This experiment was designed to assess within-ecotype genetic diversity at the haemoglobin (Hb) locus in the Tiv local chickens of Nigeria in Makurdi. Eighty-two (82) blood samples were collected from each bird using needles and vials (SARSTEDT Monovette®). Haemoglobin genotypes were determined using cellulose acetate electrophoresis. Data were analyzed using Population Genetics Simulation (PopGen.S2) software. The result revealed three haemoglobin genotypes (HbAA, HbAB and HbBB) controlled by two co-dominant haemoglobin alleles HbA and HbB in the Tiv local chicken population in Makurdi. Genotype frequencies of HbAA, HbAB and HbBB were 0.44, 0.32 and 0.24, respectively, in the entire chicken population. The gene frequencies of HbA and HbB observed in this study were 0.60 and 0.40, respectively in the entire population. There was moderate heterozygosity (0.48) at the haemoglobin locus in the local chicken population in Makurdi, which is an advantage to the local chicken population.
Nigerian local chickens are found in different parts of the country, including Benue state. The local chickens in Benue state of Nigeria are commonly called the Tiv local chickens of Nigeria, since the state is predominated by the Tiv tribe, hence the major keepers. Local chicken meat and eggs are preferred over that of exotic chickens (Dessie and Ogle, 2001). The Nigerian local chickens constitute about 80 per cent of the 120 million poultry found in Nigeria (Fayeye et al., 2005). Almost every family in rural communities cheaply raises the Nigerian local chickens as source of protein (meat and egg) and income, under extensive system of management across the country. They are left to feed on household leftovers and often move about as scavenging birds. In some places, they are provided with little or no shelter at night. Nigerian indigenous chickens constitute an indispensible Animal Genetic Resource (AnGR) to the country. The uncontrolled distribution of exotic chickens in different parts of Nigeria, coupled with uncontrolled crossbreeding of exotic chickens with Nigerian indigenous chickens leads to exchange of genetic materials between the two genetic populations. Consequently, this trend gradually dilutes the indigenous chicken genetic stock, and if it is not controlled, the Nigerian indigenous chicken gene pool could be lost in the future before they are fully characterized genetically. This is supported with the FAO report (FAO, 1999) that animal genetic resources (AnGR) in developing countries are being eroded. Nigerian local chickens are widely distributed in different geographical/ecological zones as classified by vegetation types such as rainforest zones of Southeast, South-west and South-south; Savannah zones of North-central, Northeast and Northwestern Nigeria. The Nigerian local chickens found in each of the geographical zones are believed to constitute different genetic population with limited inter-population gene flow, which could be attributed to long distances separating them. These local chickens may have evolved pronounced adaptabilities in response to combined influence of locally prevailing environmental conditions, uncontrolled breeding as well as forces of natural selection, mutation and random genetic drift. It is based on this background that some researchers have come up with phenotypic classification of Nigerian local chickens ecologically into different ecotypes, for example Fulani ecotype, Yoruba ecotype, heavy and light ecotypes, Tiv ecotypes etc. categorically, Sonaiya and Olori (1990) characterized Nigerian indigenous chickens based on ecotypes. Momoh et al. (2010) grouped Nigerian local chickens based on body size and body weight into heavy ecotype (HE) and light ecotype (LE). The heavy ecotype represents local chickens from dry savannah (Guinea and Sahel Savannah), Montane region and cattle kraals of the North whose mature body weights range from 0.9 – 2.5kg. The light ecotype represents local chickens from the swamp, rainforest and derived savannah zones with mature body weights of between 0.68 – 1.50kg.
