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AN EXAMINATION OF THE TEMPORAL AND SPATIAL EXPRESSION PATTERN OF MUSCLE PROTEINS IN THE EARLY BOVINE EMBRYO

By

CHRISTY MARIE WAITS

THIS THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2012

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© 2012 CHRISTY WAITS

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To Mom and Dad thanks for all the love and support, and to Elizabeth and Katelynn, the world is what you make of it, kisses on your faces

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ACKNOWLEDGMENTS

Firstly, 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 the members of my 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 experiments.

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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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

ABSTRACT ..................................................................................................................... 9

CHAPTER

1 INTRODUCTION .................................................................................................... 10

2 LITERATURE REVIEW .......................................................................................... 13

Breeds of Cattle ...................................................................................................... 13 Overview .......................................................................................................... 13

Bos taurus ........................................................................................................ 13 Bos indicus ....................................................................................................... 14

Advantages of using Bos indicus in Southern 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 Development .......................................................................... 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 IN BOVINE FETAL GROWTH IN EARLY GESTATION BETWEEN ANGUS AND BRANGUS CATTLE .................................................................................................................. 34

Background ............................................................................................................. 34

Materials and Methods............................................................................................ 35 Results and Discussion........................................................................................... 37

Implications ............................................................................................................. 39

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4 SPATIAL AND TEMPORAL EXPRESSION OF MYOGENIC PROTEINS IN THE EARLY BOVINE EMBRYO ..................................................................................... 41

Background ............................................................................................................. 41 Materials and Methods............................................................................................ 45

Embryo Collection ............................................................................................ 45 Whole Mount Myosin Immunostaining .............................................................. 46

Immunohistochemistry...................................................................................... 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 Embryos ........ 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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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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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 anterior to the limb bud. ............................................................................................................. 63

4-6 Day 45 Embryos. ................................................................................................ 64

4-7 Primary muscle fibers are present in the forelimb 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 muscle. .............................. 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 in 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 in 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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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 THE TEMPORAL AND SPATIAL EXPRESSION PATTERN OF

MUSCLE PROTEINS IN THE EARLY BOVINE EMBRYO

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 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 < 0.05) than Brangus fetuses. To define

early bovine myogenesis immunohistochemistry was performed for myosin heavy chain

(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 in 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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CHAPTER 1 INTRODUCTION

Florida is a subtropical region known for high temperatures and humidity. Cattle

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 lower 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

taurus breeds are preferred and selected for superior performance and meat quality

(Marshall, 1994). These breeds perform well in temperate climates but are poorly

adapted to the sub-tropical climate of Florida therefore their performance decreases.

Bos indicus cattle are more tolerant to warmer, sub-tropical climates as well as resistant

to parasites 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 consumers (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

many Florida’s beef herds contain Bos taurus x Bos indicus crossbred cattle. Through

crossbreeding 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. Approximately 42% of beef cows and 50% of the country’s cow-calf

producers are located 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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purebred breeds for the 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

will go to slaughter and produce quality meat products to be bought by consumers. A

considerable amount of research has been placed on animal growth, particularly muscle

growth. There are numerous studies evaluating how to produce a better meat product,

through 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 pre-

natal growth, particularly when comparing cattle of different breed types. Studies have

shown that different breeds have different birth weights as well as dam and sire breed

effects on birth weight (Casas et al., 2010; Reynolds et al., 1980).

The variation 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 developing 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 too are capable of migrating to further growth and development (Cossu and

Biressi, 2005). Muscle is formed through myogenesis, via the myogenic cascade, in the

vertebrate embryo somites are where skeletal muscle originates (Arnold and Braun,

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1993). Progenitor 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 the 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 hypothesis 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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CHAPTER 2 LITERATURE REVIEW

Breeds of 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 conditions 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 and

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 and 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

Hereford 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 maturing with higher muscle and protein retention capacity and better food

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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 cattle (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, sleek, 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 average 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 length 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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with a reputation for poor neonatal performance and low calf survival rates (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 more connective

tissue (Elzo et al., 2012; Whipple et al., 1990).

