Advances in Bioscience and Biotechnology, 2013, 4, 925-929 ABB
http://dx.doi.org/10.4236/abb.2013.410121 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Molecular studies on the Creole cattle breed in Mauritius
Olivier Pasnin1, V. M. Ranghoo-Sanmukhiya2*
1Mauritius Oceanography Institute, Quatre-Bornes, Mauritius
2Department of Agriculture and Food Science, Faculty of Agriculture, The University of Mauritius, Réduit, Mauritius
Received 30 June 2013; revised 15 July 2013; accepted 2 August 2013
Copyright © 2013 Olivier Pasnin, V. M. Ranghoo-Sanmukhiya. This is an open access article distributed under the Creative Com-
mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
ABSTRACT
There are three main cattle breeds in Mauritius; the
Friesian cows, the Creole cows and the Cross (Creole
and Friesians) breeds. The main objective of the stu-
dy was to differentiate the Creole breeds from the
other two breeds thus valorizing and conserving the
Creole cow which is in danger of extinction. Ran-
dom Amplified Polymorphic DNA and random mi-
crosatellite analysis were the two PCR based tech-
niques used. The populations studied consisted of 5
Friesians, 5 Creole breeds and 5 Cross breeds. The
breeds were obtained from the Curepipe Livestock
Research Station which is the only place where there
is a record of Creole cows in Mauritius. Among t he 5
Creoles breeds chosen, 2 of them could have been
impure breeds due to their morphological character-
istics. DNA extraction was carried out from blood
taken from the cows selected, and yielded DNA of
good quality and quantity. Polymorphic bands were
obtained from the Random Amplified Polymorphic
DNA primers and random microsatellite primers and
the data obtained were used for constructing a dendo-
gram. From the dendogram obtained, the breeds
were separated and the two Creole samples, which
were suspected to be impure, formed different clus-
ters from the true Creole breeds. From the results
obtained, the Creole breed was easily distinguishable
from the other breeds studied using molecular tech-
niques.
Keywords: Creole Cows; Friesian Cows; DNA
Extraction; Microsatellites; RAPDS
1. INTRODUCTION
There are three main cattle breeds in Mauritius which are
the Friesian, the Creole and the Cross (Creole and Frie-
sians) breeds which are kept on a small scale as a side
activity in some household [1]. The first cattle breed to
exist in Mauritius was probably the Zebus or Sangas
which was introduced by the Portuguese in year 1511
and afterwards the Ongle, Mysore and Hissar cattle were
imported from India by sugar estates. The breed Afri-
kanders were also introduced as Zebu herds and in 1922,
Friesian cattle were imported from South Africa to start
dairy herd. There is no proved explanation on the origin
of the Creole cow in Mauritius but everybody seems to
agree that it originates from north Europe and most
probably came through France if not from France [1].
According to Bennie [2], it is possible that this breed was
introduced in the eighteenth century although the first
introduction of cattle dated back to 1511.
The Creole cattle breed found in Mauritius has been
declining drastically mainly due to the introduction of
new breeds such as the Friesian cattle. Thus, while the
Creole cattle represented over 77% (32,332 heads out of
41,968) of the cattle population in 1964 [3] and in 2001
it represented less than 8% with 274 heads out of 3781
adult animals [1]. The Creole cows have adapted them-
selves quite perfectly to the environment and low level of
nutrition in Mauritius. They are known to be resistant to
diseases and have interesting potential both in terms of
production and reproduction. In 1944 a decision was
made to cross the Friesian breed with the Creole one
through artificial insemination leading to the production
of the Cross breeds [1].
The physical characterization of the Creole breed
showed that it is different from the other breeds found in
Mauritius. It is characterized by its white colour, an ab-
sence of hump and its polledness. The creole breeds are
actually used for milk production and used for beef pro-
duction. The breed continues to decline each year and a
molecular characterization is essential for preventing
germplasm erosion and also to obtain the information
necessary on this particular breed.
*Corresponding author.

