a; . ‘ . ‘ ‘ . s . ‘ 3%.;

. 3?.me
a ,w

 

, . x; .

.. . . . rung. i .4“.

, , .. ”firm“ a...
:Fnfi, . . . .. . . . (1

,= .H .. . _ @a gig

.mmn.

a L.
g, a.

 

 

“ma-if. !.
. « ”6:3. ,
fizkwmvd
C. 1...:

 

, 53.3...1; , . .. . . z a; :.
Bear 1;... . .

THE‘sss

4,05; \

This is to certify that the

thesis entitled

Effects of Callipyge Lamb Genotype on Skeletal
Muscle Characteristics

presented by

John D. Heller

has been accepted towards fulfillment
of the requirements for

Masters—Jegree in Animal—Science

mgfiw

Major professor

Date q'lq'O‘

0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

 

 

 

 

 

LIBRARY

Michigan State
University

 

 

 

PlACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c‘JCIRC/Dateouepes-ms

EFFECTS OF CALLIPYGE LAMB GENOTYPE ON SKELETAL MUSCLE
CHARACTERISTICS
BY
John D. Heller

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2000

ABSTRACT

EFFECTS OF CALLIPYGE LAMB GENOTYPE ON SKELETAL MUSCLE
CHARACTERISTICS

By
John D. Heller

Callipyge (CLPG) lambs are leaner, heavier muscled, and more efficient at
converting feed to body weight than non-CLPG lambs. The current study
determined the effects of lamb genotype (NN, CN, NC, and CC where C= mutant
CLPG allele, N = wild-type allele, paternal allele listed first) on skeletal muscle
characteristics. This study confirms that lambs of the CN genotype have heavier
Iongissimus dorsi (LD) and biceps femoris (BF) muscles compared to other
genotypes (NN, NC, CC), whereas infraspinatus (IS) muscle weights were similar
among genotypes. Muscle DNA and protein concentrations were generally not
different among genotypes. However, proteianNA ratios were lower and
RNAzDNA ratios of LD muscles from CN and CC lambs were higher than those of
NN and NC lambs. Type llx myosin heavy chain (MHC) was 25% higher in CN
lamb LD compared to L0 of other genotypes. An increase in the proportion of type
llx MHC may explain the previously reported increase in percentage and'size of
type llb muscle fibers in CLPG lamb LD. Western blot analysis revealed that
desmin in myofibrils isolated from CN lamb LD appeared to degrade more slowly
than desmin from myofibrils of NN lambs, when incubated with m-calpain in vitro.
This suggests that desmin of CN lambs is more resistant to calpain cleavage than
desmin of NN lambs. Collectively, these data provide evidence that changes in
myosin isoform distribution and decreased susceptibility of desmin to proteolysis

may contribute to muscle hypertrophy in ON lambs.

DEDICATION

“He said that men ought to remember those friends who were absent as well as
those who were present.”
Thales. vii., Diogenes Laertius. Circa 200 A. D.

This thesis was written in memory of those family members who have gone
before me and those who are still with me.

ACKNOWLEDGEMENTS

It should go without saying that projects such as a masters thesis require
the time and effort of a multitude of people and likewise a multitude of thanks. It is
only with the immense amount of time, support and foresight of Dr. Matthew E.
Doumit that this project can be submitted for completion of my masters degree.
His knowledge base, effort and friendship often seem unending, thank-you. Along
with Dr. Doumit, I would like to thank the members of my graduate committee, Dr.
Gale Strasburg, Dr. George Smith and Dr. H. Allen Tucker, their input and advice
throughout my degree was invaluable.

On a consistent basis, fellow laboratory workers provided insight and able
hands, making experiments both easier and more enjoyable. Important players in
this group were laboratory technicians, Sharon Debar and Jamie Sue Prater, plus
graduate students, Scott Kramer, Nick Mesires, Chuck Allison and Matt Ritter.
Despite incidents in which my stubbornness got the better of me, these individuals
helped push me through my masters. Another component of the lab to which I am
grateful is our undergraduate workers: Betsy Booren, Sally Dean, Jeannine
Grobbel and Courtney Dilley. I am thankful for their time spent performing tedious
laboratory tasks and bearing the blow of outbursts. Outside of my primary
laboratory, I would like to thank Dr. George Smith and his laboratory group for
allowing me to work with them and helping me with several experiments.

I would like to thank my many roommates/friends, Josh, J.D., Justin, Roy,
Mark and Gwyn for all the laughs, not to mention their ability to deal with my

infamous snooze button and non-changing moods. it is important for me to thank

several other friends who supported me during my graduate career, including,
Dolph, the “estrogen lab”, Charles, and Courtney.

I am grateful to the Department of Animal Science at Michigan State
University for providing the opportunity to further my education by pursuing a
masters degree. Additionally, the faculty and staff within this department have
been a pleasure to work with, making MSU a place I will never forget.

Finally, i would like to thank my family, Earl, Kay and Marty who have
always surprised me with their love and support. I will forever be grateful for what
they have done to allow me to succeed. It is through these individuals help and
support that I have not only obtained a masters degree but life skills, friends and

memories. Thank you.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... viii
LIST OF FIGURES .................................................................................... ix
LIST OF ABBREVIATIONS ...................................................................... x
INTRODUCTION ....................................................................................... 1
LITERATURE REVIEW ............................................................................. 3
SKELETAL MUSCLE GROWTH ..................................................... 3
CALLIPYGE SHEEP ...................................................................... 5
GENETIC INHERITANCE OF CALLIPYGE PHENOTYPE ............ 6
PRENATAL GROWTH ................................................................... 9
POSTNATAL GROWTH ............................................................... 10
CALLIPYGE MUSCLE FIBER TYPE CHARACTERISTICS ......... 13

POTENTIAL MECHANISMS OF CALLIPYGE GENE ACTION 14

CHARACTERISTICS OF MEAT FROM CALLIPYGE LAMBS ..... 17
MATERIALS AND METHODS ................................................................ 18
SAMPLE COLLECTION ............................................................... 18
CARCASS MEASUREMENTS ..................................................... 18
PROTEIN AND NUCLEIC ACIDS ................................................. 20
SAMPLE PREPARATION ............................................................ 20
MYOSIN HEAW CHAIN ISOFORM SEPARATION .................... 22
MYOSIN HEAW CHAIN WESTERN BLOTS .............................. 22
SARCOPLASMIC PROTEIN SEPARATION ................................ 24

vi

M-CALPAIN MEDIATED IN VITRO PROTEOLYSIS ................... 24

STATISTICAL ANALYSIS ............................................................ 27
RESULTS ................................................................................................ 28
CARCASS MEASUREMENTS AND MUSCLE DISSECTION ..... 28
QUANTIFICATION OF SKELETAL MUSCLE NUCLEIC ACIDS
AND PROTEIN ............................................................................ 29
MEASURES OF MUSCLE FIBER TYPE TRANSITION ............... 33
M-CALPAIN MEDIATED IN VITRO PROTEOLYSIS ................... 37
DISCUSSION ........................................................................................... 40
APPENDICES .......................................................................................... 48
REFERENCES ........................................................................................ 56

vii

LIST OF TABLES

Table 1.

Possible Offspring from CN Sire Mating .......................................... 7
Table 2. .

Carcass Characteristics for NN, CN, NC and CC genotypes ........ 29
Table 3.

Longissimus dorsi Muscle Measurements ..................................... 30
Table 4.

Biceps femoris Muscle Measurements .......................................... 31
Table 5.

lnfraspinatus Muscle Measurements ............................................. 32
Table 6.

Percentage of Myosin Heavy Chain lsoforrns ............................... 34
Table 7.

Percentage of Sarcoplasmic Proteins ........................................... 36

viii

LIST OF FIGURES

Figure 1.

Myosin Heavy Chain lsoforrns ....................................................... 34
Figure 2.

Gel of Sarcoplasmic Proteins ........................................................ 35
Figure 3.

Western Blot of Desmin ................................................................ 39

ix

LIST OF ABBREVIATIONS
1°: Primary
2°: Secondary
Ab: Antibody
ALD: Aldolase
AP: Alkaline phosphatase
BAA: B- Adrenergic agonists
BF: Biceps femoris
BSA: Bovine serum albumin
CC: Homozygous caliipyge, non-hypertrophied
CK: Creatine kinase
CLPG: Callipyge gene
CN: Heterozygous caliipyge, hypertrophied
ddl: Double distilled water
DNA: Deoxyribonucleic acid
EDTA: Ethylenediamine tetra acetic acid
EN: Enolase
GDF-B: Growth differentiation factor-8
G3PDH: GlyceraIdehyde—3-phosphate dehydrogenase
IS: lnfraspinatus
kD: kiIoDalton
LD: Longissimus dorsi

LDH: Lactate dehydrogenase

MHC: Myosin heavy chain

NC: Heterozygous callipyge, non-hypertrophied
NFDM: Non-fat dry milk

NN: Homozygous normal, non-hypertrophied
PAGE: Polyacrylamide gel electrophoresis
PGAM: Phosphoglycerate mutase

PGl: Phosphoglucose isomerase

PGM: Phosphoglucomutase

PHb: Phosphorylase b

PK: Pyruvate kinase

PMSF: Phenylmethylsulfonyl fluoride

PVDF: Polyvinylidene fluoride

REA: Ribeye area

RNA: Ribonucleic acid

RPM: Rotations per minute

SA: Serum albumin

SDS: Sodium dodecyl sulfate

TPI: Triose phosphate isomerase

xi

lNTRODUCTION

Growth and function of skeletal muscle, the most abundant tissue in the
animal body, is essential for locomotion and therefore survival of animals.
Skeletal muscle also provides a high quality source of protein and iron for human
diets. Gaining a better understanding of skeletal muscle growth should allow
researchers to both prevent and cure muscle atrophy from disease or injury and
produce animal protein more efficiently.

A ram with a heritable, heavy muscled phenotype was first noticed in
1983. This phenotype resulted from a novel genetic mutation. The gene
responsible for the mutation has been named callipyge (CLPG, Cockett et al.,
1994). Phenotypic CLPG lambs have 11% more muscle mass, 23% less fat, and
are more efficient converters of feed to lean than phenotypic non-CLPG lambs
(Freking et al., 1998a,b; Jackson et al., 1997a). Increases in production
efficiency and muscle mass are of great interest to the livestock industry.
Phenotypic CLPG lambs also have larger legs and loins than their normal
counterparts, which result in more desirable carcasses. However, CLPG lamb
chops are less tender and flavorful than lamb chops from non-CLPG lambs
(Cockett et al., 1994; Shackelford et al., 1998). Today’s standard consumer of
lamb does not consider these traits desirable. Overcoming these obstacles
would allow CLPG lamb to meet consumer demands for larger, leaner portions of
meat while satisfying demands for a tender, flavorful product.

The biological mechanisms resulting in muscle hypertrophy due to the

CLPG condition are unknown. Additional research is needed to address both the

CLPG muscle growth phenomenon and meat quality issues associated with the
mutation. An improved knowledge of muscle development in CLPG lambs will
allow for development of strategies to more efficiently produce meat from sheep
and other species. This information may also shed light on potential
management practices to make CLPG lamb more palatable.

The CLPG mutation is an example of paternal polar overdominance, an
inheritance pattern rarely seen in nature. This mode of inheritance restricts
expression of the characteristic CLPG muscle hypertrophy to only heterozygous
lambs inheriting the mutant CLPG allele from their sire.

Most previous experiments have evaluated CLPG lambs based solely on
phenotype. This means that up to 66% of the categorized lambs in the non-
CLPG group may be carrying the mutant CLPG allele. The objectives of the
current study are to examine the effects of lamb genotype and/or phenotype on
skeletal muscle DNA, RNA, and protein concentrations, indices of muscle fiber

type, and susceptibility of myofibrillar proteins to calpain-induced proteolysis.

LITERATURE REVIEW

Skeletal Mgsclegowm

Skeletal muscle development occurs as a result of distinct prenatal and/or
postnatal events. Muscle fiber hyperplasia, an increase in muscle fiber number,
occurs during prenatal development. Postnatal muscle growth results from
muscle fiber hypertrophy (Pearson and Dutson, 1991).