Advantages of using Bos indicus in Southern United States

Florida and the Gulf Coast region are characterized by subtropical temperatures,

with mild winters and hot summers (West, 2003). Bos taurus, 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 rises, 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 thermotolerance 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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Disadvantages of using Bos indicus in Southern United States

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

in the rest of the cattle industry in the U. S. Today’s beef industry In an industry

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 Bos 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 herds (Cundiff et al., 1986).

Approximately 42% of beef cows and 50% of the country’s cow calf producers are

located in the southern United States; an estimated 30% of cattle in the United States

contain some percentage of Bos indicus genetics (Morrison, 2005; 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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et al., 2007; Elzo et al, 2009). However, the advantages are tempered by older age at

reproductive maturity, slightly longer gestation rate when 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 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).

Muscle Growth 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 immunohistochemistry 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 Development

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 16 as

seen through IVP (in vitro-production) and SCNT (somatic cell nuclear transfer); most

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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

been examined and documented. Maddox-Hyttel et al. (2003) saw on day 9 of

gestation, embryos are devoid of zona pellucida and present a well-defined inner cell

mass (ICM) covered by a thin layer of trophoblast cells (the Rauber’s layer). At day 11,

the Rauber’s layer becomes focally interrupted and tight junctions form adjacent

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

epiblast 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 epithelium 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 what 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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Research conducted using immunohistochemistry of fetuses in the second

trimester of pregnancy provides an interesting look at muscle development using

muscles in the 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

embryonic 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 developed 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, at 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 (Martyn 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 has 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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after approximately day 50; detection 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; Holland

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 Brahman 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

Charolais 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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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 rate 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

One 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 grouping 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).

Organization 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 called 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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found between two successive 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 globular 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 contraction, 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 rely 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 with the strands of the thin filament (Lieber, 2002).

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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 induced by

signals emanating from neighboring tissues in “naϊve” proliferating cells (Cossu and

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 Rudnicki, 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). Skeletal 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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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, notochord 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 mesoderm, 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 regions the dermomyotome and the

sclerotome; the sclerotome is formed from the ventromedial portion, making it the most

ventral 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,

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 mesenchymal 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 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 Ozawa, 1995).

In mice, the myotome forms on embryonic day 9 (E9) in the mouse, followed by

fusion of myoblasts 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 gestation 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 day 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 array of muscles with many traits, the tongue is coated with sensors on the

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 mouth 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 mesenchyme, 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; Huang 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, located 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

named these cells “satellite cells” due to their 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).

27

At its discovery, the satellite cell was credited with the function of the cell

responsible for growth and maintenance of skeletal muscle, yet it was not proposed to

be a stem cell (reviewed by Péault 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 such as injury (Bischoff, 1994). Because 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 Péault 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, 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 Péault 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). 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).

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 differentiation (Tajbakhsh, 2009).

Myogenic Regulatory Factors

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 mechanisms 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). 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, and Mrf4 (Myf6;

Weintraub et al., 1991). Together the various MRF’s cooperate 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

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 removing 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 factor that is important for specification of muscle cells (Chen et al., 2007).

Cell culture and immunocytochemistry 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 progenitor 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

30

ball-shaped somites beginning in the cranial region of the mouse embryo 8 days post

conception (dpc; Ott et al., 1991). Myf-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 around 11.5 days post conception Myf5 expression

decreases rapidly throughout the embryo;

Pax3 and Pax7

Wingless and Int 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). Paired 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

regulators of the myogenic process (Relaix et al., 2005). During gestation, Pax3 and

Pax7 expression patterns diverge (Horst et al., 2006). Pax3 is essential for skeletal

myogenesis and acts upstream of MyoD during skeletal muscle development

(Ridgeway and Skerjanc, 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;

31

however in culture of primary muscle fibers Pax7 is expressed in proliferating primary

myoblasts and is 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 embryogenesis (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

dermomyotome appear to divide tangentially and form a “secondary myogenic domain”

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).

