visualized with Hoechst 33342. Pax7 positive cells are present in the lateral nasal mesenchyme and the olfactory epithelium most likely representing invading sensory neurons (B). Panel C is increased magnification of Pax7 staining. Box in nuclear stained cryosection of head i n longitude (A) for position reference. Secondary antibody only positive control yielded no staining for Pax7. Scale bar in A equals 0.5 mm and in B and C scale bar equals 50 m. A. B. C. Hoechst 33342 Pax7 Merge

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76 CHAPTER 5 CONCLUSIONS Angus cattle are characterized by shorter gesta tion lengths than other breeds of cattle which mak es th is breed highly favorable in the beef cattle industry. In t his study fetal growth was measured and compared between Angus and Brangus fetuses; results indicate Angus fetuses begin to grow at a faster rate around d 55 of gestation Breed of cattle affects birth weight of the calf; these results provide insight for those in the beef industry as to when in gestation growth rate differences between breeds occurs and set forth preliminary work to begin inv estigation for the cause or causes of variation in birth weights between different breeds of cattle. To further explore variation of growth in cattle during gestation, this study explored myogenic lineage in cattle using Holstein emb ryos as a model. The re is a time period of gestation that is lacking in bovine embryonic development studies that occurs between d 21 to d 85 of gestation. Working within that time span where the void exists, i mmunohistochemistry of d 28 and d 45 embryos yields results simil ar in mouse and avian models with a few differences; myogenesis begins in the somites and myoblasts delaminate from the dermomyotome and migrate to form limb and body muscles. This research has laid the foundation for f uture work to explore variation in m yogenic developmen t of different breeds of cattle, not only to see if there is variation among breeds in the onset of various myogenic regulatory factors but also to examine other factors involved in myogenesis such as desmin expression or the possibility of overlapping expression of Pax3 and Pax7

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88 BIOGRAPHICAL SKETCH Christy Waits is the daughter of Vernon and Theresa Waits, she was born in Enterprise, Alabama and raised in Daleville, Alabama. Upon graduation from Daleville High School in 1998, Christy attende d Enterprise State Junior College and e arned her Associate of Arts degree in 2000. She then continued her education by attending Auburn Un iversity and earned a Bachelor of Science degree in z oology in May of 2003. After graduation Christy worked various jobs to include forestry and fire work to endangered species monitoring and research and finally working as the Operations Assistant at Buck Island Ranch in Lake Placid, Florida After four years of working on the ranch, in 2010, Chri sty decided to pursue a Master of Science at the University of Florida in Gainesville. Christy is currently working as a Bio Science Technician at the Naval Air Station in Jacksonville, F lorida at the Navy Center of Entomo logy as a contractor worker for Lovelace Respiratory Research Institute