Embryonic formation of muscle fibers from mononucleated cells is called
myogenesis (Stockdale, 1992). Myogenesis begins with the formation of
myoblasts, in the myotomes of somites. Myoblasts are derived from
mesenchymal stem cells that become committed to the myogenic lineage.
Myoblasts proliferate, differentiate and fuse together to form multinucleated
myotubes. During differentiation, myoblasts permanently withdraw from the cell
cycle and begin to express muscle-specific genes. The first myotubes that
develop as elongated structures are termed “primary”. Myotubes develop with
centrally located nuclei. Myotubes then begin to accumulate myofibrillar proteins,
such as actin and myosin, and assemble them into organized contractile units.
As myotubes mature nuclei migrate to the periphery of the cell and become
displaced by myofibrils, which are the organelles of muscle contraction. These
myofibrils align to give skeletal muscle fibers their typical striated appearance.

Secondary muscle fiber formation occurs late in embryogenesis and
throughout fetal development. Secondary myotubes result from myoblast

proliferation, differentiation and fusion at the periphery of primary myotubes.

Numerous secondary myotubes form around each primary myotube. These
myotubes also undergo myofibrillogenesis and maturation into muscle fibers, as
described for primary muscle fibers. Skeletal muscle fiber numbers typically do
not change after birth in livestock species. As previously stated, postnatal growth
is considered to result primarily from hypertrophy, or growth through an increase
in cell size (Pearson and Dutson, 1991).

Muscle fiber hypertrophy occurs throughout prenatal and postnatal growth.
Hypertrophy of muscle fibers is associated with increases in both DNA and
protein. Since muscle fiber nuclei are incapable of DNA synthesis, the
accumulation of muscle fiber DNA results from proliferation, differentiation and
incorporation of satellite cells (Mauro, 1961; Moss and LeBlond 1971). Satellite
cells, which lie between the sarcolemma and the basement membrane of muscle
fibers, undergo proliferation and differentiation in response to external stimuli,
such as growth factors (reviewed by Florini and Ewton, 1996; Dodson et al.,
1996). The majority of muscle fiber nuclei accumulate postnatal, as a result of
satellite cell incorporation. It is generally accepted that each muscle fiber
nucleus accommodates a finite quantity of volume (Cheek, 1971). Consequently,
accumulation of muscle fiber DNA via satellite cells is a prerequisite for muscle
fiber hypertrophy.

In young, growing lambs myofibrillar proteins increase disproportionately
with DNA, resulting in an increased protein to DNA ratio. (Lorenzen et al., 2000).
Accumulation of muscle protein occurs when fractional protein synthesis exceeds

degradation. Muscle protein accumulation leads to increases in myofibril number

and length. Myofibrils increase in length due to addition of new sarcomeres, the
smallest functional contractile units of muscle, at the tendon ends of muscle
fibers (Williams and Goldspink, 1971). Myofibrils also increase in diameter as
myofibrillar proteins accumulate. Myofibril enlargement is followed by
longitudinal splitting, which results in formation of two myofibrils that can
subsequently increase in diameter (Goldspink, 1970). Muscle fiber size is
ultimately related to sarcomere number as well as the number and size of
myofibrils.
Callipyge Sheep

A Dorset ram, Solid Gold, from the Moffat flock in Piedmondt, Oklahoma
exhibited a unique muscle hypertrophy phenomenon in 1983 (Cockett et al.,
1999). Offspring from this heavily muscled ram soon became popular with
commercial and club lamb producers due to their muscularity and trimness. The
gene responsible for this muscular condition was mapped to ovine chromosome
18 and named callipyge (CLPG), which is Greek for beautiful buttocks (Cockett et
al., 1994). Initial studies showed that CLPG lambs have 32% more muscle mass
than non-CLPG lambs. This increase is due to hypertrophy of the Iongissimus
dorsi and muscles of the pelvic limb (Cockett et al., 1994). Callipyge lamb
carcasses were also found to be 7% leaner than normal carcasses (Cockett et
al., 1994). These findings fueled producers’ interest in callipyge sheep and
prompted numerous studies confirming the CLPG gene’s ability to increase
muscle mass and decrease fat (F reking et al., 1998b; Jackson et al., 1997a,b,c;

Koohmaraie et al., 1995). Callipyge lambs also consumed less feed, but had the

same average daily gain as normal lambs. Therefore, CLPG lambs have an
improved feed utilization efficiency (Jackson et al., 1997a).
Genetic Inheritance of CW

Initial studies on the inheritance of the callipyge gene showed segregation
of the muscular phenotype to be 49%, or 97 out of 200 lambs (Cockett et al.,
1994). This was confirmed by similar matings producing greater than 300 lambs
(Freking et al., 1998a,b; Jackson et al., 1997a). Thus, inheritance patterns of the
hypertrophied sheep suggested a single autosomal dominant gene was
responsible. However, further research demonstrated the muscle hypertrophy
phenomenon is expressed only in CN offspring, C denotes the mutant CLPG
allele and N denotes the wild-type normal allele (Cockett et al., 1996). The first
allele indicates paternal origin (Table 1). This pattern of inheritance is called
polar overdominance (Cockett et al., 1996). Polar refers to reciprocal
heterozygous genotypes expressing different phenotypes. Overdominance is
when both homozygous genotypes produce similar phenotypes (Cockett et al.,
1996). The genotypes possible from mating a heterozygous CLPG ewe with a
heterozygous CLPG ram are CC, NC, NN and CN, the paternal allele being
written first. Of these offspring, only animals of the CN genotype express the
muscle hypertrophy phenotype.

Chromosomal position of CLPG was further mapped to a 3.9cM interval
on chromosome 18 bounded by CSSM18 and OY3-OY5-BMS1561 flanking

markers (Freking et al., 1998a). Although the precise identity of the CLPG

mutation is unknown, candidate genes have been proposed. Bidwell et al.

(1999) reported GTL2 (gene trap locus 2) expression, measured at 56 days of

Table 1. Possible offspring from CN sired matings. Paternal alleles are on the
left hand side of the boxes and maternal alleles are written above. The
first allele written denotes paternal origin and C denotes the mutant
callipyge allele. Asterisk next to genotype denotes phenotypic
callipyge and percentages to the right are estimated number of
offspring that exhibit the callipyge phenotype.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N N
CN* CN” 50% CLPG
NN NN

C N
CC CN” 25% CLPG
NC NN

N C
CN" CC 25% CLPG
NN NC

C C
CC CC 0% CLPG
NC NC

 

 

 

age, was elevated in hypertrophied muscles containing at least one CLPG gene.
Furthermore, GTL2's chromosomal position makes it an ideal candidate gene for
CLPG but GTL2 lacks an open reading frame, making it a non-coding sequence
(Bidwell et al., 1999; Wylie et al., 2000). While the candidate gene identified by
Bidwell et al. (1999) did not clarify the biological mechanisms responsible for the
CLPG condition due to the non-coding sequence of GTL2, it did help researchers
solidify future findings on imprinted genes. Imprinted genes on human
chromosome 14q32, homologus to mouse distal chromosome 12 and sheep
distal chromosome 18, were identified as GTL2 and DLK1 (Wylie et al., 2000).
The maternally expressed gene, GTL2, lacks a significant open reading frame,
while the paternally expressed gene, DLK 1, encodes for a cell-surface
transmembrane protein containing epidermal growth factor-like repeats (Wylie et
al., 2000). The findings from Bidwell et al. (1999) and Wylie et al., (2000)
suggest CLPG lambs result from imprinted genes GTL2/DLK1. Furthermore,
Miyoshi et al. (2000) contends the maternally imprinted GTL2 in humans is the
same as maternally imprinted MEG3 in mice. Miyoshi’s findings along with
Fahrenkrug et al. (2000), who suggested PREP-1 (preadipocyte factor-1). and
MEG-3 (maternally expressed gene 3) as candidate genes, based on
comparative mapping of the ovine CLPG locus, lead to the same conclusion.
Depending on the preferred name, the functional candidate genes for CLPG are
the maternally expressed, non-coding MEGS (GTL2) and the paternally
expressed, adipogenesis regulator PREP-1 (DLK1, p62, ZOG; Fahrenkrug et al.,

2000; Wylie et al., 2000). These are candidates due to chromosomal position,

mode of inheritance and negative impact on adipogenesis, as seen in CLPG
lambs. As the impact of these genes on muscle growth is not understood, further
research is warranted.

Prenatal growth

There have been no studies addressing prenatal development of CLPG
lambs. However, it is assumed that prenatal growth of CLPG (CN) lambs is
similar to prenatal growth of non-CLPG (NN) lambs. The CLPG gene does not
affect birth weights, percentage of dystocia, multiple births or gender distribution
(Jackson et al., 1997a). Additionally, no increase in apparent muscle fiber
number exists (Koohmaraie et al., 1995). Since muscle fiber hyperplasia occurs
prenatally, these data suggest that increased muscle mass in CLPG lambs is due
to postnatal muscle hypertrophy.

The CLPG condition in lambs is distinct from the well-studied condition of
double muscling in cattle. Recent discoveries have identified the genetic factor
controlling double muscling in cattle. ln attempts to understand the physiological
role of transforming growth factor-8 superfamily members, McPherron et al.
(1997) “knocked out” the gene for a growth factor and subsequently produced
GDP-8 (myostatin) null mice. These mice had a ZOO-300% increase in muscle
mass compared with control mice. McPherron et al. (1997) further established
the increase in musculature was associated with an 86% increase in muscle fiber
number, as well as hypertrophy. Muscle fiber hyperplasia clearly does not occur
in CLPG sheep. It is thought that myostatin (GDP-8) does act as a negative

regulator of myoblast proliferation and/or differentiation, since myostatin (GDF-8)

null mice exhibit an increase in muscle fiber number. Myostatin null mice also
exhibit 14.50% increases in fiber diameter (McPherron et al., 1997). Thus, the
increased muscularity can be attributed to an increase in number of muscle cells,
as well as muscle fiber hypertrophy.

Belgian Blue and Piedmontese cattle have long been known for their
“double muscled” features. It has only been since McPherron's work in 1997 that
GDF-8 was identified as the mutation resulting in double muscled cattle. Belgian
Blue and Piedmontese have an 11-bp deletion and a GA transition, respectively,
in the coding region for myostatin. Therefore, it appears these breeds have
mutations within the myostatin gene and myostatin is a negative regulator of
muscle growth in cattle as well as mice (Grobet et al., 1997; Kambadur et al.,
1997). From a production standpoint the CLPG mutation is preferable to
mutations in myostatin. Increasing muscle fiber number often increases the size
and form of the fetus, thereby impairing ease of parturition in double muscled
animals. An increase in muscle fiber number does not occur in CLPG sheep and
hence. no difficulties in parturition are observed.

Postnatal Growth

Callipyge (CN) lambs phenotype is indistinguishable from normal lambs
until 4-6 weeks of age when the CLPG phenotype becomes apparent (Jackson et
al., 1997a,b). Duckett et al. (2000) reported selective muscular hypertrophy of
CLPG lambs begins between 7 and 20 kg, which was 2.7 and 14 weeks of age,
respectively. Despite discrepancies in onset of muscle hypertrophy, several

studies have shown muscles of pelvic limbs and torsos from CLPG lambs are

10

significantly larger than normal lambs. These studies also found no hypertrophy
occurred in muscles of the CLPG thoracic limb (Duckett et al., 2000; Jackson et
al., 1997a,b; Koohmaraie et al, 1995). Callipyge lambs have 32%, 42% and 39%
heavier Iongissimus, biceps femoris (BF) and semimembranosus muscles,
respectively, but no change in infraspinatus (IS) and supraspinatus muscle
weights in comparison to non-CLPG lambs at 6 months of age (Koohmaraie et al,
1995).

Hypertrophied Iongissimus, biceps femoris and semimembranosus
muscles have greater than 100% more calpastatin activity than non-
hypertrophied control muscles. In contrast, infraspinatus and supraspinatus
muscles of CLPG sheep, which do not hypertrophy, are similar in calpastatin
activity to phenotypically normal lambs (Koohmaraie et al., 1995). These authors
suggested increased calpastatin activity increases muscle mass by inhibiting the
calcium activated neutral proteases (calpains), which are thought to initiate
myofibrillar protein breakdown. lf calpastatin's inhibitory effect on calpains does
increase muscle mass, hypertrophied muscles of CLPG sheep would have less
protein turnover and a greater net protein accretion. Lorenzen et al. (2000)
evaluated protein turnover by measuring in vivo protein synthesis and accretion,
then determined degradation by difference. Callipyge lamb LD and BF muscle
fractional accretion rates were greater than those of non-CLPG lambs (Lorenzen
et al., 2000). These authors attributed the increased fractional accretion rate to a
reduction in fractional protein synthesis and an even greater reduction in

fractional protein degradation. However, this study only looked at one window of

11

time , 5-11 weeks of age, which is after CLPG lambs have already begun to
exhibit their extreme musculature. Therefore, Lorenzen et al. (2000) were unable
to conclude that the mechanisms responsible for maintaining muscle hypertrophy
are the same as those at the onset of muscle hypertrophy.

