Equine Nutrition and Physiology Society

REFEREED PAPERS FROM THE 14TH SYMPOSIUM

FIBER TYPE COMPOSITION OF THE MIDDLE

GLUTEAL MUSCLE OF MULES
Holly M. Greene, BS; t Steven J. Wickler, PhD, DVM; 1
Russell L. Tucker, DVM; 2 Craig London, DVM 3

SUMMARY
This study compared the muscle fiber type distribution
in the middle gluteal muscles of mules and horses. Nine
mules and ten Quarter Horse-type horses from a commercial pack outfit were sampled with a Bergstrtm biopsy
needle. The middle gluteal muscle was sampled at a location 10 cm dorsocaudal to the tuber coxae at an angle of 45 ~
Muscle samples were histochemically analyzed for myosin ATPase (pH 9.5 preincubation) and succinic dehydrogenase activity. Comparing myosin ATPase and succinic
dehydrogenase activities, muscle fibers were identified as
either type I, type IIA, or type IIB. For each sample, 150
fibers were counted. Mules had more type I fibers (39.7+2.3
vs 20.8+2.2%, P< .0001) but a fewer % of type IIA fibers
(39.3+1.35 vs 47.4+1.37%, P=.0007) and fewer type liB
fibers (21.1+_2.3 vs 31.5+2.4%, P =.0062). Mules did have
a greater percentage of oxidative fibers, type I plus type
IIA, (79.00-&2.3 vs 68.2+2.5%, P=.0055). The remaining
muscle samples were assayed for activity of citrate synthase and 13-hydroxyacyl-CoA dehydrogenase (HOAD).
There were no significant differences between mules and
horses for either citrate synthase (P=.49) or HOAD (P=.76).

INTRODUCTION
During the past decade, considerable research has
been conducted on the athletic ability of the horse (Equus
caballus), but literature on the mule is predominantly
anecdotal. The mule, a hybrid between Equus caballus and

Authors' address: 1California

State Polytechnic University, Pomona,

Equine Research Center, Department of Animal and Veterinary Sciences,
Pomona, CA 91768; 2Washington State Univerisity, Department of
Radiology, College of Veterinary Medicine, Pullman, WA 99164; 3Rock
Creek Pack Station, Bishop, CA 93515.
Acknowledgements:Supported by an RSCA grant to SJW. The authors
thank Dr. Lind and Dr. Talbot of Bishop Veterinary Hospital, Rock Creek
Pack Station, Bishop, CA and University of California White Mt. Research
Station, Bishop, CA.

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Equus asinus, has been particularly useful in mountain

packing environments, where work loads are heavy and an
increased endurance capacity is required. A number of
comprehensive studies comparing muscle fiber types as a
function of breed, athletic ability, and disease studies have
been done in the horse, TM however there are no data on
mules. Could the suggestion of an increased endurance
capacity in the mule be supported by a distribution of
muscle fiber types consistent with an increased endurance?
The purpose of this study was 1) to compare muscle fiber
type distribution in mules to those in horses and 2) to
establish baseline data in middle gluteal muscle from
mules.

MATERIALS AND METHODS

Nineteen equids, 9 mules and 10 Quarter Horse-type
horses from a commercial pack outfit were sampled in June
1994, after 7.5 months of free access to native pastures. The
animals were judged in good condition and in similar
athletic conditioning. The age of the animals ranged from
6 years to 12 years for the mules, and from 8 years to 15
years for the horses. The biopsy technique was performed
following the method of Andrews et al. s A mild sedation
with xylazine hydrochloride and butorphanol was administered intravenously. A 25-cm 2 area over the sampling site
was shaved and aseptically prepared. After a local 2%
lidocaine anesthesia, a 1-cm stab incision was made through
the muscle fascia, but not into the muscle. This sampling
site was located 10 cm dorsocaudal to the tuber coxae at an
angle of 45~ 6 A Bergstrom biopsy needle was passed
through the skin incision and into the middle gluteal
muscle. The insertion was standardized to a depth of 8 cm
in order to minimize possible variation of sampling depth.
Suction was applied using a 60-cc syringe and a sample
was taken. Sample sizes varied from 50 to 100 mg. After
needle withdrawal, pressure was applied to the sample site
for hemostasis.
The muscle fibers were oriented under a dissecting
microscope into parallel bunches. Muscles were mounted
onto a cork carrier, coated with embedding medium
(Cryoform|
frozen in isopentane, cooled in liquid nitrogen, and stored -70~ until histochemical analysis (approximately 2 months later).
For each muscle sample, six sequential 8-gm crosssections were cut in a cryostat at -20~ and alternately
placed onto two coverslips. These samples were used to