O. Pasnin, V. M. Ranghoo-Sanmukhiya / Advances in Bioscience and Biotechnology 4 (2013) 925-929 926
RAPD markers and analysis have many advantages
over other methods of examining polymorphism due to
their ease and simplicity compared to other marker sys-
tems. RAPD markers have actually been widely used in
the characterization of livestock breeds [4-6]. Microsa-
tellites form part of the most common markers used for
genetic characterization of cattle breeds [7-9]. In trans-
genic breeding, molecular markers can be used as refer-
ence points for the identification, isolation and manipula-
tion of relevant genes, and for identification of animals
carrying the transgenes [10]. The use of microsatellites
as a genetic appraisal tool is quite significant [11]. Mi-
crosatellites can detect rare alleles compared to other
markers [12]. Many researchers are now using microsa-
tellites due to its various advantages compared to other
markers. The detection of microsatellite polymorphism
results in the greatest expected heterozygosity [13]. Mi-
crosatellites polymorphisms enable differentiation, even
between closely related breed, more clearly and also in-
crease the accuracy of the predicted divergence [14]. Mi-
crosatellites are proved to be useful in the probability of
sample identity at population levels [15].
2. MATRIALS AND METHODS
2.1. Animal Samples
The animal breeds included 5 Friesians, 5 Creoles breeds
and 5 Cross breeds. They were obtained from the Cure-
pipe Livestock Research Station where there is a record
of Creole cows in Mauritius.
2.2. DNA Isolation Protocol and Purification
Genomic DNA was extracted from peripheral blood and
use of fresh blood was essential to obtain a good yield of
DNA. The blood was stored in sterilized tubes containing
EDTA solution as heparin can cause inhibition of PCR
amplification [16]. A two step protocol was used for the
DNA extraction. The first step was the extraction of ge-
nomic DNA from white blood cells pellet and the second
step involved the extraction of DNA from cell lysis solu-
tion using organic solvents. The first step was done on
the first day and the white blood cells were inoculated in
SDS solution at 37˚C overnight and on the following day
the second step was carried out. The DNA obtained was
stored in about 200 - 300 μl nano pure water at −20˚C.
2.3. PCR Reaction
The thermal cycle used for the RAPD PCR was 5 min-
utes for initial denaturation step at 94˚C, followed by 38
cycles at 94˚C for 30 seconds for denaturation step, 35˚C
at 30 seconds for annealing step, 2 minutes at 72˚C for
extension and finally a 10 minutes at 72˚C for final ex-
tension. 50 RAPD primers were screened. The same
protocol and thermocycling conditions as for the RAPD
PCR were used for the screening of 50 random microsa-
tellites primers, except for the annealing temperature
which was 56.5˚C.
2.4. DNA Profile Analysis
Each genotype was characterized by its banding pattern
using the DNA hyperladder 2 (Bioline) as base pair lad-
der. The microsatellite and RAPD markers as viewed
from the gels after electrophoresis and staining were
converted into a matrix of binary data, where the pres-
ence of the band corresponded to value 1 and the absence
to value 0. The statistical software NTSYS-PC [17] and
DARwin 5 software [18] were used to construct a UP-
GMA dendrogram using hierarchical clustering. Using
NTSYS software, a dissimilarity matrix was calculated
utilising Jaccard’s coefficient [19]. The matrix was con-
verted to a dissimilarity matrix corresponding to the
complement (dissimilarity = 1 − similarity). Cluster
analysis based on the dissimilarity matrix, was per-
formed using un-weighted pairgroup method arithmetic
averages (UPGMA) [20] of the NTSYS-PC version 2.2.
3. RESULTS
Maximum polymorphism was obtained using OPJ16
(CTGCTTAGGG) (Figure 1), OPA10 (GTGATCGCAG)
(Figure 2) and OPY18 (GTGGAGTCAG) (Figure 3).
Out of microsatellites primers screened maximum poly-
morphism was obtained using microsatellite primers
TTCX5 (Figure 4) and GTAGX4 (Figure 5). Both mi-
crosatellite markers and RAPD markers were found to be
suitable for the assessment of genetic diversity among
the different cattle breeds in Mauritius.
Two major clusters were observed in the dendogram
produced using RAPD and SSR markers (Figure 6)
clearly distinguishing between the Creole breed and the
Friesian breed. There was one Creole cattle which clus-
tered with the Cross breed while a cattle from the Cross
breed clustered with the Friesian breed.
LANES:
F: Friesian Breed
C: Creole Breed
X: Cross breed of
Friesian and Creole
breed
N
: Negative control
HL: Hyperladder II
Figure 1. RAPD amplification using primer OPJ16.

