Analysis of Haemoglobin Polymorphisms of Indigenous Chickens in Borno State, Nigeria

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1 NJAST Nigerian Journal of Animal Science and Technology Nig. J. Anim. Sci. Tech. Vol. 1 (): Department of Animal Science, University of Maiduguri Analysis of Haemoglobin Polymorphisms of Indigenous Chickens in Borno State, Nigeria Abdulraheem A. O., Alade N. K., Aliyu J., Raji, A. O. and Mohammed I. D. Department of Animal Science, Faculty of Agriculture, University of Maiduguri, Nigeria Abstract This research work examined the haemoglobin polymorphism of three indigenous chicken populations (Frizzle feathered, naked neck and normal feathered chickens) in the Central and Southern parts of Borno State, north eastern, Nigeria using cellulose acetate electrophoresis technique. The laboratory analyses were carried out at the Department of Animal Science Laboratory, University of Maiduguri, Nigeria. The genotype frequencies of Frizzle feathered, naked neck and normal feathered chickens for AA haemoglobin were 6.5%, 1.50% and 8.%, respectively. Those of AB haemoglobin were 78.1 %, 81.5 % and 8. % while the values for BB were 15.6%, 6.5% and 8.%. The highest frequencies of BB, AA and AB were observed in frizzle feathered, naked neck and normal feathered chickens, respectively. The genotype frequencies observed in cocks and hens were 1.89 % and 17.0 % for AA; % and 7.4 % for AB, and 9.4 % and % for BB haemoglobin, respectively. Higher frequencies of AA and BB were recorded in hens while BB is in favour of cocks. The gene frequencies of the three strains were 0.45, 0.5 and 0.50 for A haemoglobin and 0.55, 0.47 and 0.50 for B haemoglobin. Naked neck had the highest gene frequency of A, frizzle for B while normal had 50% each for A and B. In the Central and Southern parts of Borno state, AB haemoglobins were predominant for the two sexes with hens having greater frequency. There were more Haemoglobins AA and BB in hens. Thus, the most important Haemoglobin for indigenous chickens in this part of the country is the AB. Key words: Polymorphism, haemoglobin, frizzle feathered, naked neck, cellulose acetate electrophoresis Introduction Globally, poultry is by far the largest group of livestock species contributing about 0 % of all animal protein consumed (Permin and Pedersen, 000; Mengesha and Tsega, 011). In Africa, chicken species contributed around 98% of the total poultry population. (Gueye, 00) and are believed to have originated from Southeast Asia (Hillel et al., 00). The indigenous chickens or native chicken populations was about 10 million in Nigeria (RIM, 199), 85 % of which are found in the northern part (Ajayi et al., 01). These birds among other inherent advantages over their exotic counterparts are hardy, can adapt to rural environment, survive on little inputs and is better in both fertility and hatchability of egg (Ige et al., 01). In addition, they are used for strengthening marriage bond and social relationships of the society (Mengesha and Tsega, 011). The indigenous chickens are characterized by stress factors notably amongst which are the high temperature (Ibe, 199) that by consequence affects animals performance. An attempt to significantly reduce these stress problems through management practices or dietary adjustments have not been successful (Ige et al., 01). Nevertheless, these birds are important reservoirs of useful (major) genes and possess a number of adaptive traits. Among the major genes that have been found potentially useful to the tropical environment are Naked neck (Na) and Frizzle (f) genes. These genes have been associated with heat