The precise role of the calpain system in muscle protein turnover is
unclear. For example, Brahman cattle have increased levels of muscle
calpastatin, but do not exhibit muscle hypertrophy relative to other cattle (Cole et
al., 1963; Whipple et al., 1990). Thus, a causal relationship between muscle
calpastatin activity and muscle growth remains to be demonstrated. it seems
likely that several factors may contribute to CLPG muscle hypertrophy.

Protein, DNA, and RNA content as well as RNA concentration and
RNAzDNA ratio are greater in the Iongissimus and semitendinosus of CLPG
lambs compared with non-CLPG lambs. Protein concentration, DNA
concentration and proteinzDNA are not altered by the CLPG phenotype
(Koohmaraie et al., 1995). In contrast, Carpenter et al. (1996) found proteinzDNA
ratio to be greater in the semitendinosus, Iongissimus and gluteus medius
muscles but not the supraspinatus muscle of CLPG lambs compared with non-
CLPG lambs. ProteianNA and RNAzDNA ratios in the semitendinosus,
Iongissimus, gluteus medius and supraspinatus were similar between CLPG and
non-CLPG (Carpenter et al., 1996). Traditionally RNAzDNA and proteianNA
have been used as indices of transcription and translation, respectively. While
this is a crude method to measure transcriptional or translational efficiency it

does provide insight into areas where further research needs to be performed.

12

The above studies suggest the CLPG phenotype does alter the rate of
transcription and/or translation.

During postnatal growth, increases in muscle and decreases in fat imply
CLPG lambs undergo a repartitioning effect. Considering the increase in feed
efficiency previously discussed, a partitioning of more energy to muscle and less
to other metabolic functions or biological products occurs in CLPG lambs.
Energy repartitioning is also supported by the lighter fleece weights (P < .01) and
shorter staple lengths (P < .03) observed in CLPG ewes (Jackson et al., 1997a).
C_alljpyge Mgscle Fiberlype Characteristics

Muscle fiber phenotypes are influenced by intrinsic properties of
myoblasts and extrinsic factors such as innervation. Muscle fiber phenotype
alterations result in fast, slow or mixed fast/slow fiber types. Muscle hypertrophy
can be obtained by fiber type transitions, since white muscle fibers (type llb)
typically have larger diameters than red (type I) or intermediate fibers (type Ila).
Fiber type differences result from production of lsoforrns of muscle contractile
protein (Robbins et al., 1986). The most abundant skeletal muscle contractile
protein is myosin and myosin heavy chain isofonns are more abundant than
other contractile protein isoforms, making them easier to evaluate. Myosin heavy

chain isoforrn gene expression occurs in the order lellaellxellb (arrows

denote possible transitions between different MHC isoforrns within a muscle
fiber) (Schiaffino and Reggiani, 1994). Conventional actomyosin ATPase typing
does not allow distinction between type llx and llb muscle fibers (Lefaucheur et

al., 1998). A higher percentage of type llb fibers have been observed in

13

hypertrophied CLPG lamb muscles. in the Iongissimus of 24-week-old CLPG
lambs. 9% of the muscle fibers are type I (slow, oxidative), 34% are type lla (fast,
oxidative-glycolytic) and 57% are type llb (fast glycolytic) vs. normal lambs,
which have 10%, 46% and 44% of these fiber types, respectively (Koohmaraie et
al., 1995). Lorenzen et al., (2000) observed comparable values at 5, 8 and 11
weeks of age with only the type llb fibers increasing statistically in CLPG lambs.
Hypertrophied muscles (gluteus medius and semitendinosus) but not a non-
hypertrophied muscle (supraspinatus) had similar increases in the percentage of
fast, glycolytic fibers (Carpenter et al., 1996; Koohmaraie et al., 1995).
Koohmaraie et al. (1995) also demonstrated type Ila and type llb muscle fibers of
the LD muscle in CLPG undergo a 47% and 45% increase in cross-sectional
area, respectively.

The semitendinosus type I, type Ila, type llb muscle fiber area is increased
in the CLPG condition by 3%, 99% and 52%, respectively. Increases in both
fiber type distribution and fiber area occur only with type llb muscle fibers. These
characteristics and an increase in type Ila fiber diameter account for the muscle
hypertrophy in CLPG lambs (Carpenter et al., 1996; Koohmaraie et al., 1995;
Lorenzen et al., 2000).

Potential Mechanisms of Callipyge Gene Action

Although the CLPG gene has not been identified, several experiments
have characterized biological mechanisms that may be associated with CLPG
gene action. Whisnant et al., (1998) examined blood hormone profiles to

determine ifthese differed between CLPG and normal sheep. Serum

14

concentration, frequency or pulse amplitude of growth hormone secretion did not
differ between CLPG and normal lambs. Likewise, there were no differences in
insulin, lGF-l or serum thyroxine concentrations between CLPG and normal
lambs. Serum cortisol levels increased similarly with restraint in CLPG and non-
CLPG lambs (Whisnant et al., 1998). This information implies that levels of
hormones that are generally thought to regulate growth and body composition
are similar between CLPG and non-CLPG lambs.

B-adrenergic agonists (BAA) are a class of endogenous or synthetic
compounds that elicit numerous physiological responses. The physiological BAA
compounds are the catecholamines, epinephrine and norepinephrine. Synthetic
forms of BAA, similar to endogenous forms in both pharmacological and chemical
properties, include; cimaterol, clenbuterol, L 664,969, ractopamine, and
salbutamol. All of the BAA initiate physiological responses by binding to B-
adrenergic receptors. B-adrenergic agonists have been used to increase muscle
mass and decrease fat in many species (Mersmann, 1998).

Studies in lambs show biceps femoris (BF) muscle mass is increased
18.6%-32.8% with BAA treatment (Koohmaraie et al., 1991; Beermann et al.,
1987). Differences in efficacy are dependent upon the agent used and length of
administration. Increases in muscle mass are accompanied by increases in
protein and RNA content as well as concentration (Koohmaraie et al., 1991;
Beennann et al., 1987). Concentration of muscle DNA is decreased, but content
is not altered with treatment (Kim et al., 1987; Koohmaraie et al., 1991;

Beennann et al., 1987). Muscles of BAA treated lambs have an increased

15

proportion and diameter of type II muscle fibers (white) (15%-50%) (Kim et al.,
1987; Beennann et al., 1987).

The similarities between BAA treated and CLPG lambs led researchers to
examine the effects of BAA treatment on CLPG lambs. Administration of the BAA
(L-644,969) to CLPG lambs elicited no response (Koohmaraie et al., 1996).
These authors suggested CLPG lambs may not respond to BAA because muscle
growth rates may already be near maximum. Additionally the CLPG gene may
exert its effects through intracellular events similar to BAA treatments
(Koohmaraie et al., 1996).

While the biological mechanism affecting the CLPG phenotype has not
been found, evidence suggests biological differences occur in homozygous and
heterozygous CLPG sheep. Freking et al. (1998) demonstrated that
hypertrophied CN lamb muscles had the highest calpastatin activity and shear
force values (least tender), CC lambs were intermediate, and NN and NC lambs
had the lowest calpastatin activity and shear force. The study performed by
Freking et al. (1998) was the first to evaluate all genotypes from CLPG matings.
Other studies have quantified characteristics of phenotypic CLPG or non-
phenotypic normal lambs. The above information also allows for pooling of NN
and NC genotypes when there is no statistical difference. Nevertheless, a
biological difference in CC lambs provides evidence for studying skeletal muscle-

characteristics of CLGP genotypes to better understand CLPG gene action.

16

Qaracteristics of Meat from calm

The hypertrophied condition of CLPG lambs adds lean tissue to the
carcasses, but increased levels of muscle calpastatin have adverse effects on
carcass quality. Callipyge lamb loin chops are significantly tougher than normal
lamb chops and do not undergo typical tenderization during postmortem Storage.
Warner-Bratzler shear force values are 44.8%, 112.2% and 144.7% greater for
CLPG lamb loin chops than control lamb chops on days 1, 7, and 21,
respectively (Koohmaraie et al., 1995). It is important to note that even after 21
days of aging CLPG shear force values were still greater than shear force values
for day 1 normal lambs (Koohmaraie et al., 1995). Myofibril fragmentation index
values are higher for normal lambs, indicating there is less postmortem
proteolysis of myofibrillar proteins in CLPG lambs. Western blot analysis
confirmed a lack of postmortem degradation of myofibrillar proteins troponin-T,
desmin, vinculin, nebulin and titin from CLPG lambs (Koohmaraie et al., 1995).

Elevated levels of collagen and increased collagen crosslinks also
decrease meat tenderness. However, collagen content and
hydroxylysylpyridinoline crosslink concentration are lower in CLPG lambs than
non-CLPG lambs (Field et al., 1996). Collectively, these data demonstrate that

decreased tenderness in CLPG lambs results from less postmortem proteolysis.

17

MATERIALS AND METHODS
giggle Collection

Muscle samples were obtained after slaughter of market weight
(approximately 22-week-old), phenotypically CLPG (CN, n=6) and non-CLPG
(NN, NC, CC, n=3 per genotype) ram lambs. Lamb genotyping was performed
as described by Freking et al., (1998) and genotype data was provided Dr.
Freking (USDA-ARS, Clay Center, NE). Muscle samples were collected in the
abattoir at Roman L. Hruska U.S. Meat Animal Research Center (USDA-ARS,
Clay Center, NE). Skeletal muscle samples were collected within 15 minutes
after exsanguination. Longissimus dorsi, infraspinatus, and biceps femoris
samples were removed from the right side of each lamb, diced and quick-frozen
in liquid nitrogen, then stored at -80°C. Samples were kept on dry ice during
transportation to the Muscle Biology Laboratory at Michigan State University,
where they were stored at -80°C.

Carcass measurements and protein and nucleic acid measurements were
collected from all genotypes and muscles. Myofibrillar degradation analysis,
myosin heavy chain isofonn and sarcoplasmic protein separation was performed
with LD tissue of NN, NC, CC and ON lamb samples.

Carcass Measgrements

Lamb carcass weights were recorded on an in-Iine rail scale immediately
after slaughter and evisceration. To offset muscle weight removed during
sample collection 0.9 kg was added to each carcass weight. Ribeye area (REA)

was determined as the cross-sectional area of the Iongissimus dorsi. Backfat

18

was defined as the subcutaneous fat thickness measured at a point half the
lateral distance of the Iongissimus muscle from the vertical process of the
thoracic vertebra. Both measurements were taken adjacent to the 12th rib
interface after the longissimus muscle was exposed by a cut perpendicular to the
vertebral column between the 12‘” and 13‘“ rib. Tracer paper was applied to the
cut face and the outside perimeter of the loin muscle was traced onto the paper,
along with the outside perimeter of subcutaneous fat. Ribeye area was taken
from the right side of lamb carcasses. Traced REA’s and backfat thickness were
measured using a standard lamb REA grid and backfat ruler. Ribeye area and
backfat thickness measures were averaged from independent measures taken
by three evaluators.

After a 24 hour chill of the carcass, the Iongissimus dorsi (LD), biceps
femoris (BF) and infraspinatus (IS) were dissected from the left side of the
carcass and weighed. The LD was removed by making an incision perpendicular
to the long axis of the muscle and adjacent to the cranial edge of the ilium. A
knife was run along the vertical processes of the lumbar and thoracic vertebrae
to remove the medial side of the LD from the spine. This was followed by an
incision running along the transverse processes of the lumbar vertebrae, ribs,
and ending at the cervical vertebrae to detach the LD from its insertion. The LD
muscles were removed from carcasses, and then separated from attached

superficial muscles such as the spinalis or multifidus dorsi.

