Investigation on Hemoglobin Polymorphism in Different Breeds of Dog
Hemoglobin is a complex metalloprotein, iron containing pigment of erythrocytes. It plays an important role in oxygen transport and is directly proportional to the property of the animal for sustained muscular activity or ability to meet demands for sudden burst of speed. It also has a variety of other roles of gas transport and effect-modulation which vary from species to species, and are quite diverse in some invertebrates. The sequences of hemoglobin differ between species. Even within a species, different variants of hemoglobin exist. Although the existence of genetically controlled polymorphism with regard to hemoglobin in several animal species has been well established but a very few research work has been conducted on dog. Keeping in view the above aspects, the present study in dog was envisaged to investigate the hemoglobin polymorphism in different dog breeds.
Materials and Methods
The present investigation was carried out on a total of 58 apparently healthy dogs belonging to 7 different breeds of adjacent area of West Bengal University of Animal and Fishery Sciences campus, Belgachia, Kolkata, West Bengal, India. The different number of dogs used for this research purpose was Spitz : 21, German Shepherd : 6, Labrador : 9, Mongrel : 8, Golden Retriever : 5, Dalmatian : 4 and Cocker Spaniel : 5.
Blood Sample Collection
To determine the hemoglobin polymorphism about 2ml of whole blood was withdrawn aseptically from the saphenous vein of each of the dog and was kept in vials containing EDTA. The whole blood was mixed thoroughly but gently to avoid coagulation and haemolysis. Then the blood was centrifuged at 6000 rpm for about 15 minutes. When the red blood cells were sedimented at the bottom of the centrifuge tube, then the upper portion i.e. plasma was expelled gently by a glass dropper. Then the red cells were washed three times in normal saline solution (0.9%) to free the red cells from plasma proteins. After that the red cells were collected in a dry, aseptic glass vial and stored in deep freeze at about -200C until until electrophoretical investigation.
Detection of Hemoglobin Polymorphism
For the purposes of genetic characterization of the different breeds of dog, polymorphism of the haemoglobin blood protein was analyzed. The Haemoglobin types were analyzed by vertical polyacrylamide gel electrophoresis (PAGE) in discontinuous tris-citrate buffer system as described by Andrew (1986) with few modifications. Considering that Hb is a stained protein, no specific staining technique was required for analysis of HB types. Depending upon the migration rate as fast, slow and combination of two distinct bands, haemoglobin types are identified as HbAA, HbBB and HbAB, respectively. (Metin and Ceyhan, 2004).
The hemoglobin polymorphism was categorized on the basis of phenovariants. The frequency of genotypes and genes which corresponded to the transferrin genotypes were computed by direct counting method for co-dominant loci.
Results and Discussion
Hemoglobin (Hb) Phenovariants were studied in a total of 58 dogs belonging to 7 different breeds. The electrophoresis picture of different haemoglobin types of dogs and their relative mobility during Polyacrylamide gel electrophoresis were complied in a photograph and have been presented in Fig. No.1. Two different haemoglobin Phenovariants (Hb AB and Hb BB) controlled by one autosomal locus with two codominant alleles namely Hb A and Hb B were observed after Polyacrylamide Gel Electrophoresis (PAGE) according to their mobilities in the gel electrophoresis field. It was observed that Hb BB phenovariant was slower in mobility towards anode and phenovariant Hb AB with one component of Hb B with slower mobility and other component of Hb A with faster mobility. In the present study, it was found that 54 dogs had Hb AB and only 4 dogs with Hb BB type Phenovariant. The more frequent phenovariant was the heterozygous Hb AB type dog that constituted about 93 percent of the total population and remaining 7 percent were Hb BB type dogs. The research result indicated that Hb AB was the most common phenovariant and responsible for haemoglobin types in dogs. The interesting feature noticed in the present study was that Hb AA type was not found at all in the investigated population. But Tanabe et al. (1978) found that Hb AA occurred only in Japanese native breed and Korean dogs. The result indicated that the Hb AA phenovariant was not adapted in this area. The heterozygous Phenovariant Hb AB was observed in all breeds of dog but homozygous phenovariant Hb BB was found only in Mongrel and Dalmatian dog breeds. Metin and Ceyhan (2004) conducted a study on German Shepherd and Labrador dog breeds and found Hb BB genotype in these breeds which is not in agree with this study. The HbBB type phenovariant was also observed in Jindo dogs by Kim et al. (1988), Turkish dog breeds by Metin and Ceyhan (2004) and in Yugoslav Shepherd dogs by Dimitrijevic Vladimir et al. (2005).