2 stress (Horst, 1989). Thus, they serve as a good pool for genetic improvements (Mengesha and Tsega, 011). The need for characterization is a consequence of the potential rate of decrease of genetic variation among farm animals (Ige et al., 01) especially chickens. The loss of genetic variation within and between breeds is detrimental not only from the perspective of culture and conservation but also of utility since lost genes may be of future economic importance (FAO, 000b) and that genetic diversity when lost cannot be replaced (Ige et al., 01). Genetic diversity can be detected at three levels; morphological, biochemical and at DNA level. Advances in biotechnology offer possibilities of improving, utilizing and characterizing present domestic animals diversity. Characterization at biochemical level can assess the genetic diversity within and between livestock and determine genetic relationships among populations (Ige et al., 01). According to Ajayi et al. (01), blood groups and blood proteins have been widely used to characterize animal population, especially haemoglobin. Haemoglobin, a respiratory carrier is found in vertebrate red blood cells, some invertebrates and in the root nodules of legumes (Chineke et al., 007). The molecule is tetrameter of four sub units, each of which has two parts: a polypeptic chain, globin and a prosthetic group, haeme, which is an iron containing pigment that combines with oxygen and gives the molecule its oxygen-transporting ability (Chineke et al., 007). The haeme portion is alike in all forms of haemoglobin, genetic variation is restricted to the structure of the globin portion only (Peters et al., 004). Among other relevance, haemoglobins have revealed more about the molecular basis of human, animal and medical genetics than any other system (Chineke et al., 007). They illustrate mechanisms of forming new genes other than point mutation, cast light on the process of evolution of both the molecular and population levels as well as provide a model of gene action during development (Peters et al., 004). Normal and different variants of haemoglobins are identified by standard laboratory techniques. Cellulose acetate electrophoresis technique is popular for detection of serum protein and its quantitation because of its simplicity and effectiveness in detecting most variants of haemoglobin at alkaline ph (Talen, 009). The objective of this research was therefore to characterize indigenous chickens of the northeastern Nigeria through haemoglobin polymorphism using Cellulose acetate electrophoresis technique. Materials and Methods Study Area The research was conducted in Borno State, Northeastern Nigeria. This State lies between Latitudes N and Longitudes E. A greater part of the state lies on the Chad Formation. Geographically, the state could be divided into broad relief regions; hilly/mountainous area of generally over 600 m above sea level, which cover the southern parts; and plains of generally less than 600 m above sea level which dominate much of the central and northern parts of the State. This makes the rainfall to vary from 700 to 1000 mm in the Southern part and 00 to 500 mm in the North. Three seasons can be identified in the State: the cool dry (harmattan) season (October to January); the hot dry season (February - May); and wet season (June - September). The temperatures are high all year round, with hot season mean temperatures ranging between 9 0 and 40 0 C. (Ayuba et al., 00; Mohammad and Ahmad, 014) Blood collection One hundred adult chickens comprising of three selected strains (normal feathered, frizzle feathered and naked neck) were randomly collected from four Local Government Areas (Jere and Konduga from the Central part; Biu and Chibok from the Southern part) of the state. Blood samples were collected through wing 54

3 veins from these birds and placed in labeled bijou tubes containing Ethylene Diamine Tetra Acetic acid (EDTA) as anticoagulant and refrigerated. Samples were prepared for analysis and the different tubes containing the samples were centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded and samples were rewashed with 10 ml of cold distilled water. Then, m NaCl was added to wash the cell. The supernatant was then discarded and the different sediments were rewashed using ml of cold distilled water. The remaining serum and plasma were centrifuged. Cold distilled water was added to the sediment to re-suspend the cells in order to release the haemoglobin by haemolysis. The lysate was well separated after standing and stored at refrigeration temperature pending electrophoresis (Ajayi et al., 01). Cellulose acetate electrophoresis Cellulose acetate strips were prepared, labeled and thereafter soaked in EDTA borate buffer (ph 8.6) and blotted slightly with filter paper to remove excess buffer. The samples (haemolytes) were impregnated on the cellulose acetate paper using applicator and placed on the electrophoresis tank (CHIBEST electrophoresis tank) with the use of forceps. Tank was powered with the lead closed. The samples were allowed to separate for about minutes. Thereafter, the cellulose acetate papers were blotted dry using filter paper and then dried in an open air for some three minutes and the result was taken. The direct gene counting method as described by Zaragoza et al. (1987) was adopted to score the resulting haemoglobin bands based on the separation of haemoglobin variations: - The presence of a single faster band was designated as AA homozygote; - The presence of a single slower band was designated as BB homozygote; and - The presence of both bands was designated as AB heterozygote. All laboratory works were carried out at the Department of Animal Science laboratory, Faculty of Agriculture, University of Maiduguri, Maiduguri, Borno State, Nigeria. Estimation of genotype and gene frequencies Genotype frequencies were calculated as follows: P= H = Q= Gene frequencies were calculated according to Hardy-Weinberg equation as follows: p = P + ½ H q = Q + ½ H P= Genotypic frequency of allele AA H= Genotypic frequency of allele AB Q= Genotypic and Gene frequency of allele BB p= frequency of allele A q= frequency of allele B N= Total number of individuals sampled = Observed genotype number for AA = Observed genotype number for AB = Observed genotype number for BB Results and Discussion The distribution of haemoglobin types among strains and between sex of indigenous chickens found in Borno State are shown in Table 1. Out of the frizzle feathered chickens sampled, had AA Hb, 5 had AB Hb while 5 had BB Hb genotypes. Between the sexes, male birds had no record for AA Hb but had 18 and, AB and BB Hb, respectively. The female birds on the other hand had AA, 7 AB and BB Hb genotypes. Naked neck strain had, 4 AA, 6 AB and BB haemoglobin genotypes of the birds sampled. Of this, male birds had equal number of Haemoglobin AA and BB (1) and 1 AB while the females possessed AA, 5 AB and 1 BB. Three AA, 0 AB and BB haemoglobin types were obtained among the normal feathered birds sampled. 55

