Phenotypic variation and protein polymorphism of indigenous sheep breeds in eastern Ethiopia

Helen Nigussie, Sanjoy Kumar Pal¹, Solomon Diriba¹, Yoseph Mekasha², Kefelegn Kebede¹ and Solomon Abegaz³

Ambo University, Department of Animal Science, P.O.Box 19, Ambo, Ethiopia
helennigussie21@gmail.com
¹ Haramaya University, School of Animal and Range Sciences, P.O.Box 138, Dire Dawa, Ethiopia
² International Livestock Research Institute, P.O.Box 5689, Addis Ababa, Ethiopia
³ Ethiopian Institutes of Agricultural Research, P.O.Box 32, Debre Zeit, Ethiopia

Abstract

The current study was designed to assess phenotypic and genetic relationship among indigenous sheep breeds in eastern Ethiopia. Both phenotypic data and blood samples were collected from a total of one hundred and twenty-six adult ewes (42 per breed) and their protein (hemoglobin and serum albumin) polymorphism was investigated using horizontal gel electrophoresis.

A significant (P<0.5) variation was observed in most qualitative characters among eastern Ethiopia sheep breeds. According to multiple correspondence analysis BHS breed was clustered together with black head and white body coat color type, fat rump/thick at the base tail type and having tail shape both cylindrical and straight. Afar breed was closely associated with light brown coat color type and cylindrical and twisted tail shape while HHL sheep population was closely associated with dark brown, black and combination of both colors with white coat color type, having tail shape cylindrical and turned up at the end. Both hemoglobin and serum albumin loci were polymorphic and showed three phenotypes. The frequency of HbA (0.77) was higher in HHL sheep whereas HbB was higher in Afar and BHS sheep (0.70 and 0.67, respectively).The H _E ranged from 0.42±0.09 in HHL to 0.48±0.04 in Afar. The closest genetic relationship was found between the Afar and BHS (D=0.01), while Afar sheep and their HHL counterparts were more distant apart (D=0.22).These result showed that genetic relationship in the three indigenous breeds was associated with their genetic adaptation to the environment/agro ecology. Better diversity indices from two protein loci and its consistency with phenotypic variation showed that protein loci will be a promising tool for genetic diversity study along with phenotypic data in areas where DNA technology is not feasible.

Keywords: multiple correspondence analyses, hemoglobin, qualitative traits, serum albumin

Introduction

The combination of growing demand for animal products in developing world and stagnant demand in industrialized countries represents a major opportunity for livestock production in developing countries, where most demand is met by local production, and this is likely to continue well in the future (Thornton 2010). In Ethiopia, similar to other developing countries, changes in the demand for livestock products have been largely driven by human population growth, income increment and expansion of urbanization. Along with this, large export and domestic market for mutton and live animal has created opportunity for sheep production in Ethiopia. Besides, the strategic location of Ethiopia to Middle East is also an opportunity to export meat (largely from sheep and goats) and live animals to these countries. There are about 27.3 million sheep in Ethiopia, out of which 99.9% are indigenous breeds (CSA 2014).

Sheep play a major role in the food security and social well-being of rural populations living under conditions of extreme poverty (Duguma et al 2010) which is particularly the case for eastern parts of Ethiopia. Afar and Black head Somali sheep (BHS) breeds have been identified as Fat rumped sheep and considered as one breed group out of six breed groups identified so far in Ethiopia (Gizaw et al 2007). Besides, Hararghe highland (HHL) sheep, unidentified population, is also found in the highlands of eastern Ethiopia. A recent morphological characterization has been done on HHL sheep by (Shibabaw et al 2014). According to Gizaw et al (2013), further study will be required to determine the genetic distance between HHL sheep and other breeds in the adjoining areas. This is important to make conservation and improvement related decisions. Even though, the morphological characterization of eastern Ethiopia sheep were conducted for BHS sheep (Fekerte 2008), for Afar sheep (Tesfaye 2008) and for HHL (Shibabaw et al 2014), they were not comprehensive and they were conducted separately. The extent to which these sheep breeds vary genetically has not been documented.

Limited effort has been done to identify genetic diversity between Afar and BHS sheep using microsatellite markers (Gizaw et al 2007), but no attempt has been done to identify genetic diversity of HHL sheep so far. Besides, the genetic relationship of eastern Ethiopia sheep breeds in terms of protein polymorphism (hemoglobin and serum Albumin) and qualitative description is not known. Differentiating the variability of sheep breeds by integrating phenotypic description with protein polymorphism could be a basis for selection and subsequent genetic improvement of farm animals. Biochemical variants of different proteins may present higher accuracy procedures for a better measurement of genetic variation in sheep breeds because of their polymorphism and simple mode of inheritance.