19

The BF was removed from the left side of the carcass with the proximal
detachment being achieved with an incision on the caudal edge of the ilium and
sacral tuber. Distal detachment was at the distal end of the femur.

lnfraspinatus removal was performed by palpation of the shoulder to
locate the spine of the scapula. Once the spine of the scapula was located, an
incision was made on the ventral/caudal edge of the spine, partially detaching the
IS from the scapula. The IS was pulled from the infraspinous fossa. The
proximal end of the IS was detached from scapular cartilage and the distal end
from the lateral tuberosity of the humerus.

All muscles were closely trimmed of any other muscles and excess
connective tissue or fat, then weighed. The same individual performed all
trimming and weighing of muscles.

Protein and Nucleic Acids

Protein concentrations were determined by the biuret method (Gomall et
al., 1949). The DNA concentrations were determined according to Labarca and
Paigen (1980) using Hoechst 33258 reagent. Concentrations of RNA were
determined by the method of Munro and Fleck (1969). Detailed procedures for
quantifying protein and nucleic acid concentrations is in the appendix.

Sample Preparation

A list of all solutions used in the following methods is included in the
appendix. Half gram LD muscle samples were placed in 10 volumes of
homogenization buffer containing 75 mM KCL, 10 mM KHzPO4, 2 mM MgCl2, 2
mM ethylenediamine tetra acetic acid (EDTA), 50 mM NaF, 2 mM

20

phenylmethylsulfonyl flouride (PMSF), 6 mg/L leupeptin and homogenized by
Polytron® (Brinkmann, Westbury, NY) for 10 seconds at speed 6 using a 1.5 cm
generator. Sodium fluoride, PMSF and leupeptin were dissolved in stocks and
added to homogenization buffer the day of extraction. Samples were
homogenized a second time for 10 seconds at speed 6 to ensure a
homogeneous mixture. Samples were centrifuged at 10,000 xg for 15 minutes at
4°C to separate myofibrillar proteins from sarcoplasmic proteins. The
supernatant containing sarcoplasmic proteins was poured off and stored at
-80°C. Myofibrillar proteins were suspended in homogenization buffer, vortexed,
and centrifuged as indicated above. The supernatant fluid was discarded. This
wash procedure was performed two more times and the final pellet was
resuspended in 10 mL homogenization buffer without sodium flouride, PMSF or
leupeptin and stored at -80°C. A 0.5 mL aliquot of sarcoplasmic and myofibrillar
protein preparations was removed prior to freezing and mixed 1:1 vlv with 2x
Laemmli buffer (0.125 M Tris, 4% SDS, 20% glycerol) without B-mercapto-
ethanol (MCE), heated at 90°C for 5 minutes and stored at -20°C.

Protein concentrations were determined by bicinchoninic acid assay
(Pierce, Rockford, IL) for myosin heavy chain (MHC) studies and by biuret
(Gomall et al., 1949) for degradation, as well as sarcoplasmic protein studies. A
serial dilution of bovine serum albumin was used to create a standard curve from

10 mg/ml to 0 mg] ml. The biuret assay was used on non-denatured myofibrillar

protein samples to increase solubility.

21

Myosin Heavy Chain lsoforrn S_eparation

Myofibrillar proteins from the LD muscle of three lambs per genotype were
suspended in 1x Laemmli buffer plus glycerol (0.0625 M Tris, 2% SDS, 5% MCE,
40% glycerol, pH 6.8). Proteins (0.5 or 1 jig/lane) were loaded onto

discontinuous 8% gels (50:1 ratio of acrylamide to N,N'-methylene-bis-
acrylamide) with 4% stacking gels (50:1). Gels (20 cm x 20 cm x 0.75 mm) were
run at 275 volts for 24 hours at 4°C using a BIO-RAD Protean II xi cell (BIO-RAD,
Hercules, CA) with a water-cooled chamber. A two-buffer system was used. The
upper running buffer consisted of 0.1 M Tris (base), 150 mM glycine, and 0.1%
SDS, and lower running buffer contained 50 mM Tris (base), 75 mM glycine, and
0.05% 808 (T almadge and Roy, 1993). Myosin heavy chain (MHC) isoforrns
separated by SDS-polyacrylamide gel electrophoresis (PAGE) and were silver
stained (BIO-RAD, Hercules, CA). Electronic images were acquired and
analyses were performed with a BIO-RAD Gel Doc imaging system and Quantity
One, respectively (BIO-RAD, Hercules, CA). Designation of myosin heavy chain
isofonns was based on the relative migration of type Ila, llx, llb and l, as
described for rat myosin (T almadge and Roy, 1993).
Myosin Heavy Chain Western Blots

Myosin heavy chain gels were electrophoretically transferred to

polyvinylidene fluoride (PVDF, lmmobilonW-P, Millipore Corporation, Bedford,
MA) overnight at 4°C. Transfer buffer (25 mM Tris, 0.192 Glycine, 0.1% SDS, pH

8.3) was mixed gently with a stir bar during transfer. From cathode to anode, a

sandwich consisting of clamping cassette, one fiber pad, two pieces of Whatman

22

filter paper, gel, PVDF membrane, two pieces of Whatman filter paper, one fiber
pad and the anode side of transfer clamping cassette was assembled prior
transfer. Proteins were transferred from the gels to membrane at 70 mA
constant current. After removal from the transfer apparatus, membranes were
rinsed with double distilled (ddl) water, placed in 10 ml of 1% non-fat dry milk
(NFDM) blocking solution and incubated for 1 hour to minimize non-specific
antibody binding. All blocking, washing and incubations occurred on a red rocker
at speed 7 in Falcon” tissue culture plate tops. Primary mouse anti-myosin
monoclonal antibody (Na4) was diluted 1:5000 in 1% NFDM. Blocking solution
was poured off membranes and diluted Na4 Ab was poured onto membranes
and incubated for 1.5 hours at room temperature. Membranes were washed 3
times for 5 minutes each time with 10 mL 1% NFDM. After washes, 20 mL of 1%
NFDM with anti-mouse lgG alkaline phosphatase (AP) conjugated 2° Ab
(1:10.000 concentration) were dispensed onto membrane and incubated for 30
minutes. Membranes were washed 3 times for 5 minutes each time with 10 mL
1% NFDM followed by 2 washes with 10 mL Tris-buffered saline containing 0.1%
Tween-20. Membranes were exposed to BIO-RAD AP substrate working
solution (BIO-RAD, Hercules, CA) until desired band intensity appeared, but no
longer than 1 minute. Membranes were rinsed several times with ddl water to
remove residual substrate and halt the enzymatic activity. Membranes were

dried on paper towel before image acquisition.

23

Sapc_oplasmic Protein Separation

Ten micrograms of LD muscle protein per lane were loaded on a
discontinuous 10% (37.521 ratio of acrylamide to N,N’-methylene-bis-acrylamide)
separating gel poured up to 18 mm from the top of the inner plate on a Bio-Rad
mini protean ll (BIO-RAD, Hercules, CA). Gels were allowed to set-up for 1 hour
before a 4% (37.5:1) stacker was poured. Samples were electrophoresed at a
constant voltage of 180 until the dye front ran off (40-50 minutes). Gels were
stained in Coomassie blue R-250 and destained (1-hour wash in destain 1, 3-
hour wash in destain 2) before being dried between sheets of cellophane and
scanned using a Bio-Rad fluor-s image analysis system. Electronic images were
acquired and analyses were performed with a BIO-RAD Gel Doc imaging system
and Quantity One, respectively (BIO-RAD, Hercules, CA).
m-Calpain Mediated In vitm Proteolysis

Myofibrillar proteins from three NN and three CN lambs were purified
according to procedure of Goll et al., (1974). All samples were visually appraised
by light microscopy to ensure homogeneous and uniform myofibrils.

Six milligrams of protein suspended in 50 mM Tris (pH 6.7-7.0) were
dispensed in a 2 mL microcentrifuge tube. Volume was adjusted to 2 mL with 50
mM Tris and 165 mM NaCI, then centrifuged at 10,000 x g for 10 minutes.
Supernatant fluid was poured off and the pellet was resuspended in 2 mL of
digestion buffer (50 mM Tris, 165 mM NaCI, 10 mM MCE, 5 mM CaClz) by
vortexing and inversion. Microcentrifuge tubes were placed on a mechanical

inverter at 10 rpm for incubation with m-calpain. An initial 250 pl sample (time 0)

24

was drawn and added 1:1 vlv to 2x Laemmli buffer and heated to 95°C for 5
minutes. One unit of protease activity per 12 mg myofibrillar protein was then
added to the digestion buffer. At 5, 15, 30, 60, 120, 240, and 480 minutes after
m-calpain addition, samples were prepared as described for the initial sample. A
second in vitro digestion was performed using 0.5 unit of active protease (m-
calpain) per milligram myofibrillar protein for 120 minutes. One unit of m-calpain
activity was defined as the amount of enzyme that catalyzed an increase of 1.0
absorbence unit at 278nm in 60 minutes at 25°C. All m-calpain was graciously
provided by Dr. M. Koohmaraie from his laboratory at the Roman L. Hruska U.S.
Meat Animal Research Center (ARS, USDA,Clay Center, NE).

Separation of myofibrillar proteins was conducted using SDS-PAGE (10%
gel with 4% stacker 37.5:1 ratio of acrylamide to N,N'-methylene-bis-acrylamide)
at 180 volts for 55 minutes. Proteins were electrophoretically transferred to
PVDF at 150 mA for 2 hours using a BIO-RAD mini protean ll transfer unit, with
ice blocks, placed on a stir plate (BIO-RAD, Hercules, CA). After removal from
and disassembly of the transfer apparatus, a mark in pencil was made on the
membrane between the BIO—RAD broad range gel electrophoresis molecular
weight marker (BIO-RAD, Hercules, CA) lane and the sample lane. Two more
marks were made on the membrane at the 65 kiloDalton (kD) marker to aid in
removal of proteins greater than 66 kD. The molecular weight marker lane and
proteins greater than 66 kD were removed from the rest of the membrane and
stained in amido black (Figure 3). Proteins greater than 66 kD were used to

verify equal protein load amongst lanes. The far right lane was removed and

25

used as a negative control. The remaining membrane and negative control lane
were placed in 10 ml of 1% bovine serum albumin (BSA) blocking solution and
incubated for 1 hour to minimize non-specific antibody binding.

All blocking, washing and incubations occurred on a red rocker at speed 7
in Falcon” tissue culture plate tops. Primary mouse anti-desmin antibody was
produced by the D76 hybridoma cell line and harvested on day 12 of culture.
Supernatant fluid was harvested then stored in aliquots in a -20°C freezer
(growth and harvesting of cells is described in appendix). Primary antibody (1°
Ab) was thawed at 37°C prior to use. Alter blocking for at least 1 hour, 1% BSA
blocking solution was poured off and 10 mL of primary mouse anti-desmin
hybridoma supernatant fluid was applied to membranes, covered and incubated
for over 4 hours at room temperature. Primary mouse anti-desmin antibody was

removed and stored for second use (1° Ab can be used twice with no loss in
intensity). The negative control lane remained in blocking solution during 1° Ab
incubation. Membranes, including negative control, were washed three times for
5 minutes each time with 10 mL 1% BSA. After washes, 10 mL of 1% BSA was
dispensed onto the membrane and 20 pl anti-mouse IgG biotin conjugated
secondary antibody (2° Ab, 1:500 concentration) were added. Incubation plates
were covered and incubated for 45 minutes at room temperature. Membranes
were washed 3 times for 5 minutes each time with 10 mL 1% BSA, then
incubated with Extra-Avidin conjugated AP (Sigma, 1:1000 dilution in 1% BSA)
for 35 minutes. Membranes were washed three times for 5 minutes each time

with 10 mL 1% BSA followed by 2 washes with 10 mL 0.1% TTBS. Membranes

26

were exposed to BIO-RAD AP substrate working solution (BIO-RAD, Hercules,
CA) to visualize Ab binding to desmin and desmin degradation products.
Membranes were rinsed several times with ddl water to remove residual
substrate and stop the enzymatic reaction. Membranes were allowed to air dry
on a paper towel before visual appraisal.
Statistical Anamis

All data containing a numeric value of measure were analyzed using the
General Linear Model procedure of SAS (SAS Inst. Inc., Cary, NC) with the
Tukey-Kramer test for pair-wise comparisons of mean differences. Sample sets
NN and NC were pooled as non-CLPG lambs before being compared to CN and
CC genotypes.

