Muscle Fiber Types in Crioulo Horses

Ins N. de Castro,a PhD, Nadja L. Pinheiro, PhD,a Flvio De La Corte, PhD,b

Carlos Eduardo Nogueira, PhD,c and Igncio Leon, DVMd

REFEREED
SUMMARY

Sixty-three Crioulo horses were used for this work.

The horses belong to 7 different establishments in South
America (six located in the South of Brazil and one in
Uruguay) and were divided into 4 categories or groups
consisting of 19, 17, 14, and 13 animals, respectively.
Group A consisted of adult female horses of lineage
selected by endurance tests, and that had rested for at
least a year. These animals were winners or descendents
of the winners of endurance tests. Their pedigrees were
analyzed to check the absence of the blood of Chilean
horses in their lineage.
Group B consisted of adult female horses whose lineage was selected by Freio de Ouro tests, and that had
been kept out of training for at least a year. They were
winners, or were classified or descendents of animals that
had won the Freio de Ouro test. Their pedigrees were
analyzed and the blood of horses of Chilean origin was
found to be present, or of pure Chileans found in their
pedigree.
Group C consisted of female horses from 2 to 2.5
years old, whose lineage was selected by Freio de Ouro
testing. These were raised in paddocks and had received
no training. They were the descendents of Freio de
Ouro test champions, the pedigrees were analyzed, and
the presence of Chilean blood was found in their crossbreeding.
Group D consisted of adult animals of both sexes and
gelded male horses that had been kept in training through
daily work at a cattle ranch until one week before the collection of the material. These animals had no Chilean
blood in their cross-breeding.

From Dept. de Histologia e Embriologia, Instituto de Biologia, UFRRJa;

Dept. de Clnica de Grandes Animais, Hospital Veterinrio, UFSMb;
Dept. Clnicas Veterinria, UFPelc; and Santa Maria de Araras Farm.d
J Equine Vet Sci 2004;24:204-209.
0737-0806/$ - see front matter
2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jevs.2004.04.008

204

Muscular biopsies were taken from the gluteus

medius. The classifications of the different types of muscular fibers were performed exclusively through staining
for myofibrillar ATPase. At least 200 fibers in each sample were counted and identified through the changes in
their staining, and relative distributions (%) of the types
of fibers were determined for each horse.
The general mean of the muscular fibers for the
Crioulo horse are: 13.83% for type I, 31.63% for type
IIA, 54.37% for type IIB, and 0.17% for type IIC. Verification was made that general percentage type I fibers
were 13.83% and for type II were 86.17%. Fibers of the
type IIC were found only in young animals in Group C.
No statistical differences between females and geldings in exercise were found. It was observed that the
adult females in Group A were significantly different
from Group B in relation to the percentage of fiber types
IIA and IIB, and were not different from the other
groups. The females of Group B possessed more fibers of
type IIB, whereas the females of Group A possessed
more fibers of type IIA. Crioulo horses, when correctly
trained, adapt to the muscular work involving speed or
endurance. However, Crioulo horses with Chilean blood
possess a greater predisposition for tests involving more
speed such as Freio de Ouro. Crioulo horses with lineage from South Brazil, Argentina, and Uruguay that
havent been cross-bred with Chilean blood, have a larger
predisposition for endurance tests.
INTRODUCTION

The Crioulo Horse is a breed of Spanish origin,

largely bred in the South of Brazil, Uruguay, Argentina,
Paraguay, and Chile. These horses have been used for
work in farms and cattle ranches for more than 200 years.
Their main characteristics are rusticity, fertility, and
longevity. For four centuries the Crioulo breed has been
adapting to its environment by way of a very strict natural selection. The Associao Brasileira de Criadores de
Cavalos Crioulos (ABCCC) was founded in 1932. The
basis for the Crioulo horse in Southern Brazil was exempt from Chilean blood cross-breeding. Currently, its