The present investigation revealed that only Hb AB and Hb BB type phenovariants were observed in the investigated population. The overall genotype frequency of Hb AA, Hb AB and Hb BB in the population were 0.00, 0.93 and 0.07, respectively. The frequency of heterozygous genotype Hb AB was found to be the highest in the population. The results indicated that the heterozygous genotype was predominant over the homozygous genotype in the investigated population. The cent percent heterozygous genotype Hb AB was observed in Spitz, Labrador, German shepherd, Cocker Spaniel and Golden Retriever dog breed. The frequency of Hb AB genotype in Mongrel and Dalmatian breeds was 0.63 and 0.75, respectively, whereas the frequency of Hb AA genotype in these seven dog breed was zero. It was observed from the result that the genotype
frequency of Hb BB in Spitz, Labrador, German Shepherd, Cocker Spaniel and Golden Retriever dog breed was also zero. But the respective genotype frequency in Mongrel and Dalmatian breeds were 0.38 and 0.25 respectively. The Hb BB type phenovariant was also observed in Jindo dogs by Kim et al. (1988), Turkish dog breeds by Metin and Ceyhan (2004) and in Yugoslav Shepherd dogs by Dimitrijevic Vladimir et al. (2005) which is in agreement with this present finding. But Metin and Ceyhan (2004) conducted a study on German shepherd and Labrador dog breeds and found Hb BB genotype in these breeds which is not in agree with this study.
Table 1: Genotype and Gene frequencies of hemoglobin of different investigated dog breeds.
|Sample size||Genotype frequencies||Gene frequencies|
|Hb AA||Hb AB||Hb BB||Hb A||Hb B|
Figures in the parenthesis indicate the number of dog.
The gene frequency of Hb A and Hb B were estimated and presented in the Table no. 1. The overall gene frequency of Hb A and Hb B in the investigation population were estimated as 0.47 and 0.53 respectively. The gene frequency of Hb A was estimated as 0.50 in Spitz, Labrador, German shepherd, Cocker spaniel and Golden retriever breeds of dog. But respective gene frequency was estimated as 0.31 in mongrel and 0.38 in Dalmatian breed. The gene frequency of Hb B was also estimated as 0.50 in Spitz, Labrador, German shepherd, Cocker Spaniel and Golden retriever breeds of dog. But respective gene frequency was estimated as 0.69 in mongrel and 0.63 in Dalmatian breed. From the result it was observed that Hb B gene was predominant over the HB A allele in the overall population.
The investigated population was characterized by two alleles Hb A and Hb B which produced only two phenovariants namely Hb BB and Hb AB. Another phenovariant HB AA was not found at all. It is suggested that more extensive study based on a large number of observations should be taken up in future on these traits to draw a definite conclusion and its implication in terms of genetic improvement in performance of dogs.
Authors are thankful to the Vice-Chancellor, West Bengal University of Animal and Fishery Sciences, 37 & 68 K.B. Sarani, Belgachia, Kol -37, West Bengal for providing the necessary facilities and co-operation during the course of this study.
Andrew, A. T. (1986). Electrophoresis: Theory, Techniques and Biochemical And Clinical Application. Clavender Press, 2: 79-92
Dimitrijevic Vladimir M., Jovanovic Slobodan J., Savic Mila S., Trailovic Ruzica D. (2007) Genetic polymorphism of blood proteins in Yugoslav shepherd dog. Acta veterinaria. (Beograd), Vol. 57. no.1, 81-86.
Kim, W.K.; Han, B.K; Kim, J.S. (1988). Studies on hematologic values and types of blood protein in [Korean] Jindo dogs, 2. Types of blood protein of Jindo dogs. Korean Journal of Veterinary Research (Korea R.)., 28(2) : 299-305
Metin, Erdogan and Ceyhan, Ozbeyaz. (2004). Investigation of Blood Protein polymorphism and estimation of Genetic Distances in some dog breeds in Turkey. Turk J Vet. Anim Sci., 28: 583-590
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