O. Pasnin, V. M. Ranghoo-Sanmukhiya / Advances in Bioscience and Biotechnology 4 (2013) 925-929 927
LANES:
F: Friesian Breed
C: Creole Breed
X: Cross breed of
Friesian and Creole
breed
N
: Negative control
HL: Hyperladder II
Figure 2. RAPD amplification using primer OPA10.
LANES:
F: Friesian Breed
C: Creole Breed
X: Cross breed of
Friesian and Creole
breed
N
: Negative control
HL: Hyperladder II
Figure 3. RAPD amplification using primer OPY18.
Figure 4. Microsatellite amplifications us-
ing primer TTCX5.
Figure 5. Microsatellite amplifications us-
ing primer GTAGX4.
Figure 6. Dendogram showing relationships between the cattle
breed studied using RAPD and SSR markers.
4. DISCUSSION
Phenotypically the three cattle breeds are different, show-
ing different morphological characters. The body coat of
the Creole cow is white with a pink muzzle, white ears
and white eyelids. The body coat of the Friesian is how-
ever black with a black muzzle, black ears and black
eyelids. The body coat of the Cross breed is however
intermediate white and black with a pigmented muzzle,
brown ears and white eyelids. The most striking mor-
phological feature is the presence of horn in the Frie-
sian breeds and the cross-breed and this feature is absent
in the Creole cows.
The dendogram (Figure 6) obtained from both RAPD
and microsatellite primers confirm the difference which
exists between the cattle breeds, particularly the Friesian
and Creole cows. The Friesian cows as expected formed
a different cluster from the Creole breed and Cross breed
confirming genetically the difference which is seen in the
morphological characteristics.
Ideally the expected results for the classification of the
Creole breed would have been a cluster of all the 5 Cre-
oles in a single cluster. This would have shown that the
Creole breeds are genetically different from the other
breeds. However, 2 clusters were formed (Figure 6) one
containing 4 individuals of the Creoles breed and another
with one Creole breed and the Cross breed. The fact that
the Creole cows were classified in two separate clusters
can be explained by the following reasons: There are few

O. Pasnin, V. M. Ranghoo-Sanmukhiya / Advances in Bioscience and Biotechnology 4 (2013) 925-929 928
Creole cows in Mauritius and among the limited number
remaining only a few of them may be really pure breed.
Moreover, the Creole cows are crossed with the Cross
breeds of Creole and Friesian, therefore the new born
calves cannot be said to be pure Creole breeds as they
contained some of the characteristics of the Cross breeds,
either expressed or not. From the selected Creole cows at
the experimentation station, it was easy to distinguish if
the Creole cows were potentially pure breed or not.
Some good samples were unavailable and other cows
were selected as alternatives but they were not pure
breeds. Observing the morphological characteristics and
the dendogram obtained from RAPD primers, one of the
Creole cows which formed a different cluster from the
other three samples was not pure breed. Presence of
horns in that Creole cow was one of the characteristics
which showed that it was not a pure breed.
From the result obtained above it can be hypothesized
that existing Creole Cattle breed are not pure breed and
that there may have been considerable crossing which
have inadvertedly happened over time explaining why
the Creole cattle breed and the Cross breed cluster to-
gether in the same clade. Therefore there is an urgent
need to salvage the remaining pure cattle breed if we
wish to preserve the beneficial reproductive and produc-
tive traits in the cattle gene pool.
5. CONCLUSION
The cattle breeds are phenotypically different from each
other and they can be easily differentiated by their phy-
sical characteristics. Assessing the differences at the mo-
lecular level was a more tedious work. The RAPD and
microsatellite primers, as expected, gave good amplifica-
tion and, sharp and clear polymorphic bands were ob-
served and these markers have shown that the Creole
cows are different from the Friesian cows. The creole
cattle breed has good agronomic traits namely production
and reproduction and there is an urgent need that a solid
conservation programme be implemented if this breed is
to be saved from extinction as a large amount of crossing
has been noted and it is difficult to say whether there still
exist a pure Creole Cattle Breed in Mauritius [1].
6. ACKNOWLEDGEMENTS
We wish to acknowledge the Faculty of Agriculture, University of
Mauritius for the laboratory facilities provided and the Agricultural
Research and Extension Unit for providing the cow samples.
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