Journal of Equine Veterinary Science

May 2004

performance has been evaluated through endurance tests

and functional tests with reins with a test called the
Freio de Ouro test, created 22 years ago with the introduction of animals with Chilean blood cross-breeding.
Both tests check speed, resistance, and flexibility.1-3
Previous studies show that the horses skeletal muscle can be histochemically differentiated through ATPase
myosin activity in three fiber types. These fibers are classified as type I, those which have slow-twitch (ST) and
high oxidative, type IIA with fast twitch and high oxidative (FTH), and type IIB with fast-twitch and low oxidative (FT).4-10
Based on the ATPase myosin activity, two types of
fibers are distinguished at a pH of 9.4. Those of slowtwitch type I, having low activity in this pH and therefore
appearing in lighter staining than the fast-twitch fibers,
are called type II. The latter can be divided into an acid
pH, in types IIA, IIB, and IIC. The requested pH for this
differentiation varies between 4.3 and 4.5. The type IIC
fibers are found in small quantities in foals, they are
rarely found in adult animals, and are referred to as transitional fibers, when type I fibers are transformed into
type II fibers and vice versa.11-13
The ratio of fiber types in the muscle varies with
breed, age, sex, and training.4,14-17 There is no difference in
the rear side locations for the percentage of types of muscular fibers,18 but there are variations in the fiber types
when the biopsies are performed at different depths.16-18
Quarter horses that perform over a shorter distance
(400 m) showed a larger ratio (90-95%) of type II fibers.
Thoroughbred horses, which run distances of 1000 to
3000 m, had approximately 85% to 90% of type II fibers,
and Standardbred horses, walking or trotting at distances
from 1600 to 2600 m had 75% to 85% of type II
fibers.8,11,15,19,20 Endurance breeds, like Arabian or
Andaluz horses, showed 70% of type II fibers.14,21,22 In
endurance horses, a large proportion of types I and IIA
were found, whereas horses with high-speed performances, such as Quarter horses and Thoroughbreds, tend
to have a higher ratio of types IIA and IIB fibers.23,24
In previous studies with adult Chilean Crioulo horses,25
it was ascertained that these animals show a large proportion of type II fibers and a smaller proportion of type
I fibers. The percentage of type IIB fibers was 42.5%, and
type IIA was 39%.
Age and training lead to modifications in the muscular characteristics, such as a percentage increase of type I
fibers, as well as an increase in the type IIA/IIB ratio, and
in the oxidative ability.11,26,27 The regular training of
Standardbred horses at 18 months of age prompts an increase in the oxidative ability and a reduction in type IIB
fiber percentage.4,28

Volume 24, Number 5

The samples must be collected at the same location

and depth, 18 cm along an imaginary straight line drawn
from the tuber coxae to the tail head. The biopsy is obtained at a depth of 4 to 6 cm and, in young animals, at a
depth of 2 to 4 cm.6
At least 200 fibers in each sample must be identified
to obtain the relative ratio for each muscular fiber type
(%).10,12,15,27 It was noted that there is no significant percentage difference in the three fiber types when the overall counting is done with 200, 600, or 800 fibers.8
Due to the significant relation between heritage and
performance, fiber percentage determination may be advantageous for the establishment of the horses fitness
and characteristics. Muscular biopsy, in addition to other
factors, can have a big influence on the selection of the
best stallions and mares.
Although there is much information about muscular
biopsy in various breeds around the world, not a great
deal is available on Brazilian breeds.29
The object of this work was to define the different
types and percentages of each type of muscle fibers in
Crioulo horses, and to determine whether there is a relationship between the percentage of these fibers, and the
usages these animals are destined for.
MATERIALS AND METHODS