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Equine Nutrition and Physiology Society

REFEREED PAPERS FROM THE 12TH SYMPOSIUM

'k

50

~Mule

~"~ Mule

50-

I Horse
40

c 40E

m 30

m 30-

Horse

C
ID

P 20

"6 20
E

Q
O.

10.
O,

10
lype i

Type

IIA

lyp~

=J~

Figure 1. Percentage of muscle fibers for type I (Slow

twitch, high oxidative), type I IA (fast twitch, high oxidative),
and type liB (fast twitch, low oxidative). Values are
means+SE. Asterisks indicate differences between mules
and horses (P<.0001).

Figure 3. Citrate synthase activity for mules and horses, as

described in Figure 1, presented as lamoles per gram of
muscle per minute.

J Mule
B
80r
O)

20-

Horse

~'~ Mule
I

c
E

Horse

15-

6010-

tO

P 4O

Q
a.

E
=L

20
0

Figure 2. Percentage of oxidative fibers (type I plus type IIA

fibers) for mules and horses. Asterisk indicates difference
between mules and horses (P=.0055).

Figure 4. fS-hydroxyacyI-CoA dehydrogenase activity for

mules and horses, presented as iamoles per gram of
muscle per minute.

estimate the activity of myosin adenosine triphosphatase

(ATPase) after pH 9.5 preincubation and succinate dehydrogenase (SDH). The ATPase stained samples allowed
for identification of type I (light staining) and type II (dark
staining) fibers.
To help subclassify the type II fibers, SDH histochemical stain was combined with the ATPase activity
using an overlay technique. Fibers from the ATPase stain
were outlined using an image capture system (NIH Image | to distinguish between type I and type II. Then
successive SDH stained cross-sections were matched onto
the traced fiber outlines, thus enabling identification of
type IIA (high oxidative) and type IIB (low oxidative)
fibers. 7,8 For each muscle sample, at least 150 fibers were

scored.
Tissue homogenates of the remaining biopsy were
used to determine the activity levels of citrate synthase and
b-hydroxyacyl-CoA dehydrogenase in accordance with
the techniques of Srere 9 and Bass, 1~respectively, as modified by Wickler? 1 Tissue samples were homogenized in
buffer [0.1 M phosphate and 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.3]. Homogenates were frozen
at -20~ thawed and refrozen, ensuring disruption of the
mitochondria.
Citrate synthase activity mixture totaled 1 ml which
consisted of 100 mM tris (hydroxymethyl) aminomethaneHCL, 2.5 mM EDTA,. 1 mM 5,5'-dithiobis-2-nitrobenzoic
acid, 0.2 mM acetylCoA, .5 mM oxaloacetate, and ho-

Volume 15, Number 9, 1995

389

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REFEREED PAPERS FROM THE 14TH SYMPOSIUM

mogenate combined into a cuvette. The reaction mixture

for HOAD totaled 1 ml and contained 100 mM triethanolamine-HCL, 5 mM EDTA, .225 mM b-NADH, .1 mM
acetoacetyl-CoA, and homogenate. Measurements of the
activity were determined as a change in absorbance at 412
nm for citrate synthase and 340 nm for HOAD, both at
25~ using a Hitachi U-2000 spectrophotometer. Duplicates were run on all samples, and if a difference of more
than 5% occurred, a third sample was analyzed.
Comparisons between fiber types within one species
and between species were done using analysis of variance.
Student's unpaired t-test was used to compare citrate
synthase and HOAD. Statistical analysis results are expressed as means + SEM.

RESULTS

Mules had more type I fibers (39.7+2.3 vs 20.8+2.2%,

P < .0001), fewer type IIA fibers (39.3+1.4 vs 47.4+1.4%,
P = .0007) and fewer type IIB fibers (21.1+2.3 vs 31.5+_2.4%,
P =.0062; Figure 1). The mules had a greater percentage of
oxidative fibers, type I plus type IIA, (79.0+2.3 vs
68.2+2.5%, P =.0055; Figure 2).
Significant differences were not observed in the activity levels of citrate synthase (29.1+_.2.1 vs 34.0-&-_3.2,I.tmoles
of substrate converted per gram tissue per minute, P=.2354;
Figure 3) and HOAD (17.0-2-_1.4 vs 17.6+1.2, P=.7625;
Figure 4).