27

RESULTS

Effects of lamb genotype on carcass traits and skeletal muscle
characteristics were examined. Three lamb genotypes (CN, CC, NC) contain at
least one mutant CLPG allele, but only the CN genotype exhibits the phenotypic
muscular hypertrophy of the CLPG condition. Freking et al. (1998) demonstrated
that hypertrophied CN lamb muscles had the highest calpastatin activity and
shear force values (least tender), CC lambs were intermediate, and NN and NC
lambs had the lowest calpastatin activity and shear force. Although lambs with
the CC genotype do not exhibit muscular hypertrophy, they are distinct from
lambs of NN and NC genotypes regarding some charateristics. Therefore, CN
(referred to as CLPG) and CC genotypes were compared to the pooled NN and
NC genotypes (referred to as non-CLPG) in the current study.
Carcass Measurements and Muscle WM

Carcass traits and dissected muscle weights expressed as a percent of
carcass weight are listed in Table 2 for all lamb genotypes. Carcass weight and
backfat thickness were similar among genotypes (Table 2). Callipyge lambs had
higher leg scores and larger REAs than the average of non-CLPG. Longissimus
dorsi and BF muscles were 26% and 22% heavier, respectively in CLPG lambs
compared with non-CLPG (Tables 3 and 4). Additionally, LD and BF muscles
comprised a higher percentage of carcass weight in CN lambs than non-CLPG
lambs (T able 2). lnfraspinatus muscle weights were similar among genotypes
(Table 5) and the IS made up a comparable proportion of carcass weight in all

genotypes (Tables 2).

28

Table 2. Carcass characteristics of ram lambs of NN, NC, CN and CC
genotypes. The mutant callipyge allele is represented by C and N
represents the wild-type normal allele. The paternal allele is listed first.
The only animals expressing the phenotypic callipyge muscle hypertrophy
are genetically annotated as CN, while NN, NC, and CC genotypes are

 

 

 

 

 

 

 

phenotypic normal.
Pooled

Trait NN NC CN cc SEM
Carcass Weight, kg 27.7 26.2 27.6 21.8 1.1
12111 Rib Backfat, mm 4.2 4.2 4.2 3.3 0.4
Egg=sfiggf'ge';:asbfiz'e 11.0 10.7 14.8‘ 11.3 0.3
REA, cm2 15.1 13.5 20.4' 14.0 0.3
figggfi‘m 2.4 2.6 3.2' 2.7 0.1
gfigf’czgmzigm 1.4 1.5 1.8’ 1.4 0.03
afggfégzgfieigm 0.6 0.7 0.6 0.6 0.1

 

 

 

 

 

 

 

 

a = difierent frofii pool of NN and NC (i=<0.05).

fiantification of Skeletal Muscle Nucleic Acids and Protein

Muscle weight, protein, DNA and RNA concentrations are shown for the

LD, BF and IS muscles in Tables 3, 4 & 5, respectively. Concentration of protein

was not statistically different for LD and IS muscle groups amongst genotypes,

but BF protein concentration was lower in CLPG and genotypic CC lambs

compared with non-CLPG lambs. Concentrations of DNA and RNA for the three

muscles were similar amongst genotypes (Tables 3, 4 & 5). However, LD

muscles of CLPG lambs had more total DNA than those of non-CLPG lambs

(Table 3). Longissimus and BF muscles of CLPG lambs also had more RNA

than those of non-CLPG lambs. No difference in nucleic acid content of IS

29

muscles were observed among genotypes. Thus, increased nucleic acid content

is attributable to hypertrophy of muscles in CLPG lambs.

The RNA:DNA ratio in LD muscles of non-CLPG lambs is 20% less than
the mean RNA:DNA ratio of the ON or CC genotypes (Table 3). Protein:RNA
ratios of the LD were higher for non-CLPG lambs than CLPG or genotypic CC
lambs. Protein:DNA ratio for all three muscles as well as proteianNA and
RNA:DNA ratios for the BF and IS muscle were similar among genotypes (Tables
3, 4 and 5).

Table 3. Longissimus dorsi muscle weight, protein and nucleic acid
measurements of ram lambs of from NN, NC, CN and CC genotypes. The
mutant callipyge allele is represented by C and N represents the wild-type
normal allele. The paternal allele is listed first. The only animals

expressing the phenotypic callipyge muscle hypertrophy are genetically
annotated as CN, while NN, NC, and CC genotypes are phenotypic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

normal.

Pooled
flaimus Dorsi Trait NN NC CN CC SEM
Wet tissue weight, 9 654.7 659.2 892.1‘ 592.7 29.8
Protein, mg/g 222.7 257.9 199.6 215.3 7.8
Protein, 9 145.8 168.5 180.8 128.9 9.9
DNA, uglg 177.2 210.5 187.7 200.6 47
DNA, mg 116.0 138.2 167.6’ 119.5 6.6
RNA uglg 333.7 481.1 469.7 531.7 21.2
RNA, mg 218.1 316.2 414.8' 317.2 17.3
Protein:DNA 1259.7 1237.0 1062.5 1067.0 35.0
Protein:RNA 683.6 553.2 429.7‘I 404.3a 27.5
RNA:DNA 1.8 2.2 2.5“ 2.6a 0.1
a = different from pool of N N and NC (P<0.05).

 

30

 

Table 4. Biceps femoris muscle weight, protein and nucleic acid measurements
of ram lambs of from NN, NC, CN and CC genotypes. The mutant
callipyge allele is represented by C and N represents the wild-type normal
allele. The paternal allele is listed first. The only animals expressing the
characteristic phenotypic callipyge muscle hypertrophy are genetically
annotated as CN, while NN, NC, and CC genotypes are phenotypic

 

 

 

 

 

 

 

 

 

 

normal.
Pooled
Biceps Femorls Trait NN NC CN CC SEM
Wet tissue weight, 9 378.0 393.1 494.4‘I 308.4 16.9
Protein, mglg 274.6 283.6 232.7“ 224.1' 6.7
Protein, 9 104.3 111.2 116.1 69.0 5.7
DNA, uglg 198.7 223.8 207.7 21 1.3 5.4
DNA, mg 75.0 87.7 102.4 65.1 3.7
RNA uglg 432.7 512.2 514.0 535.5 17.9
RNA, mg 162.6 200.7 252.8‘ 164.4 8.7
Protein:DNA 1381.5 1265.1 1131.5 1065.3 35.9
Protein:RNA 645.8 558.2 455.9 433.2 25.8
RNA:DNA 2.1 2.2 2.4 2.5 0.1

 

 

 

 

 

 

 

 

a = different from pool of NI

N and NC (P<0.05).

31

Table 5. lnfraspinatus muscle weight, protein and nucleic acid measurements of
ram lambs of from NN, NC, CN and CC genotypes. The mutant callipyge
allele is represented by C and N represents the wild-type normal allele.
The paternal allele is listed first. The only animals expressing the
phenotypic callipyge muscle hypertrophy are genetically annotated as CN,
while NN, NC, and CC genotypes are phenotypic normal.

 

 

 

 

 

 

 

 

 

 

Pooled
lnfraspinatus Trait NN NC CN CC SEM
Wet tissue weight, 9 172.4 169.3 161.0 139.1 5.8
Protein, mglg 222.6 226.1 228.2 237.8 2.7
'ifiitein, g 38.3 38.3 36.9 33.0 1.5
DNA, uglg 21W 228.6 211.8 218.1 3.7
bNA, mg 37.8 38.8 34.3 30.1 1.4
RNA uglg 538.2 544.8 504.4 537.3 28.2
RNA, mg 92.3 92.3 80.6 72.7 4.1
FroteinTJWA 1014.9 991.8 10788 10957) 17.7
ProteirFRWA 418.3 420.4 480.5 483.8 22.1
WDNA 2.4 2.3 2.3 2.4 0.1

 

 

 

 

 

 

 

 

32

Mgappres offiggle FiMTransition

Gel electrophoresis of muscle proteins from lambs of all genotypes
provided a quantitative measure for all four myosin heavy chain isoforms and
several sarcoplasmic proteins. Myosin heavy chain lsoforrns type I, llb, llx and
Ila were separated using SDSPAGE (Figure 1). All bands were confirmed to be
myosin lsoforrns using a monoclonal Ab (Na4) that recognizes all forms of
sarcomeric myosin. However, specific myosin isoforms were not identified by
Western blotting, since available isoforrn specific antibodies either did not
recognize lamb myosin or cross-reacted with multiple myosin isoforrns (results
not shown). Designation of myosin heavy chain lsoforrns was based on the
relative migration of type Ila, llx, llb and l, as described for rat myosin (T almadge
and Roy, 1993). Type I, type Ila and llb myosin heavy chain isoforrns did not
change in either CN or CC genotypes in comparison to non-CLPG lambs (Table
6). In contrast, type llx of CLPG lambs increased 25% in comparison with the
mean of non-CLPG lambs (Table 6).

Sarcoplasmic proteins were separated by SDS-PAGE and several bands
were assigned putative identities (Figure 2). No differences among genotypes

were detected in the proportion of specific protein bands (Table 7).

33

Figure 1. Analysis of myosin heavy chain isofonns (MHC). Separation of MHC
from phenotypic non-callipyge (NN) lambs (left lane) and phenotypic
callipyge (CN) lambs (right lane) was accomplished by SDS-PAGE.

 

Myosin heavy chain isoforrns were visualized by silver staining as

described in methods. Designation of myosin heavy chain isoforms was
based on the relative migration of type Ila, llx, llb and l, as described for
rat myosin (Talmadge and Roy, 1993).

Table 6. Percentage of myosin heavy chain isofonn in the longissimus muscle of
ram lambs of from NN, NC, CN, or CC lambs. C represents the mutant
callipyge allele and N represents the wild-type normal allele. The paternal
allele is listed first. The only animals expressing the phenotypic callipyge
muscle hypertrophy are genetically annotated as CN, while NN, NC, and
CC genotypes are phenotypic normal.

 

 

 

 

Pooled
lsoform NN NC CN CC SEM
% Type Ila 39.9 37.4 35.0 35.5 1.3
% Type llx 42.9 38.5 54.3‘3 46.6 0.5
% Type llb 5.2 5.5 5.5 5.3 0.9
% Type I 12.0 18.6 5.2 12.5 1.3

 

 

 

 

 

 

 

a = different from pool of NN and NC (P<0.05).

34

 

 

Figure 2. Representative lane showing separation of sarcoplasmic proteins.
Left arrows indicate bands detected, right arrows indicate potential
proteins as identified by McCormick et al. (1988) in swine. Proteins were
separated by SDS-PAGE and visualized by Coomassie blue staining as

described in materials and methods.

35

Table 7. Percentage of sarcoplasmic protein band within lane from NN, NC, CN
or CC ram lambs was quantified according to Materials and Methods. The
mutant callipyge allele is represented by C and N represents the wild-type
normal allele. The paternal allele is listed first. The only animals
expressing the characteristic phenotypic callipyge muscle hypertrophy are
genetically annotated as CN, while NN, NC, and CC genotypes are
phenotypic normal. Protein bands were identified as described in Figure 2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sarcoplasmic NN NC CN CC Pooled
Protein Band SEM
1 5.3 5.0 5.4 5.4 0.07
2 0.6 0.7 0.5 0.6 0.20
3 3.3 2.6 2.9 2.5 0.02
4 6.2 5.9 5.6 5.7 0.19
5 . 2.4 2.2 2.5 2.3 0.04
6 5.0 4.7 5.5 5.3 0.11
7 1.6 1.1 1.9 1.2 0.27
8 1.2 1.2 1.3 0.8‘I 0.11
9 0.3 0.3 0.2 0.2 0.33
10 0.8 0.6 0.8 1.4 0.15
11 6.7 6.8 6.9 6.6 0.05
12 0.2 1.0 0.9 0.6 0.02
13 11.8 15.4 11.7 14.2 0.17
14 7.6 8.6 8.0 9.2 0.32
15 11.9 12.8 11.9 13.4 0.14
16 4.2 4.5 4.1 4.6 0.79
17 3.5 3.6 3.3 2.3 0.28
18 4.8 3.7 4.0 2.4 0.38
19 r 4.2 2.9 L 4.0 4.0 0.12
a = different from pool of NN and NC (P<0.05).