4 Table 1. Distribution of haemoglobin genotypes by strain and sex of indigenous chickens Genotype Sex AA AB BB Total Frizzle feathered Male Female Total Naked neck Male Female Total Normal feathered Male Female Total Males of normal birds were not observed to have AA haemoglobin but 8 AB and BB haemoglobin genotypes. Female birds on the other hand had AA, AB and 1 BB genotypes. In the indigenous chickens of Borno State, three haemoglobin variants were identified with corresponding detectable genotype haemoglobin AA, AB and BB which are under the control of two codominant alleles (HB A and HB B ). This is in conformity with the reports of other researchers who had observed same in chickens and other livestock (Al- Samarrae et al., 010). The detection of these haemoglobin types showed that the population of these fowls is heterogeneous. This thus suggests that no pure breeds of indigenous fowls in Borno State with respect to haemoglobin locus. This is in accordance with Ibe (1990) who reported that chickens in Nigeria consist of two categories: improved and unimproved. The author further explained that the unimproved are pure indigenous breeds while the improved are those derived as a result of controlled random mating between indigenous chickens and improved varieties with no visible destruction. This was further stressed by Sonaiya (1990) that local chickens have crossed to various degrees so much that pure indigenous chickens are rare. The result of this study is similar to the findings of Ajayi et al. (01) conducted on frizzle feathered, naked neck and normal feathered chickens in the Niger Delta region of Nigeria. The genotype and gene frequencies of indigenous chickens of Borno State are presented in Tables and. Frizzle feathered chickens had the highest value of BB genotype (15.6 %). This was followed by normal feathered chickens (8. %) while the least was recorded in naked neck (6.5 %). The highest number of AA genotype was observed for naked neck (1.50 %) while this is followed by normal feathered (8. %) and the least was recorded for frizzle feathered chickens (6.5 %). Normal feathered chickens had highest value for AB haemoglobin (8. %). From this study, the genotype frequency across sexes revealed that male chickens had highest value for AB (88.68 %) but least of AA (1.89 %). In contrary, female chickens had highest gene frequency for AA (17.0 %). The gene frequencies of frizzle feathered chickens were highest for Hb B (0.55), naked neck had highest gene frequency (0.5) for Hb A while normal feathered chicken gene frequency for both Hb A and Hb B is 50:50 (0.50 and 0.50). Between sexes, higher frequency of haemoglobin B gene was recorded in the male chickens while the females had higher frequency for haemoglobin A. 56