Animals and Establishment

Four groups made up of 20 animals each were the
basis for this work. The first group (Group A) was represented by adult mares with selected lineage for the endurance test that had rested for at least a year. The second
group (Group B) was made up of adult mares with selected lineage for the Freio de Ouro test, and were also
kept out of training for at least a year. The third group
(Group C) consisted of young female animals with ages
varying from 2 to 2.5 years with selected lineage for the
Freio de Ouro test, and were unbroken, having being
entirely raised in the fields. Finally, a fourth group
(Group D) was made up of adult animals of both sexes,
the males all geldings. All the animals in this group were
in training for as long as a week before the collection of
samples.
Due to a variety of problems, some samples were
discarded and the laboratory was able to process a total
of 63 samples for the assessment. The groups were made
up of 19, 17, 14, and 13 animals, respectively.
Description of Tests for Which the Animals are
Trained and Definition of Groups
Endurance test. Thirty days before the competition,
the horses are taken to a field with water and native grass

205

Figure 1. Muscular biopsy from the gluteus medius.

only, without the ingestion of any medication. The animals perform a journey of 750 km in 15 days with an average of 30 to 60 km/day, at a pace of approximately 10
km/hour. Scoring is then initiated on the performance
time and clinical standard of the animals.
The majority of the animals selected for this type of
test were of Riograndense (horses from Rio Grande do
Sul State in South Brazil), Uruguayan, and Argentinian
lineage, and with no Chilean blood (Group A).
Freio de Ouro Test. In the Freio de Ouro competition, the taming, velocity, docility, vitality, courage,
and flexibility of the animals are assessed. These traits
show the animals fitness and its capacity to function. Its
ability is demonstrated as the horse immediately reacts to
the riders demands.
There are also tests of conformation, gait, figura (similar to barrel racing), tossing (similar to reining), and
paleteada (similar to the AQHAs working cow horse
competition). The majority of these animals are the product
of cross-breeding Riograndense animals with those with
Chilean blood (Group B).
The exercise performed by Group D animals consisted of daily and continuous work on a cattle breeding
ranch located in Uruguay. These horses worked daily, in
the morning or in the afternoon, covering an average of 40
km/day during 7 days. After this period they were rested
for 3 weeks and then put back to work for 7 more days.
The pattern was repeated for an entire year, during which
time the horses were fed native grass only (Group D).
The Group C horses were raised free in the field
since they were young, and were not yet broke.

206

Figure 2. Myofibrillar ATPase staining of gluteus medius

muscle in pH 4.52.

Muscular Biopsy
Muscular biopsy samples were collected from the
gluteus medius through a biopsy needle (6 mm internal
diameter and 3 mm cannula [Fig.1]). To establish the
biopsy collection location and depth for this breed, a previous measurement of the gluteus medius depth was performed using Ausonic-Impact VF1 ultrasound with a linear probe of 10 MHz. The imaginary line from the tuber
coxae to the tails base defined the location for the biopsy
sample collection. The biopsy was performed at the first
third of the straight line, approximately 18 cm from the
tuber coxae, the thickest point of the gluteus medius. All
samples were obtained at the same location and at the
same depth. In adult animals, the samples were collected
at a depth of 6 cm marked on the biopsy needle, and in
young animals at a depth of 4 cm.
The freezing of the samples with methylbutane was
done quickly to avoid ice crystal formation. The storage
and transportation to the laboratory was carried out in a
nitrogen container at 90 C.

Laboratorial Processing and Histochemical

Analysis
Samples were sliced to a thickness of 10 m using a
cryostat microtome at 20 C. The qualification of fibers
was exclusively based on the staining of the myofibrillar
ATPase with a pH of 9.414 at 37 C during 17 minutes
and 45 seconds.
First the sections (slices) were submitted to pre-incubation in three solutions: acid with a pH of 4.25 (3 min);
acid with a pH of 4.528 (Fig 2) (2.5 min) (both acid so-

Journal of Equine Veterinary Science

May 2004

Table 1

Fiber types (%) presented as general mean, range, and standard error figures in each group
Group A