 

36

 

m_-Cglp§in Mediated In Vitro Proteolvsis

The disappearance of the myofibrillar protein desmin on Western blots
was used to determine degradation by m-calpain. Desmin is a 55 kD
intermediate filament protein, which is known to be degraded by calpains. A ratio
of 1:12 (unit m-calpain to mg protein) was determined by titration to slowly
degrade several myofibrillar proteins (results not shown). These conditions were
used to determine the degradation rate of desmin from NN and CN lamb
myofibrils. Desmin from either genotype did not completely degrade within 480
minutes (results not shown). However, there appeared to be less intact desmin
remaining from NN myofibrils compared with CN myofibrils after 480 minutes of
digestion. Several bands representing desmin degradation products between 40
and 50 kD were darker after digestion of myofibrils from CN lambs compared
with myofibrils of NN lambs. Desmin or desmin degradation products did not
appear in the negative control lane containing no 1° Ab. This indicates that
bands on desmin western blots were specific to 1° Ab binding to its antigen
(results not shown).

To achieve complete degradation of intact desmin a 1:2 ratio (unitactive
calpain to mg protein) was used in an in vitro digestion of samples for 120
minutes. Intact desmin (55 kD) was not distinctly visible after a 120 minute
digest of either NN or CN lamb myofibrils (Figure 3). Intact desmin degraded
more rapidly in myofibrils of NN lambs compared with myofibrils of CN lambs.
Furthermore, Desmin degradation products of 48-50 kD appeared transiently

until 30 minutes of digestion, but were more abundant in myofibrils of CN lambs

37

compared to NN lambs (Figure 3). Additionally, desmin degradation products of
~38-40 kD were readily apparent after 15 minutes of CN myofibril incubation with
m-calpain, and persisted throughout the 120 minute digest. These ~38-40 kD

products were barely detectable in digests of NN myofibrils (Figure 3).

38

 

 

    

Minutes
Figure 3. Western blot of desmin after m-calpain induced proteolysis of

myofibrils (1:2 unit m-calpain to pg protein). Top blot shows NN myofibrils,
while bottom blot shows CN myofibrils after digestion for 0 to 120 minutes.
Molecular weight marker and proteins larger than 65 kD (not shown) were
removed and stained with amido black to verify size of proteins and equal
protein loads among lanes. Bands visible on membrane are specific
immunoreactive desmin or desmin degradation products. The arrows
notate desmin and desmin degradation products. The 55 kD band is
intact desmin, while the bands between 50 and 40 kD are degradation

products. Phenotypic normal is annotated NN and CN is phenotypic
callipyge.

39

 

DISCUSSION

Callipyge lambs are leaner, heavier muscled, and more efficient at
converting feed to body weight than non-CLPG lambs. Improved knowledge of
muscle development in CLPG lambs will lead to strategies in more efficient
production of trim, muscular sheep or other species. In the current study, I
determined the effects of callipyge lamb phenotype (CN genotype) on skeletal
muscle DNA, RNA, and protein concentrations, as well as indices of muscle fiber
type, and susceptibility of myofibrillar proteins to calpain-induced proteolysis.

Previous studies have identified CLPG lambs based solely upon
phenotype (Koohmaraie et al., 1995; Carpenter et al., 1996; Jackson et al., 1997;
Taylor and Koohmaraie, 1998; Lorenzen et al., 2000). The study herein and
Freking et al. (1998b) confirm that lambs of the CN genotype have significantly
greater leg scores than the other three genotypes (NN, NC, CC). Phenotypic
selection based on leg scores seems to accurately separate CN genotypes from
NN, NC and CC genotypes. Still, separation based on leg score can leave up to
66% of the lambs in the non-CLPG group earring the mutant CLPG allele.
Freking et al. (1998b) suggested evaluation of all four CLPG genotypes is
needed to provide complete information on this mutation. In support of the need
to genotype these authors demonstrated hypertrophied CN lamb muscles had
the highest calpastatin activity and shear force values (least tender), CC lambs
were intermediate, and NN and NC lambs had the lowest calpastatin activity and
shear force. This implies that while CN lambs have noticable increases in

muscle hypertrophy, other CLPG genotypes may not outwardly show phenotypic

40

differences, but can exhibit biological characteristics compared to genotypically
normal lambs. My study reinforces the need to characterize CLGP genotypes to
better understand CLPG gene action. Increased dressing percentage
(percentage of animal remaining alter skinning, head removal and evisceration)
in CLPG lambs can be attributed to heavier carcass weight, lighter internal organ
weights or a combination of both (Jackson et al., 1997b, Koohmaraie et al.,
1995). There is a 30% increase in carcass weight due to increased muscle
weights of the pelvic limb and torso of CLPG lambs, compared with non-CLPG
lambs (Jackson et al., 1997c). Therefore, an increase in carcass weight is
attributable to individual muscle weights. In my study a similar increase occurred
in muscles of the torso (Iongissimus dorsi) and pelvic limb (biceps femoris), with
no change in a muscle of the thoracic limb (infraspinatus). The ability of CLPG
lambs to increase muscle mass in specific muscle groups or regions is very
perplexing and is yet to be explained. Nevertheless, the increased dressing
percentage an muscle mass of the loin and leg make CLPG carcasses extremely
desirable to the packer.

The LD and BF muscles of CLPG lambs have been shown to increase an
average of 33% and 49% in weight, respectively, compared with non-CLPG
lambs (Jackson et al., 1997c, Koohmaraie et al., 1995). Genotypic CN lambs
also had heavier LD and BF muscle weights in my study (Table 2). Although
average carcass weights among genotypes used in this study were not
statistically different, CC lambs had 20% lighter carcass weights than the

average of the other three genotypes (Table 2). Therefore muscle weights were

41

 

also expressed as a percentage of carcass weight to decrease variation among
genotypes. The percentage of total carcass weight of LD and BF muscles was
larger for CN lambs than those pooled from NN and NC lambs. The IS muscle,
which is not hypertrophied in CLPG lambs, was a similar percentage of carcass
weight for all genotypes (T able 2). The observation that muscles of the leg and
loin, but not the shoulder, undergo hypertrophy in CN lambs relative to other
genotypes is consistent with other studies comparing phenotypic callipyge and
non-callipyge lambs (Koohmaraie et al., 1995; Jackson et al., 1997c). Ehile the
reason for differential effects of the CLPG gene on these muscles is unclear. My
study demonstrated that the muscle hypertrophy is associated with increased
muscle RNA content.

Protein, DNA and RNA concentrations were measured in the LD, BF and
IS muscles (Tables 3, 4 8. 5) to gain a better understanding of muscle
characteristics influenced by the CLPG gene. Muscle DNA and protein
concentrations were generally not statistically different among genotypes (Tables
3, 4 & 5). These results are in agreement with previous studies on phenotypic
callipyge lambs (Koohmaraie et al., 1995; Lorenzen et al., 2000). The RNA
concentrations among genotypes were not statistically different (analysis not
presented). Nevertheless, my study did find RNA concentrations, on average, to
be lower in the LD and BF muscles of NN lambs than the average of the three
genotypes that contained at least one CLPG allele. RNA concentrations in LD
and BF muscles of these latter genotypes were similar. It is currently unclear

why this trend is observed in LD and BF muscles of phenotypic normal lambs

42

that carry a CLPG allele, but not in non-hypertrophied IS muscle of any lambs
carrying a CLPG allele. Koohmaraie et al., (1995) also demonstrated an
increase in RNA concentration in phenotypic CLPG lambs compared with control
lambs. In contrast, Carpenter et al. (1996) found no difference in RNA
concentration among phenotypic CLPG and non-CLPG lambs. The protein, DNA
and RNA concentrations of the IS muscle were similar across all four genotypes
in my study. These data are consistent with previous characterization of non-
hypertrophied muscles in CLPG lambs (Carpenter et al., 1996).

Koohmaraie et al. (1995) demonstrated that proteianNA ratios decreased
and RNA:DNA ratios increased in hypertrophied muscles of CLPG lambs. I also
found the RNA:DNA ratio to be higher in LD muscle of CN and CC lambs
compared with NN and NC lambs. This ratio supports the trend of increased
RNA concentration and suggests that there is either less RNA degraded or an
increase in transcription in CLPG lambs. Protein:RNA ratios of LD and BF
muscles tended to be lower for lambs with a mutant CLPG allele than NN lambs
for my study. Previous studies have suggested that CLPG muscle hypertrophy is
associated with a decline in muscle protein degradation (Koohmaraie et al.,
1995; Carpenter et al., 1996; Lorenzen et al., 2000). If this proves to be true,
then decreased proteianNA ratio observed hereinsuggests that translational
efficiency in CLPG lamb muscle may be low relative to non-CLPG lamb muscle.
These possibilities warrant further evaluation of protein and nucleic acid

concentrations of more genotypic CLPG and non-CLPG lambs. If the current

43

 

findings are confirmed, future studies on RNA stability, gene transcription and
translational efficiency in these lamb genotypes may be informative.

Muscle fiber phenotype alterations result in type I (slow), type llb (fast) or
type Ila (fast/slow) fiber types. Ultimately, fiber type differences result from
production of muscle contractile proteins of differing lsoforrns (Robbins et al.,
1986). My study was the first to measure myosin heavy chain lsoforrns
associated with muscle fiber types of the LD muscle from CLPG lamb genotypes.
There was a 25% increase in type llx of CLPG lambs compared with the mean of
non-CLPG lambs. The type llx myosin heavy chain lsoforrn is considered to be
associated with fast-twitch muscles and is a transitional isoforrn between type Ila
and llb. While other fiber types analyzed did not show statistical differences, the
increased proportion of type llx MHC isoform was coincident with numerical
decreases in both type I and Ila. This information is consistent with the general
idea that MHC gene expression occurs in the order lellaellxellb (arrows
denote possible transitions between different MHC isoforms within a muscle
fiber) (Schiaffino and Reggiani, 1994). Conventional actomyosin ATPase typing
does not allow distinction between type llx and llb muscle fibers (Lefaucheur et
al., 1998). Still, type llb muscle fibers of CLPG LD muscles increase in size and
distribution (Carpenter et al., 1996, Koohmaraie et al., 1995, Lorenzen et al.,
2000). My data indicate the increased percent and size of type llb muscle fibers
in the LD of CLPG lambs is due primarily to an increase in proportion of type llx
MHC isoform. Increased percent and size of type llb muscle fibers, along with an

increase in diameter of type Ila muscle fibers in the LD of CLPG lambs, accounts

for the muscle hypertrophy (Carpenter et al., 1996, Koohmaraie et al., 1995).

Type llb muscle fibers have also been shown to have a greater capacity
for glycolytic metabolism and a decreased capacity for oxidative metabolism than
type I muscle fibers. I expected these changes to be reflected as differences in
the proportions of sarcoplasmic proteins, which include glycolytic enzymes.
However, no significant differences among genotypes were apparent.

Jackson et al. (1997a, b) observed that feed intake (kg/d) in CLPG lambs
was decreased an average of 14% whereas feed conversion (lb of gain/lb of
feed) of the same group increased 10% over their non-CLPG contemporary
group. An increase in percentage of type llb muscle fibers in CLPG lambs may
contribute to improved production efficiency of these sheep (Lorenzen et al.,
2000). Type llb muscle fibers have lower protein turnover rates than type I
muscle fibers, making them potentially more efficient (Garlick et al., 1989).
Decreased protein turnover via elevated calpastatin activity was speculated by
Koohmaraie et al. (1995) to decrease proteolysis of myofibrillar proteins and
subsequently increases muscle mass in CLPG lambs. However, calpastatin r
activity is also elevated in CC lambs (Freking et al., 1998) and Bos Indicus cattle

(Whipple et al., 1990), which do not exhibit muscle hypertrophy. Therefore, I

 

believe additional factors, such as translational rate and/or phosphorylation of
myofibrillar proteins contribute to either decreased myofibrillar proteolysis and
increased efficiency of CLPG lambs.

A reduced susceptibility of CLPG proteins to proteolysis could help explain

the decrease in protein turnover observed in CLPG muscle tissue (Lorenzen et

45

al., 2000) and the reduced rate and extent of postmortem proteolysis seen in
CLPG meat (Koohmaraie et al., 1995). My study focused on susceptibility of CN
and NN myofibrillar proteins to m-calpain-mediated in vitro proteolysis. The
disappearance of the myofibrillar protein desmin on Western blots was used to
determine degradation by m-calpain. Desmin is a 55 kD intermediate filament
protein, which is known to be degraded by calpains. Desmin from both CN and
NN lambs was susceptible to calpain-induced degradation. Differences in rate of
calpain-mediated proteolysis were evident between CN and NN lamb muscle
proteins. Desmin degradation products between 38 to 40 kD, persisted longer
during digestion of CN myofibrils compared with NN myofibrils. It is my belief
that these degradation products are more readily digested in NN lamb myofibrils
compared with CN lamb myofibrils and subsequently undetectable by my
detection method. Results from my experiment suggest desmin and other
myofibrillar proteins of CN lambs are more resistant to degradation than those of
NN lambs. Although the reason for differential sensitivity of myofibrillar proteins
to calpain cleavage has not been determined, this may partially explain the
observation that muscle protein fractional breakdown rate is reduced in CLPG
lambs, resulting in muscle hypertrophy (Lorenzen et al., 2000). Subsequently,
this information supports the conclusion that CLPG lambs have reduced
postmortem proteolysis.

