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ABSTRACT

The water buffalo is an important farm animal raised all over the world but mostly in Asia. They were distributed from southern Asia to Europe during the Pleistocene Epoch. They are classifi ed according to their chromosome number and morphological data either as river type (2n=50) or swamp type (2n=48). Anatolian water buffalo was classifi ed as river type and no further karyotyping specifi cation was made up to now. Previous studies have shown that classifi cation based solely on morphological data is not adequate. Therefore we have applied a procedure in order to identify the chromosome number of the Anatolian water buffalo for the purpose of proving them to be of the river type. Our study has shown that Anatolian water buffalo possess 50 chromosomes which proves their classifi cation as river type buffalos.

Keywords: Anatolian water buffalo, river type, swamp type, karyotype

INTRODUCTION

Most of the farm animals were domesticated in Fertile Crescent around 10.000 years ago as supported by archeological data and mtDNA analysis studies (Driscoll et al., 2009). In contrast, the water buffalo was domesticated in southern Asia far from the Fertile Crescent about 3000-6000 years ago (Kierstein et al., 2004; Yindee et al., 2010). They were distributed from southern Asia to Europe during the Pleistocene Epoch. Over 168 million water buffalos are raised in the world, most of them (161 million) in Asia (Iannuzzi and Di Meo, 2009). There are around 100.000 water buffalos in Turkey distributed in the northwestern part of the country (Figure 1) and their number has decreased sharply in recent years (Oztabak et al., 2009). Water buffaloes provide over 10% of the annual milk production in the world. The milk of buffaloes is known to be high in fat, minerals, proteins and lactose and low in water. With this content, it has a notable value in making butter, high-quality cheeses, and other dairy products. It is known that there exist two main types of Asiatic water buffaloes (Bubalus bubalis): the

KARYOTYPE OF ANATOLIAN WATER BUFFALO (BUBALUS BUBALIS)

N. Ulku Karabay Yavasoglu1, Cinel Koksal2, Yalcin Yaman3 and Cemal Un4

1Ege University, Faculty of Science, Department of Biology, Division of General Biology, Bornova, Izmir, Turkey, E-mail: ulku.karabay@ege.edu.tr2Ege University, Center for Drug Research and Pharmacokinetic Applications, Bornova, Izmir, Turkey3Marmara Animal Research Centre, Bandırma, Balıkesir, Turkey4Ege University, Faculty of Science, Department of Biology, Division of Molecular Biology, Bornova, Izmir, Turkey

Original Article

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river buffalo with 50 chromosomes in the genome and the swamp buffalo with 48 chromosomes in the genome (Fischer and Ulbrich, 1968; Iannuzzi, 1994). The river buffaloes are by far the most numerous of the speciesand include the Murrah, Jafarabadi, Surthi, Mehsana, Egyptian and Mediterranean breeds (about 80% of the world’s buffalo population). They are raised in a region extending from India in the east to Italy in the west that includes Pakistan, Iran, Turkmenistan, Iraq, Syria, Egypt, Turkey, Greece, Bulgaria and the former Yugoslavia (Kikkawa et al., 1997; Rosati and Van Vleck, 2002). River buffaloes are found in many countries, while swamp buffaloes are limited to a few Far Eastern countries. Water buffaloes are believed to have been introduced into Turkey in the seventh century. Native Anatolian water buffalos have typically half crescent shape horns directed backwards and black hair and skin(Soysal et al., 2005). They are classifi ed as a river type of water buffalo and believed to have 25 pairs of chromosomes (2n = 50). It was reported before, that the Sri Lanka or Indian Chilika wild buffaloes combine swamp type morphology with river type karyotype and DNA sequences (Barker et al., 1997; Lau et al., 1998; Mishra et al., 2009). Therefore, morphologic data alone are not suffi cient to decide if a given buffalo belongs to the river or swamp types. To the best of our knowledge, no karyotyping study was conducted in Anatolian water buffalo up to now. The aim of the study was to determine the chromosome numbers of Anatolian water buffalo in order to prove them to be of the river type.