Fiber

Type I 19 13.69
Type IIA 19 33.14
Type IIB 19 53.17
Type IIC 19 00.00
General mean
Type I
Type IIA
Type IIB
Type IIC

Group B

Group C

Group D

Range

SE

Range

SE

Range

SE

Range

SE

5.94-22.89
20.16-46.15
41.02-63.64
00.00-00.00

0.87
1.53
1.54
0.00

17
17
17
17

12.12
27.65
60.23
00.00

8.11-20.55
20.00-37.84
52.21-67.10
00.00-00.00

0.88
1.24
1.09
0.00

14
14
14
14

16.89
31.74
50.61
0.17

7.39-29.11
25.53-39.46
37.13-62.56
00.00-5.13

1.58
1.23
1.94
0.38

13
13
13
13

12.97
34.49
52.53
00.00

5.11-25.00
25.00-43.65
44.35-61.95
00.00-00.00

1.51
1.63
1.35
0.00

63
63
63
63

13.83
31.63
54.37
0.17

5.11-29.11
20.00-46.15
37.13-67.10
00.00-5.13

0.61
0.77
0.87
0.09

General mean refers to all the groups of horses combined.

Table 2

Mean values, Tuckey test and standard error of fibers type I, IIA and IIB in the four groups of animals
Fibers type (%)
I

Group

A
B
C
D
General
Mean
F
P<

19
17
14
13

13.69ab
12.12b
16.89a
12.97a

63

13.83

IIA
SE

0.87
0.88
1.58
1.51

33.14a
27.65b
31.74ab
34.49a

0.61

31.63

2.92
0.04

IIB
SE

SE

1.53
1.24
1.23
1.63

53.17b
60.23a
50.61b
52.53b

1.54
1.09
1.94
1.35

0.77

54.37

0.87

4.33
0.01

7.97
0.000195

a,b, In the column, means followed by different characters are significantly different under Tuckey test analysis (P < .05).

lutions were at room temperature); and alkaline with a

pH of 9.8 (for 20 min at 37 C).
At least 200 fibers were counted in each sample and
identified through the changes in their staining. A relative
(%) distribution of muscular fiber types was determined
for each horse. The counting was done through projection
on a video monitor.

Statistical Analysis
The experimental plan adopted was made entirely
casual through a variable number of repetitions. The statistical method used variance analysis, with F test classification criteria and the Tukey test, where significant differences were detected by the F test at the same level of
5%.
RESULTS

It was determined (Table 1) that the general mean

of the muscular fibers for the Crioulo breed are 13.83%

Volume 24, Number 5

for type I, 31.63% for type IIA, 54.37% for type IIB,
and 0.17% for type IIC. Therefore, the general percentage for type I fibers was 13.83% and for type II was
86.17%.
Table 1 shows data referring to general mean, range,
and standard error of the mean for types I, IIA, IIB, and
IIC fibers, in the four different animal categories. In general, the mean difference in values between type IIA fibers
and type IIB fibers was of 22.74%.
In young females in Group C the presence was observed of transition fibers or type IIC in 5 animals with a
mean of 0.17%.
In Group D, which comprises mares and geldings in
training, no significant difference was found in the t test
between sexes.
As shown in Table 2, Group A (adult females selected for endurance tests) was significantly different in
fiber types IIA and IIB from Group B (adult females selected for the Freio de Ouro test. Freio de Ouro fe-

207

males (Group B) had more type IIB fibers, whereas the

endurance females (Group A) had more type IIA fibers.
Group A was not different from the other groups.
The young females (Group C) were different from
the adult females (Group B), both groups selected for the
Freio de Ouro test, as regards type I fiber percentage
(16.89 and 12.12%) and IIB (50.61 and 60.23%). They
did not show a difference as per type IIA fiber percentage. The other groups did not show significant differences
as to type I fiber percentage.
It was also found that the adult animals selected for
the Freio de Ouro test (Group B) showed significant
differences in relation to type IIB fiber percentage (larger
IIB type fiber percentage) from the other groups.
Group D, formed by animals in training, was different from Group B (Freio de Ouro animals) with reference to type IIA and IIB fibers.
The groups which carried out predominantly aerobic
work, and with no Chilean blood in their pedigrees (Groups
A and D), showed similar characteristics concerning the
three muscular fibers types.
During the processing of samples at the laboratory it
was found that the best time for incubation with ATPase
was 17 minutes and 45 seconds, in a solution with a pH
of 9.414 at 37 C.
DISCUSSION