Although DNA-based technologies are now the method of choice for genetic characterization of livestock, protein polymorphisms remain tremendously useful, especially in developing countries like Ethiopia. This is because of their utility, ease, cost, and amount of genetic information accessed, or simplicity of data interpretation (Akinyemi and Salako 2012). The role or potential of these alternatives approach in animal genetic diversity study should not be underplayed since genetic research in Africa is less fully developed than in Europe (Gifford-Gonzalez and Hanotte 2011). Mwacharo et al (2002) reported that for populations whose genetic status is unknown, protein polymorphism may be used first to verify the degree of genetic relationship and to prioritize breeds to be analyzed using microsatellites. Thus, a study was designed to assess phenotypic and genetic relationship among indigenous sheep breeds in eastern Ethiopia using hemoglobin and serum albumin polymorphism and phenotypic description as a basis for further characterization to set up sustainable genetic improvement and conservation program.

Materials and methods

Location of the study area

The experiment was conducted at Animal Genetic Laboratory, School of Animal and Range Sciences, Haramaya University, located 505 km east of Addis Ababa. The site is situated at an altitude of 1980 m.a.s.l, 9 ⁰ 26' N latitude and 42⁰ 3' E longitude. The study area and approximate sampling site for blood sample collection is shown in Figure 1.

Sampling strategy and blood sample collection

Sheep population primarily targeted based on the information from previous genetic diversity study, group discussion with elders and experts at zonal and district level. Major ecological zones and phenotypic distinctness were also considered in sampling. Detailed information about sampling strategy and description of the production system was reported in (Nigussie et al 2015). The major three breeds, agro ecology and production systems were included. Blood sample were collected from a total of 126 matured individual ewes (2-3 individual per flock) from nine different locations (Table 1). Blood samples were collected with plain test tube and kept refrigerated during transportation to Animal Genetics Laboratory of the School of Animal and Range Sciences, Haramaya University for protein polymorphism determinations.

Figure 1. Sampling area (·) for qualitative data and blood sample collection

Qualitative data collection

According to (FAO 2012) general phenotypic characterization guideline, selected qualitative data on coat color, coat pattern, tail type, tail shape were collected (FAO 2012) (Table S1).

Analysis of samples

Serum and erythrocyte (red blood cell) samples were separated from the whole blood by centrifugation (at 3000r/min) for 10 minute. Separate aliquots of serum and erythrocytes were stored at -20°C until they were further analyzed. Gel electrophoresis was carried out on1% agarose gel to analyze inherited biochemical differences in Hemoglobin (Hb) and serum albumin (Alb). Hemoglobin separated by the use of Tris-EDTA-Borate pH 8.6 and Serum albumin separated by the use of Tris-citrate, pH 6.2 (RIKEN 2006). The analysis was done in Animal Genetics Laboratory of the School of Animal and Range Sciences, Haramaya University.

Determination of Hemoglobin (Hb) and Serum Albumin (Alb) types

The identification of Hb phenotypes was achieved depending on the migrating speed of the electrophoretic bands detected from the start line towards the cathodal zone (Agaviezor, et al 2013). After completion of the electrophoretic run the hemoglobin pattern could be read directly on the gel without staining. The Hb polymorphism was pointed out by detection of three migration zones. A single faster band was designated as the AA homozygote (HbAA).The presence of a single slower band was represented as BB homozygote (HbBB), and the presence of both bands was also designated AB heterozygote (HbAB). The identification of Alb phenotypes was achieved similar to that of Hb, however, the albumin bands were visible after staining with amido black. The Alb which presented the fastest migrating speed in the substrate electrophoretic was named as Alb of “fast type” (AlbFF), and the one whose band is closest to the application point of serum samples was designed as Alb of “slow type” (AlbSS). The presence of both types of Alb in the same animal determines the appearance of bands with intermediate visibility after staining with a moving electrophoretic field, representing albumin of “fast/slow” type (Albs)(Hrincă 2009).

Data Analysis

Qualitative data were analyzed using frequency and correspondence analysis procedure of SAS (SAS 2008). Allele frequency for Hb and Alb phenotype were determined by direct count from the phenotype. Allele frequencies of Hb and Alb locus as well as their frequencies of expected genotype were calculated. Chi-square test was done to check whether the loci are within Hardy-Weinberg equilibrium using frequency analysis procedure (SAS 2008). Observed (H _O) and expected (H_E) heterozygosity estimates were calculated using PopGene 1.31 (Yeh et al 1999). Nei’s standard genetic distance (Nei et al 1972) between breeds was calculated.