Pax3+/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

and 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

MyoD 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

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

associated 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-potent 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

and 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

Myf5 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

frontonasal 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

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

(Murdoch 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

examination 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 insight to bovine fetal development.

34

CHAPTER 3 DIFFERENCES MEASURED BY ULTRASONOGRAPHY IN BOVINE FETAL GROWTH

IN EARLY GESTATION BETWEEN ANGUS AND BRANGUS CATTLE

Background

The variation that exists in biological traits important for beef production is vast;

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 curve 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

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 250g/day (Burfening et al., 1978; 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).

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 by determining when variation in growth rates occurs

between developing Angus and Brangus fetuses.

Materials and Methods

Angus (n=43) and Brangus (n=33) cows housed at the University of Florida’s

Santa Fe River Ranch Unit were artificially inseminated to pre-assigned multiple sires

within their respective breeds. The average age for Angus cows was 53 years (range

36

2-11 yrs) and Brangus cows was 52 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. At 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) for 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 bermudagrass (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 cows 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 F2α (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 cows in both treatment groups; all cows received 25 mg FGF at CIDR

removal and 25 mg PGF approximately 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.

37

Beginning on d 33, each pregnancy was evaluated by trans rectal ultrasonography

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 day 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 measurement.

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. Crown-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 fetuses

with CSLs of 2.08 ± 0.03 cm and 1.95 ± 0.03 cm, respectively. Calculation of CRL of d

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 length 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 fetuses 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 percent 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 inheritance 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;

39

Brahman sires increase birth weights compared with Bos taurus sire breeds when bred

to Bos taurus cows (Roberson et al., 1986; Comerford 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 in gestational development of the fetus to begin tracing the myogenic

lineage to examine muscle growth and development.

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

Cro

wn

-ru

mp

len

gth

, cm

Gestation, days

Angus

Brangus

#

*

0

10

20

30

40

50

60

5 6 7

Perc

en

t g

row

th

Gestation, wks

Angus

Brangus

*

41

CHAPTER 4 SPATIAL AND TEMPORAL EXPRESSION OF MYOGENIC PROTEINS IN THE EARLY

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 gestation

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 embryo sizes

and in what 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. Beginning around day 120 muscle fiber types are being discerned into

Type I or Type II fibers (Martyn et al., 2004). The average sizes of primary 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 differentiation from day 180 onwards (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-

42

lateral lips of the dermomyotome respond 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 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).

Somites differentiate into two regions the dermomyotome and the sclerotome; 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 Ozawa,

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). 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

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

secondary 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, temperature, 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 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;

Huang et al., 1999).

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 Péault 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,

vascular cell adhesion molecule-1 (VCAM-1), c-met (receptor for hepatocytes growth

44

factor), neural cell adhesion molecule-1 (CD56), Foxk1, and syndecans 3 and 4

(reviewed by Péault 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). 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 embryonic 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, and Mrf4 (Myf6; Weintraub et al.,

1991). Together the various MRF’s cooperate 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).

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 also some differences from mouse development.

Materials and Methods

Embryo Collection

Non-lactating Holstein cows (n=20; 3 ± 0.4 yr) were injected with 100 μg GnRH

(Cystorelin; Merial) and fitted with a vaginal progesterone insert (CIDR; Pfizer). After 7

d, the CIDR was removed and cows were injected with 25 mg PGF2α. Fifty-six hours

post PGF2α treatment; the cows received an injection of 100 μg GnRH and were

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 captive-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 minutes 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.

46

Whole Mount Myosin Immunostaining

Embryos (d28) were fixed sequentially in 4% paraformaldehyde for 60 min and

4% paraformaldehyde containing hydrogen 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. Embryos 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) hybridoma 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

47

with epifluorescence, a digital camera (CoolSnap EF, Photometris) and NIS Elements

software.