Previous studies have demonstrated that it is possible

to individually classify a horse and establish its training
category based on its muscular physiology.22
Type II fiber percentages found in Crioulo horses
(86.17%) in this study were similar to the minimum values
found in Thoroughbred horses (85-90%) and was higher
than the percentage found in Standardbred horses (7585%), Chilean Crioulo horses (81.5%)8,11,15,18,19,25 and in
endurance breeds like Andaluz and Arabian (70%).14,21,22
The presence of type IIC fibers occurred only in the
groups containing young animals and was not higher than
2%, confirming data presented in previous research.19,23,27
Unlike previous research with other breeds,26,27 no significant increase in type I fibers occurred with increased
age, comparing young animals in Group C to the other
groups. Type IIA fibers did not show any increase, with
consequent reductions in type IIB fibers as age increased.
Perhaps this is due to the fact that these younger animals
are already 2 to 2.5 years old.
The group of Crioulo horses selected for the endurance test showed a larger ratio of type IIA fibers and
a smaller ratio of type IIB fibers compared with Crioulo
horses selected for the Freio de Ouro test. In this group,
a smaller ratio of type IIA fibers and a larger ratio of type
IIB fibers was observed (Table 2). Andaluz horses se-

208

lected for the endurance test also showed a larger percentage of type IIA fibers.21
Previous research carried out with Chilean Crioulo
horses noted a larger ratio of type IIB fibers (42.5%) than
type IIA (39%),25 confirming the results achieved in this
study. However, Table 2 shows that Groups A and B differ statistically regarding type II fiber percentage. Accordingly, it is possible to suggest that the Crioulo horse
lineage with Chilean (Group B) origins may be selected
for muscular work, which demands greater speed. Group
A animals free of Chilean blood adapt perfectly to the endurance tests.
The technique used for the histochemical analysis
performance was the same previously described,28,30 but
modifications in pH and incubation time of solutions did
occur. One factor explaining this modification would be
breed variation at the muscular metabolism.4
CONCLUSION

The muscular biopsy is a relatively simple method

which serves to establish the percentage of the different
muscular fiber types found in Crioulo horses. From these
results, one can determine each animals fitness, as well
as its selection for future crossbreeding.
The adult females selected for the endurance test
were significantly different from the females chosen for
the Freio de Ouro test, as far as type IIA and IIB fibers
are concerned. Freio de Ouro females showed more
type IIB fibers and the endurance females showed more
type IIA fibers.
When properly trained, Crioulo horses with Chilean
blood have a greater predisposition to tests that demand
more speed and thrust (Freio de Ouro). Crioulo horses
with Riograndense, Uruguayan, and Argentinean lineage
(without Chilean blood in their crossbreeding) have a
greater adaptability for endurance tests.
The histochemical technique of myofibrillar ATPase
could be adapted to the pH and solution incubation time
depending on the different breeds being studied.
Acknowledgments
We thank the owners for the use of their dear animals, Dr
Stephanie Valberg for her orientation during the laboratorial analysis, Dr Jos Henrique Souza for his orientation
during the statistical analyses, Cristina Zambrano, Srgio
Scherer Filho, Susana Rieck, Marcelo Napoleo, Joaquin
Lopez de Alda, Carlos Eduardo Veiga, Antonio Augusto
Fernandes, Beatriz Hetzel, Marta Lopez, and Julio Bozano.

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