DISCUSSION

Mules have a high percentage of type I fibers, a high

percentage of oxidative fibers (type I plus type IIA), and
relatively high citr/tte synthase activity. Mules are quite
frequently utilized as packing animals, which entail long
term, low intensity bouts of exercise. These indicators of
aerobic capacity of muscle all support the perception that
mules can sustain these protracted levels of muscle activity
and resist fatigue.
The nomenclatures for fiber types can be, at times,
confusing and are reviewed by Snow and Guy? 2 Type I
fibers are slow twitch, high oxidative; type IIA fibers are
fast twitch, high oxidative; and type liB fibers are fast
twitch, low oxidative. In humans, sprinters have a higher
percentage of type II fibers, whereas elite, long distance
runners possess a higher proportion of type I fibers, lz
390

Similarly, in the horse, there is a relationship between the

fiber composition of the middle gluteal muscle and the type
of work for which the breed was selected? 3
The percentage of type I fibers observed in our Quarter
Horse-type animals (20.8+2.2%) is greater than that observed by Snow and Guy la (6.8+1.0). This difference may
be due to our use of cross-bred Quarter Horses as opposed
to the American Racing Quarter Horses used by Snow and
Guy? a
Oxidative capacity of muscle can also be indicated by
enzyme activities, such as citrate synthase and HOAD. TM
The citrate synthase values for muscle samples from our
mules and horses have relatively high activity (29.1+2.1 vs
34.0-2_3.2~moles converted per gram per minute, for mules
and horses, respectively), and are similar to values calculated from Valberg and Essen-Guatavsson 15for the gluteal
muscle of Thoroughbred horses (24.6) and Standardbred
horses (33.4).
Previous studies have indicated an increase in citrate
synthase and HOAD activity corresponding with higher
percentages of Type I fibers, 8 while in our study, the
horses, although possessing fewer percentage of type I
fibers, had similar activity for citrate synthase and HOAD.
While the percentage of type I fibers is correlated with
breed? z the aerobic capacity of muscle is a function of
athletic conditioning. In one study? citrate synthase of the
middle gluteal muscle changed by over 50% during a 15wk training program. The animals used in this study had
been at rest (i.e., no formal work) on pasture for the
previous 7.5 months. It would be of interest to follow
changes in the muscle during their conditioning period in
the summer.

REFERENCES
1. Guy PS, Snow DJ: The effect of training and detraining of
muscle composition in the horse. J Physio11977;269:33-51.
2. Lindholm A, Piehl K: Fibre composition, enzyme activity
and concentrations of metabolites and electrolytes in muscle of
standardbred horses. Acta Vet Scand 1974;15:287-309.
3. Lopez-Rivero JL, Aguera E, Monterde JG, Vivo J,
Rodriquez-Barbudo MV: Skeletal muscle fiber size in untrained
and endurance-trained horses. Am J Vet Res 1992;53:847-850.
4. Snow DH, Guy PS: Percutaneous needle muscle biopsy
in the horse. Equine Vet J 1976;8:150-155.
5. Andrews FM, Reed SM, Johnson GC: Muscle biopsy in
the horse: Its indications, techniques, and complications. VetMed
1993;88:357-365.
6. Kline KH, Lawrence LM, Novakofski J: Changes in muscle
fiber type variation within the middle gluteal of young and mature
horses as a function of sampling depth. Equine Exercise

JOURNAL OF EQUINE VETERINARY SCIENCE

Equine Nutrition and Physiology Society

REFEREED PAPERS FROM THE 12TH SYMPOSIUM

Physiology 2, ICEEP Publications 1987;271-277.