Collectively, results from my experiment demonstrate that LD and BF
muscles are heavier in CN lambs than other genotypes. No differences were

detected in characteristics of IS muscles among genotypes. In LD muscles of

46

CN and CC genotypes the RNA:DNA ratios are higher than in LD muscles of
non-CLPG lambs. This suggests CN and CC lambs either have a decrease in
RNA degradation or a more efficient transcription process. Greater muscle mass
in CN lambs is associated with an increase in the proportion of type llx myosin
heavy chain isoforrn. A decrease in susceptibility of myofibrillar proteins to
degradation may also contribute to CN lamb muscle hypertrophy, reduced

fractional breakdown of muscle protein, and increased efficiency of lean growth.

47

 

APPENDIX

Homegenlzation buffer

75 mM KCL

10 ITIM KH2P04

2 mM MgCl2

2 mM EDTA
*Sodium Fluoride, Phenylmethylsulfonyl flouride (PMSF) and Leupeptin were
dissolved in stocks and added to homogenization buffer the day of protein
extraction.

50 mM NaF
2 mM PMSF
6 mglL Leupeptin
Digestion Buffer
50 mM Tris
165 mM NaCl
10 mM B-mercaptoethanol (MCE)
5 mM CaClz
pH = 6.7 - 7.0
Myosin Heavy Chain lsoforrns Lower Running Buffer
1L 2_|_- 4_L
50 mM Tris Base 6.05 g 12.1 g 24.2 g
75 mM Glycine 5.63 g 11.26 g 22.52 g
0.05% SDS 0.50 g 1.0 g 2.0 9
ddeo fill to 1 L fill to 2 L fill to 4 L

*pH adjustment is not necessary.

Myosin Heavy Chain lsoforrns Upper Running Buffer
3 2L LL
0.1M Tris Base 12.11 g 24.22 g 48.44 g
150 mM Glycine 11.26 g 22.52 g 45.04 g
0.1% SDS 1.0 g 2.0 g 4.0 g
ddH20 fill to 1 L fill to 2 L fill to 4 L
*pH adjustment is not necessary.

Standard Running Buffer, pH 8.3

1 L 4 L
0.025 M Tris (F.W. 121.1) 3.0 g 6.0 g 12.1 g
0.192 M Glycine (F.W. 75.07) 14.4 g 28.8 g 57.6 g
0.1% SDS 1.0 g 2.0 4.0 g
ddH20 fill to 1 L fill to 2 L fill to 4 L

*This solution can be made directly in a large jug. Check the pH and store at
room temperature. However, it is usually a better idea to make the solution in a
beaker and then pour it into a jug alter everything has dissolved.

48

Standard Transfer Buffer

1_L 2.1.. &
Glycine 14.42 g 28.83 g 57.66 g
Tris Base 3.03 g 6.06 g 12.12 g
15% Methanol 150 mL 300 mL 600 mL
ddH20 fill to 1 L fill to 2 L fill to 4 L

*pH 8.1-8.3, adjustment is not necessary. This solution may be reused 4-5 times
with filtering (#1 Whatman) between each use. Dispose in Methanol Hazardous
Waste container.

1% Blocking Solution Bovine Serum Albumin (BSA)

ddH20 360 mL
10XTris Buffer Solution 40 mL
0.05% Tween 20 0.2 mL

1% BSA (Sigma A2153) 4.0 g

TTBS (0.05% Tween 20), pH 7.4
Add 250 pL of Tween 20 to 500 mL TBS

2X Laemmli buffer minus MCE, pH 6.8

0.125 M Tris

4 % SDS

20% Glycerol

*Cut concentrations of Tris and SDS in half for 1X

Use the 2X Treatment Buffer minus MCE from above to soluble proteins
prior to BCA Protein Assay. Add MCE and 4 mg/mL Bromophenol Blue in
the proportions shown below.

10 mL 30 mL 50 mL
2X Treatment Buffer Minus MCE 9.0 mL 27.0 mL 45.0. mL
MCE 1.0 mL 3.0 mL 5.0 mL

4 mg/mL Bromophenol Blue 200 pL 600 uL 1.0 mL

*Make in hood due to smell and toxicity of MCE. Make fresh daily (or use
aliquots that have been frozen). Any solution not used may be aliquoted and
frozen for future use. Disposal is in the Hazardous Waste container. If the dye is
not dark enough or the samples are not dense enough, add 50% more
Bromophenol Blue.

Coomassie Blue Stein Rm
(0.025% Coomassie Blue R250, 40% Methanol, 7% Acetic Acid)

u 2L 1L
Coomassie Blue R250 0.25 g 0.5 g 1.0 g
Methanol 400 mL 800 mL 1600 mL

49

Acetic Acid 70 mL 140 mL 280 mL

ddH20 fill to 1 L fill to 2 L fill to 4 L
*Dissolve Coomassie Blue in Methanol only. Stir Methanol and Coomassie Blue
until it dissolves then add ddeo and Acetic Acid. Filter solution with a Whatman
#1 filter.

Destainlng Solution l (40% Methanol, 7% Acetic Acid)

LI. 2!. LL
Methanol 400 mL 800 mL 1600 mL
Acetic Acid 70 mL 140 mL 280 mL
ddH20 fill to 1 L fill to 2 L fill to 4 L

*Mix in hood and store at room temperature.

Destaining Solution ll (5% Methanol, 7% Acetic Acid)

2 A 511-.
Methanol 50 mL 100 mL 200 mL
Acetic Acid 70 mL 140 mL 280 mL
ddH20 fill to 1 L fill to 2 L fill to 4 L

*Mix in hood and store at room temperature.

TISSUE PROTEINIDNAIRNA DETERMINATION

HOMOGENIZATION (polytron homogenizer)
1. Place 0.5 to 1.0 gram of muscle a 50 ml conical centrifuge tube (orange cap).
Add 25 x volume by weight of cold extraction buffer.

EXTRACTION BUFFER 1 liter
Tris (10 mM) 1.21 9
EDTA (5 mM) 1.86 g

pH 8.0

2. Homogenize sample for 2 min at setting 4 (30 sec on, 30 sec off 4 times;
keep sample tube in beaker containing ice slush)

3. Take aliquots of homogenate for RNA, DNA, and protein determinations.
250 pL for protein x 3
100 uL for DNA x 3
3 mL for RNA x 3

50

WW

1. In 3 ml methacrylate cuvets (pre-selected for uniformitY). add 100 pl of
homogenate and 2900 pl of DNA assay buffer.

2. DNA standard curve: Calf thymus DNA stock = 200 pglml
Prepare standards by serial dilution.

pg DNA/ml DNA stock Extraction buffer (Tris, EDTA)
100 500 pl 500 pl mix, remove 500 pl, dilute 1:1
50 M
25 "
12.5 "
6.25 “
3.125 "
O Extraction buffer only

Place 100 pl of each standard in duplicate cuvets.

3. In a dimly lit room, add 3 ml of Hoechst 33258 Reagent (diluted to 1pglml in
DNA Assay Buffer) to all cuvets. Incubate in the dark for 15 min. Record
time that Hoechst reagent is added to standards and samples.

4. Turn on DYNAQUANT fluorometer and allow 15 min warm-up. FOLLOW
THIS SEQUENCE: SETUP, PROMPT OFF, UNITS nglml, ESC, place 0
standard in fluorometer, READ, ZERO, place 25 pg/ml standard in
fluorometer, CALIBRATE, 2500 nglml (this actually represents 2500 ng in the
cuvet), ENTER.

Read all standards (duplicate) and samples (triplicate) in sequence that
Hoescht Reagent was added. Record time that standards and samples
are read peiodically (each 9—12 tubes). Time from addition of Hoechst
reagent to sample read should be kept constant (1 3 min).

5. Use the standard curve to determine the concentration of the samples. Divide

by 1000 to get the pg DNA per cuvet, and divide that value by .004 g fresh
tissue/cuvet to get pg DNA/g muscle. (or multiply by 250 dilution factor)

51

 

DNA REAGENTS

 

DNA Assay buffer 1 liter
0.05 M NaH2P04 "' HOH (FW 138.0) 6.90 g
2 M NaCl (FW 58.45) 116.90 9
2 mM EDTA‘Na (FW 372.2) 0.744 9
pH to 7.4
Hoechst 33258 reagent

1 mglml stock solution in ddi water
Store in the dark at 4 C
Dilute fresh 1:1000 in DNA Assay buffer (H33258 = 1 pg/ml)

DNA Stock Solution
Calf thymus DNA 2.0 mg

1. Dissolve 2.0 mg of calf thymus DNA in 8 ml Extraction Buffer in original DNA
bottle. Allow 2.5 to 3 hr at room temp.

2. To find the purity of the solution, dilute it 1:10 and read its absorbence at
280nm and 260 nm.

Divide A260 by A280 to get the purity. It should be around 1.8

3. To find the concentration, multiply the A260 reading by 10. It is known that at
A260, 50pglml DNA will read 1.0, therefore divide 50 by 1 and multiply by
your A260 reading.

4. Dilute DNA stock solution to 200 pg/ml. Freeze in aliquots at -80° C.

52

 

PROTEIN DETERMINATION

1. Place 250 pl aliquots (in triplicate) of homogenate in 16 x 100 mm borosilicate
glass tubes. Add 750 pl 1 N NaOH to each.

Prepare duplicate BSA standards in 25% extraction buffer, 75% 1 N
NaOH @ 8, 4, 2, 1, 0.5 and 0 mglmL. Use 1 ml of standard for
readings.

2. Parafilm tubes, vortex briefly and let incubate at 37 C for 3 to 4 hours. Turbid
samples should become clear.

3. Add 4 ml of biuret reagent to each tube, parafilm and vortex. Place in dark for
30 min at room temp.

4. Remove 200 pL from each standard or sample, dispense in microtiter plate
and read at 540 nm. Determine protein concentration of samples based on
standard curve.

BIURET REAGENT
Dissolve the following in about 500 ml of 0D. H20 in a 2 liter volumetric-
CuSO4 - 5 H20 1-5 9
Sodium potassium tartrate 6.0 9

Add with constant stirring 300 ml of fresh carbonate free 10% NaOH.

Make up to 1 liter and store in a dark polyethylene bottle. Discard if black or red
precipitate appears.

53

 

RNA DETERMINATION

. Place 1 ml of homogenate in 1.5 ml micro-centrifuge tubes and add 330 pl of
1.2 N perchloric acid (PCA). Allow to sit on ice for 10 min.

. Centrifuge at 10,000xg at 0-4°C for 5 min. (F-20/micro rotor = 8,900 rpm.)
Decent and discard supernatant.

. Add 700 pl cold .2 N PCA. Break up pellet with a glass rod and vortex.
Centrifuge as above. Discard supernatant. Repeat 2X's using 700 pl of .2 N
PCA.

. Add 1.0 ml of .3 N KOH (room temp). Break pellet and vortex. Heat at 37° C
for 60 min. Place in incubated shaker after heating in water bath. (DNA
determination may be done during the incubation.)

. Add 330 pl of cold 2.4 N PCA and allow tubes to sit in an ice bath for 10 min.
Centrifuge at 10,000xg at 04°C for 5 min. (F-20/micro rotor = 8,900 rpm.)
Decant (retain supernatant.)

. Wash precipitate with 500 pl of cold .2N PCA. Break pellet and vortex.
Centrifuge at 10,000xg at 0-4°C for 5 min (F-201micro rotor = 8,900 rpm.) and
mix the supernatant with the supernatant from step 5. Repeat.

. RNA Measurement- Measure O.D. of RNA hydrosylate at
260 nm and 232 nm.