MATERIALS AND METHODS

The protocol was approved by Ege

University, Local Ethical Committee of Animal Experiment (number: 2011-70). Peripheral blood samples were collected in vacutainers containing sodium heparin from fi ve healthy male buffaloes maintained at the Marmara Animal Research Centre, Bandırma, Balıkesir, Turkey. Briefl y, 0.5 ml of whole heparinized peripheral blood was cultured in 10 ml of RPMI 1640 medium enriched with fetal bovine serum (15%), L-glutamine, antibiotics (gentamicin) and Phytohemagglutinin M as mitogen. The cultures were incubated at 37ºC in 5% a CO2 atmosphere for 72 h. 0.2 ml of Colcemid solution (10 μg/ml) was inoculated into each culture tube and the cultures were incubated for an additional 15 minutes. The cultures were harvested with hypotonic treatment (0.075 M KCl) and fi xed in methanol: acetic acid (3:1). The cell suspension was dropped on cleaned, cold and wet slides. Air-dried slides were stained in 2% Giemsa (Iannuzzi et al., 1990; Kumar and Yadav, 1991). Twenty slides were prepared for each animal. About 25 metaphase spreads were screened for chromosome complement. Those spreads with clear staining and non-overlapping chromosomes were photographed (x 1000) for the preparation of karyotypes.

RESULTS AND DISCUSSION

Metaphase spread and karyotype of the Anatolian water buffalo was demonstrated in Figures 2a and 2b, respectively. The chromosomes were clearly distinguishable and sorted from chromosome 1 to chromosome 24 for somatic pairs. Similarly sex chromosomes X and Y were identifi ed clearly. The Anatolian water buffalo has fi ve pairs submetacentric and 19 pairs acrocentric, including both the X and Y chromosomes. We have

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Figure 1. Anatolian water buffalo.

Figure 2. a) Metaphase spread (x 1000) of the Anatolian buffalo (male) (above). b) Karyotype of the male Anatolian buffalo (right).

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demonstrated that Anatolian water buffalo has 48 somatic chromosomes and two sex chromosomes, a total 50 of chromosomes, which is suitable to river type of water buffalo. The Asiatic water buffalo (Bubalus bubalis) has two main subspecies; the river buffalo (2n=50) and the swamp buffalo (2n=48). These two subspecies differ by one chromosome pair, where a tandem fusion translocation between river buffalo chromosomes 4 and 9 is comparable to swamp buffalo chromosome 1 (Di Berardino and Iannuzzi, 1981). The river buffalo has fi ve pairs submetacentric and 19 pairs acrocentric, including both the X (the largest acrocentric) and Y (small acrocentric) chromosomes (Nair et al., 1986; Iannuzzi, 1994). Since previous studies have shown that the Sri Lanka or Indian Chilika wild buffaloes combine swamp type morphology with river type karyotype and DNA sequences (Barker et al., 1997; Lau et al., 1998; Mishra et al., 2009), morphological data may be deceptive for classifi cation of water buffalos. There was no karyotyping study in Anatolian water buffalo, which is classifi ed as river type based on morphological data. Our study has proved Anatolian water buffalo to be of the river type. Buffalo cytogenetics could serve as an essential tool in implementation of breeding programmes, particularly in screening bulls used for artifi cial insemination programmes. Systematic cytogenetic investigation of breeding problems of buffaloes is still lacking, and hence most of the aberrations have escaped our attention (Murali et al., 2009). Clinical cytogenetics has only recently developed in river buffalo, especially in Italy where both bulls used for reproduction and many females with reproductive problems have been investigated cytogenetically by using both banding and FSH-mapping techniques (Iannuzzi, 2007).

Anatolian water buffaloes may play an important role in animal husbandry in Turkey since they are resistant to many diseases and their milk has special composition suited for making cheese and cream. In a study it was reported that Anatolian water buffalo have ahigh frequency of the PRNP gene in alleles (indel 12 and indel 23) which are associated with resistance to BSE (bovine spongiform encephalopathie) in cattle (Oztabak et al., 2009). It was also determined in the same study that Anatolian water buffalo population was subjected to a bottle neck effect recently because of a sharp decrease in the number of individuals which causes losse of some gene alleles. The genome of the Anatolian water buffalo is poorly analyzed. In conclusion, further studies should be conducted in order to characterize genetic structure of the Anatolian water buffalo.

Confl ict of Interest The authors have declared that there is no confl ict of interest.

REFERENCES

Barker, J.S., S.S. Moore, D.J. Hetzel, D. Evans, S.G. Tan and K. Byrne. 1997. Genetic diversity of Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with protein-coding loci. Anim. Genet., 28: 103-115.