5 The diversity observed for both the genotype and gene frequencies is explained by Hardy- Weinberg law which states: Gene and hence genotype frequencies in a large randomlymating population that is not subjected to selection, migration and mutation, remains constant every generation (Ibe, 1998). Table : Genotype frequency of indigenous chickens of Borno State Genotype AA (%) AB (%) BB (%) Total (%) Strain Frizzle feathered Naked neck Normal feathered Sex Male Female Table : Gene frequency of three Borno State indigenous chicken strains Allele A B Total Strain Frizzle feathered Naked neck Normal feathered Sex Male Female The values of genotype and gene frequencies recorded in this study are in line with the report of Ajayi et al. (01) for frizzle feathered, normal feathered and naked neck chickens of the Niger Delta indigenous chickens. These are however different from the values (0.4, 0.77 and 0.68, 0.7) observed by Ige et al. (01) for Yoruba and Fulani ecotype chickens in south western Nigeria. Mazunder et al. (1989) had equally reported varied frequencies of 0.96 (HB A ) and 0.04 (HB B ) for White Leghorn chickens. The discrepancy observed in the results could be attributable to the genetic background of the breeds/chicken types under various studies. Frequency of haemoglobin B was predominant in frizzle feathered chickens while normal feathered chickens were in 50:50. Mazumder et al. (1989) identified only genotype haemoglobin AA in their study. More so, Lee et al. (000) had reported the Korea native chickens to be monomorphic at haemoglobin locus. In another study, Washburn et al. (1971) reported that chickens with homozygous mutant haemoglobin genotypes were approximately 0 % less susceptible to Marek disease. Haemoglobin polymorphism had equally been reported to influence growth rate and hatchability in chickens. Hatchability was reported to be highest in haemoglobin AA, followed by AB and then BB. 57

6 The distribution of haemoglobin genotypes between cocks and hens of Borno State were shown in Table 4. In the Central part of the state (Jere and Konduga LGAs), no AA haemoglobin was observed for either the cocks or hens. On the other hand, 1 AA haemoglobin was detected for cocks and 8 AA for the hens in the Southern part (Biu and Chibok LGAs) of the state. In the Central part as well, 0 AB haemoglobin types were recorded for cocks and 0 (AB) for hens. This is against 15 AB Hb genotypes for cocks and 16 AB Hb for hens in the Southern part. In this study, the BB Hb types followed the same pattern with AA Hb in the Central part as there were no BB Hb genotypes obtained among the chicken populations in this part of the state. However, BB Hb genotypes were recorded for the cocks and 8 BB Hb for the hens. It is observed that haemoglobin AB were predominant in the two sexes (cocks and hens) though hens had greater value of this Hb type than the cocks in both the Central and the Southern parts of the state. Haemoglobin AA and BB were predominant in hens than cocks. The account for the absence of homozygous haemoglobin genotype AA and BB in the Central part could not be unconnected with uncontrolled mating among the chicken populations of this part of the state (Central) and those of the others especially from the Southern part where homozygous haemoglobin (AA and BB) abound. This could have resulted from the influx of people with their chickens and other livestock from other parts of the state to the Central (Jere, MMC and Koduga LGAs) as a result of insurgency. Ajayi et al. (01) reported higher haemoglobin number of AA for female chickens which is in line with the result of this study. The absence of some genotypes in some sexes (cocks) as observed from this study could be an indication that the population is not in Hardy-Wein berg equilibrium. In this study also, hens were seen to have more of haemoglobin AA (8) than the cocks (1) while the cocks were predominantly of AB haemoglobin types. This result showed that sexual dimorphism is not an important factor in the inheritance of haemoglobin. Contrary to this result, Ajayi et al. (01) observed that male chickens were predominantly of BB haemoglobin types in the Niger Delta indigenous chickens. The differences in the results of this study and that of the authors may be linked with environmental difference and genetic drift of the birds in the two regions. Table 4: Distribution of haemoglobin types between cock and hen of Borno State indigenous chickens Genotype Central Southern Total Jere Konduga Biu Chibok AA - - 0(4) 1(4) 9 AB 11(14) 9(16) 8(10) 7(6) 81 BB - - 0() (5) 10 Total 11(14) 9(16) 8(7) 10(15) 100 Values for hens in parentheses Conclusion The indigenous chickens of Borno State, north eastern Nigeria were identified to have three haemoglobin variants with corresponding detectable genotype haemoglobin AA, AB and BB which are under the control of two codominant alleles (HB A and HB B ). Haemoglobin AB is the most important haemoglobin component. The chicken strains and their sexes were detected to be predominant in different haemoglobin types. The indigenous chicken population is not in Hardy-Weinberg equilibrium with respect to sex as some genotypes are absent in cocks indicating that sexual dimorphism is not an important factor in the inheritance of haemoglobin. 58

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