7. Essen B, Lindholm A, Thornton J: Histochemical
properties of muscle fiber types and enzyme activities in skeletal
muscles of Standardbred trotters of different ages. J Equine Vet
Sci 1980;12:175-180.
8. Snow DJ, Valberg SJ: Muscle anatomy, physiology, and
adaptations to exercise and training. In: The Athletic Horse
Hodson DR, Rose RJ (Eds.) 1974:145-179.
9. Srere PA: Citrate synthase in rat liver. Methods in
Enzymology 1969; JM Lowenstein (Ed.) 13:3-16.
10. Bass AD, Brdicska D, Eyer P, Hofer S, Pette D: Metabolic
differentiation of distinct muscle types at the level of enzymatic
organization. Eur J Biochem 1969;10:198-206
11. Wickler SJ: Seasonal changes in enzymes of aerobic heat
production in the white-footed mouse. Am J Physiol

DIGESTION OF SOYBEAN MEAL PROTEIN

IN THE EQUINE SMALL AND LARGE
INTESTINE AT VARIOUS LEVELS OF INTAKE
E. B. Farley MS; 1 G. D. Potter PhD; 1
P. G. Gibbs PhD; 1 J. Schumacher DVM; 2
M. Murray-Gerzik MS ~

1981 ;240: R289-294.

12. Snow DH and Guy PS, Muscle fibre type composition of
a number of limb muscles in different types of horses. Res Vet Sci
1980;28:137-144.
13. Snow DH, Baxter P: Muscle fibre composition and
glycogen depletion in horses competing in an endurance ride. Vet
Rec 1981; 108:374-378.
14. Gollnick PD, Armstrong RB, Saltin B, Saubert CW,
Sembrowich WL, Shepherd RE: Effect of training on enzyme
activity and fiber composition of human skeletal muscle. J Appl
Physiol 1973;34:107-111.
15. Valberg S, Essen-Gustavsson B: Metabolic response to
racing determined in pools of type I, IIA and lib fibers. Equine
Exercise Physiology 2, ICEEP Publications 1987:290-301.

tract digestion of nitrogen was 95.7%. True digestibility of

nitrogen in the small intestine over the range of linearity
was 72.2%, while true digestibility of nitrogen reaching the
large intestine was 89.8%. These data indicate that the
protein in SBM was almost completely digested in the
equine digestive tract. Furthermore, approximately 75% of
the digestible protein was digested prececally when nitrogen intake was less than approximately 125 mg/kg of body
weight per feeding.

INTRODUCTION
SUMMARY
Four mature pony geldings weighing an average of
134 kg and fitted with ileal cannulas were used in a 4 x 4
Latin square experiment to determine the digestibility of
soybean meal (SBM) protein in different segments of the
equine digestive tract at various levels of protein intake. A
complete basal corn-based diet was supplemented with
SBM to formulate four diets with increasing crude protein.
The diets, labeled A (basal), B, C and D, contained 4.9%,
9.5%, 14% and 16.5% crude protein (as fed), and provided
nitrogen, per feeding, at approximately 44.8, 84.3, 123.9
and 146.3 mg/kg of body weight, respectively. Chromic
oxide was fed to measure ileal flow and fecal excretion.
Digestion and absorption of nitrogen was determined from
changes in nitrogen:chromium ratios, and true digestion of
nitrogen was computed by regression analysis. True total

Authors' address: 1Equine Science Program, Departmentof Animal

Science, Texas Agricultural ExperimentStation,Texas A&M University,
College Station, TX 77843; 2Departmentof Large Animal Medicineand
Surgery, Texas A&M University,CollegeStation,TX 77843.

Volume 15, Number 9, 1995

While the protein digestion coefficient of the total

digestive tract is sometimes used in the assessment of
nutritive value of feeds, it is not a reliable indicator of the
quality of feed protein available to the equine. The protein
in most feedstuffs, from forages to protein supplements, is
approximately 90% truly digestible over the digestive tract
of the equine. 1This coefficient does not reveal the site nor
the extent of the absorption of the end product of digestion.
Protein digested in the small intestine is primarily absorbed
as amino acids, while the protein digested in the cecum and
large intestine is primarily absorbed as ammonia? ,3Amino
acids essential for growth of muscle tissue, enzyme production and other physiologic functions are important to
the equine, particularly during lactation and growth. 4Therefore, protein digestibility in the small intestine is a more
desirable measure of the value of feed protein than is total
tract protein digestibility, which includes digestion in the
cecum and large intestine.
Knowledge of where a particular feed protein is digested and absorbed would be advantageous when choos-

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