However, characterization of Nigerian local chickens based on their mean phenotypic values provides only a crude estimate of the average effects of the functional variants of genes possessed by the local chicken genetic resource. A better approach to characterization of Nigerian local chicken populations is the use of molecular genetic techniques. Our central hypothesis is that genetic analysis of Nigerian local chicken populations using molecular genetics techniques could uncover genetic polymorphism existing among the local chicken populations across different regions in Nigeria. If such polymorphisms at the level of the gene or DNA are uncovered, they can be exploited in marker-assisted selection (MAS) and gene-assisted selection (GAS) for the purpose of genetic improvement of Nigerian local chickens. Genetic characterization is also useful to help conserve the valuable genetic variants inherent in the local chicken genetic resource. Perhaps, a preliminary approach to molecular characterization of local chickens is the use of blood protein polymorphisms such as haemoglobin polymorphism. Haemoglobin is a complex Iron containing conjugated protein, globin, with four polypeptide chains and each of the four chains units with ‘heme-group’ to produce haemoglobin molecule (Bhattacharyya, 2015). Haemoglobin is responsible for transporting oxygen (O2) and carbon (iv) oxide (CO2) in the blood of vertebrates. Haemoglobin has been one of the most studied polymorphisms in vertebrate species since the infancy of both the population and evolutionary genetics (Pieragostini et al., 2010). Haemoglobin polymorphism had been investigated in some farm animal species in Nigeria. For example, Akinyemi and Salako (2010) reported two haemoglobin genotypes HbAA and HbBB in West African Dwarf Sheep in Southwest Nigeria. Essien et al. (2011) found three haemoglobin genotypes HbAA, HbAB and HbBB in the Bunaji cattle in Zaria, Nigeria. Akinyemi et al. (2014) investigated haemoglobin polymorphism in Mallard and Muscovy ducks and reported three haemoglobin genotypes HbAA, HbAB and HbBB controlled by two co-dominant alleles HbA and HbB. A similar result was reported by Oguntunji and Ayorinde (2015) with HBAA being the prevalent genotype across three ecotypes of locally adapted Muscovy duck (Cairina moschata) in Nigeria. Some researchers have attempted genetic diversity study of Nigerian local chickens in different regions of Nigeria using haemoglobin polymorphism. For example, Salako and Ige (2006) reported two haemoglobin genotypes HbAA and HbAB in Nigerian indigenous chickens in Southwestern Nigeria. Yakubu and Aya (2012) reported three haemoglobin genotypes HbAA, HbAB and HbBB in Nigerian indigenous chickens in Nasarawa state. Ajayi et al. (2013) found three haemoglobin genotypes HbAA, HbAB and HbBB in Nigerian indigenous chickens in Niger Delta region, with HbBB as the most prevalent, while HbAA was the rarest.
There is need to assess the genetic diversity of Nigerian local chickens in Benue state. A preliminary approach to genetic diversity study of Nigerian local chickens in Benue state is the use of blood protein polymorphism. Therefore, the objective of this study was to assess the genetic diversity at the haemoglobin locus in the Tiv local chickens in Makurdi, Benue state.
Materials and Methods
Location of Study
The study was carried out in Makurdi area of Benue state, Nigeria. Benue state lies within the Guinea Savannah region of middle belt of Nigeria. It geographic coordinate are longitude 7° 471 and 10° 01 East; latitude 6° 251 and 8° 81 North. The area is characterized by two seasons – a period of dry season from October to March, and a period of rainy season from April to September. Annual rainfall ranges from 973 mm to 1324 mm.
Blood Sample Collection
Fresh blood samples were collected individually from 82 local chickens on free range in Makurdi, Benue state. About 1ml of blood was collected from the brachial vein of each bird using needles and vials (SARSTEDT Monovette®). The samples were properly labeled according to the sex and location of the birds. Haemoglobin genotyping was carried out using the facilities at TOSEMA Laboratory in High-level, Makurdi, Nigeria.
Determination of Haemoglobin Genotypes
Haemoglobin genotypes were determined using cellulose acetate electrophoresis method. About 0.5ml of whole blood was placed into a centrifuge tube and spun for 30 minutes. Then, 10mls of cold 0.155M NACl was added to wash the red cells. Then, about 4 volumes of Hb-Genotype lysing fluid was mixed with 1 volume of saline washed packed red cell in a clean test tube, and allowed to stand for 20minutes. Equal volumes of Tris-EDTA-borate buffer with pH between 8.5 – 8.6 was added to the anode and cathode compartment of the electrophoresis tank. Cellulose acetate strips (77 x 150mm) were prepared and labelled. They were soaked in Tris EDTA-Boric acid buffer (TEB) at a pH of 8.6 and blotted slightly with a filter paper to remove excess buffer. 5ml of each of the haemolysate samples (tests and controls) was transferred into the well plate, carefully applying the haemolysate samples including controls on a slightly blotted strip using an applicator. The strip was placed on a bridge and then lowered into the compartments containing the buffer in the electrophoresis tank. It was allowed to run at 300V for 30 minutes. After completion of the electrophoresis run, the haemoglobin patterns, in order of motility were detected (HbAA, HbAB and HbBB) directly without drying or staining. The direct gene counting method was used to score Hb bands based on their mobility. Human control haemoglobin (Hb-AA and Hb-AS) were used to develop chicken haemoglobin control for the 82 blood samples drawn from the Tiv local chickens.
Data on haemoglobin genotype numbers were analyzed using Population Genetics Simulation (PopGene.S2) Software, to estimate the gene and genotype frequencies at the haemoglobin locus. Data were also subjected to Chi-square test using PopGene.S2 software. Estimates of Heterozygosity were calculated according to Nei (1978) as shown by the expression below-
Heterozygosity (He) = 1 –
Where, n = number of loci; Xi = frequencies of the ith alleles
Results and Discussion
The gene and genotype frequencies at the haemoglobin locus in the Tiv local chickens in Makurdi are shown in Table 1, while observed and expected numbers of haemoglobin genotypes in the Tiv local chicken cocks are shown in Table 2.