Calculation: pgRNA= (39.1xO.D. 260) - (15.5 x OD. 232)
X2.3 (volume of supernatant)

 

x 25
RNA Reagents
1.2 N PCA 20.49 ml of 70% PCA bring up to 200 ml with dd water.
0.2 N PCA 1 part 1.2 N PCA + 5 parts dd water
0.3 N KOH KOH-4.21gramsl250 ml dd water

Growth and harvest of hyigidoma cell lineage, D76

1. Hybridoma cells were grown in 75 cm2 tissue culture flasks (#3376, Coming
Incorporated, Corning NY) in Dulbecco’s Modified Eagle Medium

Dulbecco’s Modified Eagle Medium

25 mM HEPES (#23700-040, Gibco BRL, Grand Island NY)

10% fetal bovine serum

0.1 mM Non-Essential Amino Acids (#11140-050, Gibco BRL)

1 mM sodium pyruvate (S-8636, Sigma Chemical Co., St. Louis MO)
2 mM L-glutamine (G-5763, Sigma Chemical Co.)

0.5% antibiotic/antimycotic.

2. Frozen cell suspensions were thawed in a warm water bath and re-
suspended in 10 ml of media to dilute freezing medium and centrifuged at 300
xg for 2 minutes. The cell pellet was then re-suspended and plated at
2.5x10° eels/75 cm2 flask in 20 ml of medium (125,000 cells/ml).

3. Cells were supplied with new medium every 48 hours at a ratio of 1:4
(existing cell suspension to fresh media) and cell suspensions were
transferred into new flasks when necessary. Feeding was continued until the
desired volume of supernatant was attained. Cultures were then left
undisturbed for 12 days.

4. Cultures were harvested, pooled and centrifuged at 2000 xg for 15 minutes to

pellet cells. The clarified supernatant was then collected and stored in
aliquots at -20°C until use.

55

REFERENCES

Beermann D.H., W.R. Butler, D.E. Hogue, V.K. Fishell, R.H. Dalrymple, C.A.
Ricks, and CG. Scanes. 1987. Cimaterol-induced muscle hypertrophy
and altered endocrine status in lambs. J Anim Sci. 65:6 1514-24

Bidwell, C.A., T.L. Shay, M. Georges, J.E. Beever, S. Berghmans, K. Segers, C.
Charlier, and NE. Cockett. 1999. Differential expression of the GTL2
gene from the callipyge region of the ovine chromosome 18. 27‘"
lntemational Conference on Animal Genetics.

Carpenter, CE, 0.0. Rice, N.E. Cockett, and GD. Snowder. 1996. Histology
and composition of muscles from normal and callipyge lambs. J Anim Sci.
74:388-393.

Cheek, D.B., A.B. Holt, D.E. Hill, and J.L. Talbert. 1971. Skeletal muscle cell
mass and growth: the concept of the deoxyribonucleic acid unit. Pediat
Res. 5:312-328.

Cockett, N.E., S.P. Jackson, T.L. Shay, F. Famir, S. Berghmans, G.D. Snowder,
D. M. Nielsen, and M. Georges. 1996. Polar overdominance at the ovine
callipyge locus. Science. 273: 236-238.

Cockett, N.E., S.P. Jackson, T.L. Shay, D. Nielsen, S.S. Moore, M.R. Steele, W.
Barendse, R.D. Green, and M. Georges. 1994. Chromosomal localization
of the callipyge gene in sheep (Ovis an'es) using bovine DNA markers.
Proc. Natl. Acad. Sci. 91:3019-3023.

Cockett, N.E., S.P. Jackson, G.D. Snowder, T.L. Shay, S. Berghmans, J.E.
Beever, C. Carpenter, and M. Georges. 1999. The callipyge
phenomenon: evidence for unusual genetic inheritance. J Anim Sci. 77:
(Suppl 2/J) 221-227.

Cole, J.W., C.B. Ramsey, C.S. Hobbs, and RS. Temple. 1963. Effects of type
and breed of British, Zebu and dariy cattle on production, palatability, and
compostion. I. Rate of gain, feed efficiency and factors affecting market
value. J Anim Sci. 22:702.

Duckett, S.K., G.D. Snowder, and NE. Cockett. 2000. Effect of the callipyge
gene on muscle growth, calpastatin activity, and tenderness of three
muscles across the growth curve. J Anim Sci. 78:2836-2841.

Fahrenkrug. S.C., B.A. Freking, C.E. Rexroad Ill, K.A. Leymaster, and SM.

Kappes. 2000. Comparative mapping of the ovine clpg locus.
Mammalian Genome. 11:871-876.

56

‘ m
Hm" I.“ r—cw-v-t- . q

Field, R.A., R.J. McCormick, D.R. Brown, PC Hinds, and G.D.Snowder. 1996.
Collagen crosslinks in longissimus muscle from lambs expressmg the
callipyge gene. J Anim Sci. 74:2943-2947.

Florini, J.R., 0.2. Ewton, S.A. Coolican. 1996. Growth hormone and the insulin- ‘
like growth factor system in myogenesis. Endocrine Rev. 17(5): 481-517.

Freking, B.A., J.W. Keele, C.W. Beattie, S.M. Kappes. T.P.L. Smith, T.S.
Sonstegard, M.K. Nielsen, and K.A. Leymaster. 1998a. Evaluation of the

Ovine callipyge locus: l. Relative chromosomal position and gene action.
J Anim Sci. 76:2062-2071.

F reking, B.A., J.W. Keele, M.K. Nielsen, and K.A. Leymaster. 1998b. Evaluation
of the Ovine callipyge locus: ll. Genotypic effects on growth, slaughter &
carcass traits. J Anim Sci. 76:2549-2559.

Garlick, P.J., CA. Maltin, A.G.S. Bailie, M.I. Delday, and DA. Grubb. 1989.
Fiber-type composition of nine rat muscles. ll. Relationship to protein
turnover. Am J Physiol. 257(Endocrinol. Metab.20):E828:832.

Goldspink, D.F., V.M. Cox, S.K. Smith, LA. Eaves, N.J. Osbaldeston, D.M. Lee,
and D. Mantle. 1995. Muscle growth in response to mechanical stimuli.
Amer. Phys. Soc. pp. E288-E297.

Goldspink, G. 1970. The proliferation of myofibrils during muscle fibre growth. J
Cell Sci. 62593-603.

Goll, DE. 1991. Role of proteinases and protein turnover in muscle growth and
meat quality. Proc Recip Meat Conf. 37:250.

Gomall, A.G., C.J. Bardawill, and MM. David. 1949. Determination of serum
proteins by means of the biuret reaction. J Biol Chem. 177: 751-766.

Grobet, L., L.J.R. Martin, D. Poncelet, D. Pirottin, B. Brouwers, J. Riquet, A.
Schoeberlin, S. Dunner, F. Menissier, J. Massabanda, R. Fries, R.
Hanset, M. Georges. 1997. A deletion in the bovine myostatin gene
causes the double-muscled phenotype in cattle. Nature 17:1, 71-74

Jackson, S.P., R.D.Green, and M.F. Miller. 1997a. Phenotypic characterization
of Rambouillet sheep expressing the callipyge gene: l. Inheritance of the
condition and production characteristics. J Anim Sci. 75:14-18.

Jackson, S.P., M.F. Miller, and RD. Green. 1997b. Phenotypic characterization

of Rambouillet sheep expressing the callipyge gene: ll. Carcass
characteristics & retail yield. J Anim Sci. 75:125-132.

57

Jackson, S.P., M.F. Miller, and RD. Green. 1997c. Phenotypic characterization
of Rambouillet sheep expressing the callipyge gene: Ill. Muscle weights
and muscle weight distribution. J Anim Sci. 75:133-138.

Kambadur, R., M. Shame, T.P. Smith, J.J. Bass. 1997. Mutations in myostatin
(GDF8) in double-muscled Belgian Blue and Piedmontese cattle.
Genome Res 7:9 910-6

Koohmaraie M., SD, Shackelford, N.E. Muggli-Cockett, and RT. Stone. 1991.
Effect of the beta-adrenergic agonist L644,969 on muscle growth,
endogenous proteinase activities, and postmortem proteolysis in wether
lambs. J Anim Sci. 69:12 4823-35.

Koohmaraie, M., SD. Shackelford, T.L. Wheeler, S.M. Lonergan, and ME.
Doumit. 1995. A muscle hypertrophy condition in lamb (callipyge):
characterization of effects on muscle growth and meat quality traits. J
Anim Sci. 73:3596—3607.

Koohmaraie, M., SD. Shackelford, and T.L. Wheeler. 1996. Effects of a B-
adrenergic agonist (L644,949) and male sex condition on muscle growth
and meat quality of callipyge lambs. J Anim Sci. 74:70-79.

Labarca, C., and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay
procedure. Anal. Biochem. 102:344

Lefaucheur, L., R.K. Hoffman, D.E. Gerrad, C.S. Okamura, N. Rubinstein, and A.
Kelly. 1998. Evidence for three adult fast myosin heavy chain isofonns in
type II skeletal muscle fibers in pigs. J Anim Sci. 76:1584-1593.

Lorenzen, C.L., M. Koohmaraie, S.D. Shackelford, F. Jahoor, H.C. Freetly, T.L.
Wheeler, J.W. Savell, and ML Fiorotto. 2000. Protein kinetics in
callipyge lambs. J Anim Sci. 78:78-87.

Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J Biophys Biochem
Cytol. 9: 493-495.

McCormick, R.J. G.R. Reeck, and DH. Kropf. 1988. Separation and
identification of porcine sarcoplasmic proteins by reversed-phase high-
performance liquid chromatography and polyacrylamide gel
electrophoresis. J Agric Food Chem 36:1193-1196.

McPherron, A.C., A.M. Lawler, and S. Lee. 1997. Regulation of skeletal muscle
mass in mice by a new TGF-B superfamily member. Nature. 387: 83-90.

58

 

Mersmann, H.J., 1998. Overview of the effects of beta-adrenergic receptor _
agonists on animal growth including mechanisms of action. J Anim Sol.
76:160-172.

Miyoshi, N., H. Wagatsuma, S. Wakana, T. Shiroishi, M. Nomura, K. Aisaka, T.
Kohda, M.A. Surani, T. Kaneko-lshino, and F. lshino. 2000. Identification
of an imprinted gene, Mega/thz and its human homologue MEG3, first
mapped on mouse distal chromosome 12 and human chromosome 14q.
Genes to Cells. 5(3):211-220.

Moss, PP. and OR LeBlond. 1971. Satellite cells as the source of nuclei in
muscles of growing rats. Anat Rec. 170: 421-436.

Munro, H.N., and A. Fleck. 1969. Analysis of tissue and body fluids for
nitrogenous constituents. In: H.N. Munro (Ed.) Mammalian Protein
Metabolism. Vol. 3. p 423. Academic Press, New York.

Pearson, AM. and TR. Dutson. 1991. Growth regulation in farm animals.
Advances in meat research volume 7. Elsevier Applied Science, London.

Schianffino, S., and C. Reggiani. 1994. Myosin isofonns in mammalian skeletal
muscle. J Appl Physiol. 77:493-501

Shackelford, S.D., T.L. Wheeler and M. Koohmaraie. 1998. Can the genetic
antagonisms of callipyge lamb be overcome? Reciprocal Meat
Conference Proceedings. 51:125-132.

Stockdale, PE 1992. Myogenic cell lineages. Dev Biol. 154: 284-298.

Talmadge, R.J. and RR. Roy. 1993. Electrophoretic separation of rat skeletal
muscle myosin heavy-chain isofonns. J Appl Physiol. 75(5):2337-2340.

Whipple, G., M. Koohmaraie, M.E. Dikeman, J.D. Crouse, M.C. Hunt, and RD.
Klemm. 1990. Evaluation of attributes that affect longissimus muscle
tenderness in Bos teams and Bos indicus cattle. J Anim Sci. 68:2716-
2728.

Whisnant, C.S., R.S. Kline, J.C. Branum, G.M. Zaunbrecher, M.Z. Khan, and SP
Jackson. 1998. Hormonal profiles of callipyge and normal sheep. J Anim
Sci. 76:1443-1447.

Williams, PE. and G. Goldspink. 1971. Longitudinal growth of striated muscle
fibres. J Cell Sci. 9:751-767.

59

 

Wylie, A.A., S.K. Murphy, T.C. Orton, and R.L. Jirtle. 2000. Novel imprinted
DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic
those implicated in IGF2/H19 regulation. Genome Research. 1021711-
1718.

60