Results and Discussion

Morphological Features of Bovine Embryos at d 28 Gestation

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 recorded. 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 somite pairs were enumerated (C and

D), and panel E shows the 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 hindbrain and isthmus formation at rhomboid 2-

4 in panel G and there are three visible pharyngeal arches panel H. Morphometric for

the embryos are recorded in Table 4-1. The body length of the d 28 embryos averaged

6.0 0.5 mm and 40 2 somite pairs were noted.

Whole Mount Myosin Immunostaining Indicates Presence of Skeletal Muscle in Myotome

Muscle represents the largest tissue in the body. The extent of myogenesis was

examined in early bovine embryos. In brief, intact embryos (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 muscle within the myotome

48

compartment of the somite (Figure 4-2). Extensive myosin immunoreactivity is found in

the somites and heart. Rostral somites exhibit more robust myosin immunostaining

than caudal somites (Figure 4-2A). Myotomes adjacent to the presumptive limb bud

(LB) contain 5-10 primary muscle fibers (Figure 4-2B 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 demonstrate that the

myotome muscle fibers are positioned immediately below the dermamyotome (Figure 4-

2D). 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 mammals and birds.

Immunofluorescence Staining Reveals Myogenic Cells in d 28 Embryos

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 approximately 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 (Figure 4-3A). The tissue sections were

immunostained with anti-MyHC, anti-Myf5 and the appropriate fluorescent secondary

antibody (Figure 4-3B). Myoblasts, as defined by Myf5 immunoreactivity, represent a

discrete cell population that does not overlap with 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 Myf-5 transcripts visible between

49

10.5 and 11 days of development in the mouse (Ott et al., 1991). In d 28 bovine

embryos co-staining for MyHC and Myf5 reveal myoblasts positioned near dorsal lip of

dermomyotome with primary muscle fibers in medial and ventral regions below somite

structure this is consistent with new myoblasts forming from 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 distinctive somite structure

(Figure 4-3C). 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 and 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 have 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 epithelial 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 cells 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 4-3C). The heightened fluorescent intensity also is noted in the lateral plate

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 well 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 specific 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 with 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 decreases

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 Features of Bovine Embryos at d 45 Gestation

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.

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

easily distinguishable features characteristic of bovidae was evident.

Myogenesis in the d45 Limb and Intercostal Muscles

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 4-7A, B). It does not 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

4-7C). 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. Substantial 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 indicate 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,

52

contributing progenitors to the intercostal and ventral body wall muscles (reviewed in

Buchberger et al., 2003). Embryonic limb Pax7 transcripts are first detected in the

mouse at E11.5 in the 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 equivalent 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 and 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 primary 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 al., 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-Pax7 (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-expressing 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

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 4-10). 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 restricted 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 forming 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 Muscle Development in Tongue of d 45 Embryo

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 arch 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

immunocytochemistry reveals that the anterior tip of the tongue contains at least two

distinct muscle groups with fiber orientation primarily in the horizontal anterior-posterior

54

axis (Figure 4-11). Sporadic MyHC positive fibers that appear to align horizontal to the

transverse 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 4-12).

The cellular origins of the tongue are a hybrid, it is derived 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 of fibers running

perpendicular to the anterior-posterior axis (Figure 4-12C). 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). Cryosections 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 4-13). 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 show no apparent

differences. Desmin is present throughout the fiber with no obvious inconsistencies in

spatial intensity.

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. Unlike 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 cells 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 differentiation in the mouse and appears to be

specific to the satellite cell myogenic lineage (Seale et al., 2000).

As shown in Figure 4-14, Pax7 expression is located in all regions of the tongue.