Table 1: Gene and genotype frequencies at the haemoglobin locus in the Tiv local chickens in Makurdi
|Sex N||Genotype Number||Genotype frequencies||Allele frequencies|
|HbAA HbAB HbBB||HbAA HbAB HbBB||HbA HbB|
Table 2: Observed and expected number of haemoglobin genotypes in the Tiv local chicken cocks in Makurdi
|Hb genotypes||Observed||Expected||X2 df=1|
** (P< 0.05)
The observed and expected numbers of haemoglobin genotypes in the Tiv local chicken hens are shown in Table 3.
Table 3: Observed and expected number of haemoglobin genotypes in the Tiv local chicken hens
|Hb genotypes||Observed||Expected||X2 df=1|
The observed and expected numbers of haemoglobin genotypes in the entire sample of the Tiv local chickens are shown in Table 4, while the estimated heterozygosities at the haemoglobin locus in the Tiv local chickens are shown in Table 5.
Table 4: Observed and expected number of haemoglobin genotypes in the Tiv local chickens
|Hb genotypes||Observed||Expected||X2 df=1|
Table 5: Heterozygosities at haemoglobin locus in the Tiv local chickens in Makurdi
N = sample size
This study revealed the presence of two haemoglobin alleles HbA and HbB in the Tiv local chickens. Thus, three distinct haemoglobin genotypes HbAA, HbAB and HbBB, which are controlled by the two co-dominant alleles HbA and HbB were observed in this study due to their banding patterns. The most frequent haemoglobin genotype observed in this study was HbAA with genotype frequency of 0.44, while the genotype frequencies of HbAB and HbBB were 0.32 and 0.24, respectively, in the entire chicken population irrespective of sex. The gene frequencies of HbA and HbB observed in this study were 0.60 and 0.40, respectively, with HbA allele being the most prevalent in the entire population.
Chi-square test showed that the haemoglobin locus in the Tiv local chickens in Makurdi was not in Hardy-Weinberg equilibrium. This implies that the observed and expected genotype frequencies at the haemoglobin locus in the Tiv local chickens in Makurdi were not in Hardy-Weinberg proportion. This could be due to systemic forces such as migration, selection etc., as well as the size of population sampled. The estimated heterozygosity in the entire population was 0.48. Heterozygosity is a measure of genetic diversity in a population. The heterozygosity value of 0.48 observed in this study is an indication of moderate degree of genetic diversity at the haemoglobin locus in the Tiv local chickens in Makurdi area of Benue state.
The three haemoglobin genotypes observed in this study were similar with the findings of Ige et al. (2013) in Yoruba ecotype chickens and Fulani ecotype chickens in Nigeria. Similar finding was reported by Yakubu and Aya (2012) in normal feathered, naked neck and Fulani ecotype Nigerian local chickens in Nasarawa state, Nigeria. Ajayi et al. (2013) also reported a similar result for Nigerian local chickens in Niger Delta region of Nigeria. The predominance of HbAA genotype over HbAB and Hb BB genotypes observed in this study was in line with the findings of Yakubu and Aya (2012) who reported higher genotype frequencies of 0.54 (HbAA) and 0.62 (HbAA) in normal feathered and Fulani ecotype chickens respectively, than HbAB and HbBB. However, Ajayi et al. (2013) found HbBB most prevalent in Nigerian local chickens in Niger Delta region with frequency of 50% (HbBB) as compared to the other genotypes HbAA (20.37%) and HbAB (26.32%). The prevalence of HbA allele in the Tiv local chickens of Nigeria, in Makurdi was in agreement with the findings of Al-Samarrae et al. (2010) who reported preponderance of HbA allele in White Leghorn (0.65) and native Iraqi chickens (0.54). However, the estimated heterozygosity (0.48) at the haemoglobin locus obtained in this study was higher than the value (0.41) reported by Yakubu and Aya (2012) for Nigerian local chickens in Nasarawa state, Nigeria.
Genetic diversity within and among populations occurs if there are differences in allele and genotype frequencies between those populations The moderate genetic diversity at the haemoglobin locus is an advantage to the local chicken population since genetic diversity provides a means for populations to adapt to changing environmental conditions. Therefore, the Tiv local chickens of Nigeria need to be further evaluated to ascertain the effect of haemoglobin polymorphism on the productive performance of the chickens.
It could be observed from this study that the haemoglobin locus in the Tiv local chickens of Nigeria is controlled by two co-dominant alleles HbA and HbB, which led to the existence of three haemoglobin genotypes – HbAA, HbAB and HbBB in the Tiv local chickens of Nigeria. The most frequent haemoglobin allele in the Tiv local chickens in Makurdi is HbA. The moderate genetic diversity at the haemoglobin locus is an advantage to the population of the Tiv local chickens of Nigeria, since heterozygosity serves as a means for a population to adapt to changing climatic conditions.