Di Berardino, D. and L. Iannuzzi. 1981. Chromosome banding homologies in swamp and Murrah buffalo. J. Hered, 72: 183-188.

Driscoll, C.A., D. Macdonald and M.S. O’Brien. 2009. From wild animals to domestic pets, an evolutionary view of domestication. P. Natl. Acad. Sci. USA. 106: 9971-9978.

Buffalo Bulletin (December 2014) Vol.33 No.4

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Fischer, H. and F. Ulbrich. 1968. Chromosomes of the Murrah buffalo and its crossbreds with the Asiatic swamp buffalo (Bubalus bubalis). Z. Tierz. Zuchtungsbio., 84: 110-114.

Iannuzzi, L. 1994. Standard karyotype of the river buffalo (Bubalus bubalis L., 2n = 50). Report of the committee for the standardization of banded karyotypes of the river buffalo. Cytogenet. Cell Genet., 67: 102-113.

Iannuzzi, L. 2007. The water buffalo: evolutionary, clinical and molecular cytogenetics. Italian J. Anim. Sci., 6(2): 227-236.

Iannuzzi, L. and G.P. Di Meo. 2009. Water buffalo, p. 19. In Cockett, N.E. and C. Kole (eds). Genome Mapping and Genomics in Domestic Animals, Springer Verlag, Berlin, Heidelberg.

Iannuzzi L., G.P. Di Meo, A. Perucatti and L. Ferrara. 1990. The high resolution G- and R-banding pattern in chromosomes of river buffalo (Bubalus bubalis L.). Hereditas, 112: 209-215.

Kierstein, G., M. Vallinoto, A. Silva, M.P. Schneider, L. Iannuzzi and B. Brenig. 2004. Analysis of mitochondrial D-loop region casts new light on domestic water buffalo (Bubalus bubalis) phylogeny. Mol. Phylogenet. Evol., 30: 308-324.

Kikkawa, Y., H. Yonekawa, H. Suzuki and T. Amano. 1997. Analysis of genetic diversity of domestic water buffaloes and anoas based on variations in the mitochondrial gene for cytochrome b. Anim. Genet., 28: 195-201.

Kumar, P. and B.R. Yadav. 1991. Comparative cytogenetic studies in Mehasana, Murrah and Surti buffaloes. Indian J. Dairy Sci., 44: 2.

Lau, C.H., R.D. Drinkwater, K. Yusoff, S.G. Tan,

D.J. Hetzel and J.S. Barker. 1998. Genetic diversity of Asian water buffalo (Bubalus bubalis): mitochondrial DNA D-loop and cytochrome b sequence variation. Anim. Genet., 29: 253-264.

Mishra, B.P., R.S. Kataria, S.S. Bulandi, B. Prakash, P. Kathiravan, M. Mukesh and D.K. Sadana. 2009. Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis. J. Anim. Breed. Genet., 126: 69-79.

Murali, N., P. Devendran and S. Panneerselvam. 2009. Cytogenetic Studies On The Chromosomes of Toda Buffaloes. Buffalo Bull. 28(2): 95-100.

Nair, P.G., M. Balakrishnan and B.R. Yadav. 1986. The Toda buffaloes of Nilgiris. Buffalo J. 2: 167-178.

Oztabak, K., E. Ozkan, I. Soysal, I. Paya and C. Un. 2009. Detection of prion gene promoter and intron1 indel polymorphisms in Anatolian water buffalo (Bubalus bubalis). J. Anim. Breed. Genet., 126: 463-467.

Rosati, A. and L.D. Van Vleck. 2002. Estimation of genetic parameters for milk, fat, protein and mozzarella cheese production for the Italian river buffalo Bubalus bubalis population. Livest. Prod. Sci., 74: 185-190.

Soysal, M.I., Y.T. Tuna and E.K. Gurcan. 2005. An investigation on the water buffalo breeding in Danamandira village of Silivri district of Istanbul province of Turkey. J. Tekirdag Agric. Fac., 2: 73-78.

Yindee, M., B.H. Vlamings, W. Wajjwalku, M. Techakumphu, C. Lohachit, S. Sirivaidyapong, C. Thitaram, A.A.A.W.K. Amarasinghe, P.A.B.D.A. Alexander, B. Colenbrander and J.A. Lenstra. 2010.

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Y-chromosomal variation confi rms independent domestications of swamp and river buffalo. Anim. Genet., 41(4): 433-435.