In the anterior tip, the Pax7 cells are found as a cluster within 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 MyHC or Pax7

and incubated with Hoechst 33245 to detect nuclei (Figure 4-15). 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 presence of these cells further

56

supports the contention that these are neofibers elongating by addition of nuclei at the

tips.

Pax7 Expression in the Olfactory System

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 4-16). Regionalized expression of the transcription factor is also

found in the olfactory 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 the

early fetal period in the bovine embryo. 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

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 gestation. 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 differences; 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.

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 developing 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.

59

Table 4-1. Body Measurements and Means of D28 Embryos

Embryo Body length1,

mm Head length2, mm

Number of somite pairs3

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 to the tip of the snout 3 Number of somites on one side of the body were counted based on segmentation

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 µm.

S

H

LB

B. C. A.

D.

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 were stained using Hoechst 33342 (blue). Secondary antibody only positive control yielded not staining for MyHC or Myf5. Scale bar in row A equals 100 µm, and in rows B and C equals 50 µm.

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 µm.

MyHC Hoechst 33342

Merge Myf-5

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) immunoreactivity 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. Scale bar in whole embryo equals 100 µm, and in rows B and C equals 50 µm.

Hoechst 33342 MyHC Myf-5 Merge

A.

B.

C.

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 the embryo A - Crown-rump length, B - Crown-snout length, C Forelimb, D- Hind limb, E- Body width, F - Longest rib, G - Shortest rib, H - Tail.

65

Table 4-2. D45 Embryo Measurements

Embryo Crown rump

length1,

mm

Crown snout

length2,

mm

Forelimb3,

mm Hindlimb

4,

mm

Body width

5,

mm

5th rib6,

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 measurement 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

66

Figure 4-7. Primary muscle fibers are present in the forelimb upon completion of

embryogenesis in d45 embryo. Ten micron cryosection were immunostained 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 µm.

B.

C.

1 mm

Hoechst 33342 Myf-5 Merge

Hoechst 33342 MyHC Merge A.

67

Figure 4-8. Primary skeletal muscle fiber formation in 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 nuclear 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 µm.

Hoechst 33342

Pax7

Myf-5

Merge

68

Figure 4-9. Satellite cells positive for Pax7 migrating throughout primary skeletal

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 µm.

B.

C.

A.

Phalloidin Pax 7 Merge

69

Figure 4-10. Satellite cells positive for Pax7 migrating throughout primary skeletal

muscle in the developing limb and intercostal muscles. Embryos (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. Secondary antibody only positive control yielded no staining for Pax7. Scale bar in A equals 0.5 mm and in B equals 50 µm.

Phalloidin Pax 7 Merge

A.

B.

70

Figure 4-11. 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 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 µm.

A.

B.

C.

Hoechst 33342 MyHC Merge

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 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 µm.

A.

B.

C.

Hoechst 33342 MyHC Merge

72

Figure 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 µm.

A.

B.

C.

Hoechst 33342 Desmin Merge

73

Figure 4-14. Pax7 positive 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). Pax7 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 equals 0.5 mm and in B and C scale bar equals 50 µm.

10x

A.

B.

Hoechst 33342 Pax7 Merge

C.

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 the 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 staining, 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 µm.

Hoechst 33342 MyHC Pax7 Merge

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 in 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 µm.

A.

B.

C.

Hoechst 33342 Pax7 Merge

76

CHAPTER 5 CONCLUSIONS

Angus cattle are characterized by shorter gestation lengths than other breeds of

cattle which makes this breed highly favorable in the beef cattle industry. In this 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 investigation 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 embryos as a model. There 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,

immunohistochemistry of d 28 and d 45 embryos yields results similar 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 future work to explore variation in myogenic

development 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.

77

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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 attended Enterprise State Junior College and earned her

Associate of Arts degree in 2000. She then continued her education by attending

Auburn University and earned a Bachelor of Science degree in zoology 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, Christy 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, Florida at the Navy Center of Entomology as a

contractor worker for Lovelace Respiratory Research Institute.