Result

Qualitative character

Proportion of sheep exhibiting specific descriptor states of qualitative characters observed in the sample sheep breeds of eastern Ethiopia is shown in Table S2. A significant (P<0.5) variation was observed in most qualitative characters among eastern Ethiopia sheep breeds. (Table S2). Most of HHL sheep showed heterogeneity in most characters observed while Afar and BHS sheep breeds were showed uniformity within their color pattern, coat hair type, ear orientation and tail type (Table S2). Majority of BHS sheep were patchy (96.3%), Black head and white body (88.1%), short and smooth hair (85%) while Afar sheep were patchy (61.1), had light brown coat color, short and smooth hair (83.9 %), erect ear orientation ( 91.1%) and fat tail (66.9%) sheep (Table S2). On average, almost half of the observed HHL sheep population exhibited plain coat patterns of which black light brown with white were the most common colors. Most of sheep also had short and coarse hair (79.5%), semi pendulous ear orientation (70.9%) and fat tail (77.9%)(Table S2). A multiple correspondence analysis was carried out on the selected qualitative traits recorded and a bi-dimensional graph representing the associations among the different categories of qualitative traits is presented in Figure 2. About 30.9% of the total variation is explained by the first two dimensions (20.2% by the first and 10.7% by the second dimensions) (Figure 2). On the identified dimensions, BHS breed was clustered together with black head and white body coat color type, patchy coat pattern, fat rump/thick at the base tail type and having tail shape both cylindrical and straight. Hararghe highland sheep population was closely associated with dark brown, black and combination of both colors with white coat color type, having tail shape cylindrical and turned up at the end while Afar breed was closely associated with light brown coat color type and cylindrical and twisted tail shape. On the dimensions identified, sheep populations from HHL and Afar breed were shared some common characteristics (Figure 2).

Figure 2. Bi-dimensional plot showing the associations among the categories of the different qualitative variables

Hemoglobin (Hb) and Serum albumin (Alb) polymorphism

The Hb and Alb allelic and genotypic frequency and expected genotype for three indigenous breed is given in Table 2 and 3, respectively. Hemoglobin showed three phenotypes (HbAA, HbAA and HbBB) in three sheep breeds, but segregating with different frequencies. The frequency of HbA was the most predominant allele in HHL sheep; however, it was lower in Afar sheep. The frequency of HbB was higher in Afar and BHS sheep (Table 2). Serum albumin were also followed the same trend like that of Hb and it showed three phenotypes (AlbFF, Albs and AlbSS) (Table 3). Allele AlbS were occurred relatively at high frequency in Afar sheep (0.63) followed by HHL (0.60) whereas it was lower in BHS (0.52) sheep. Chi square test revealed that indigenous sheep were under Hardy-Weinberg equilibrium for Hb and Alb locus (Table 2 and 3).

Hemoglobin and serum albumin loci were found to be polymorphic in all three sheep breeds. The levels of genetic variability between and within population are given in Table 4. Expected heterozygosity (H_E) estimates within breeds at the blood protein loci analyzed showed that all the three breeds have similar H_E. The observed hetrozygosity (H_O) was lower than the H_E.. A positive correlation was observed between the effective numbers of alleles per locus and mean H_E. Afar and BHS sheep had better effective number of alleles per locus; they also had better mean hetrozygosity compared to HHL sheep (Table 4).

Nei's (1972) standard genetic distances between indigenous sheep breeds are shown in Table 5. Standard genetic distance between sheep breeds pairs was estimated in order to assess the presence of genetic similarity and dissimilarly among the three indigenous sheep breeds. The shortest genetic distance between Afar and BHS was quite low, while HHL sheep is distant from Afar and BHS sheep though the largest genetic distance value was found between Afar and HHL sheep breeds (Table 5).

Discussion

According to the González et al (2011) who clearly demonstrated that morphological (phenotypic) diversity is a good reflector of ecological selection regimes and history of a breed. Besides, Yakubu et al (2010) pointed out that phenotypes are an expression of genetic characteristics, modified by environmental conditions and variance in both genetics and environment may affect phenotypic variance. This is therefore, in the current study, we tried to explore the phenotypic and genetic relationship among eastern Ethiopia sheep using phenotypic description and protein polymorphism as a base for further characterization of indigenous sheep breeds. This preliminary work was reported for the first time for Ethiopian indigenous sheep as it took the case of eastern Ethiopia. Protein polymorphism study together with phenotypic description have become imperative because of its importance in the improvement of farm animals, and the fact that some polymorphic alleles may be connected or linked with traits of economic importance due to pleiotropic effect, or general heterozygosity (Egena and Alao 2014).

Even though there were a significant variation among three breeds of eastern Ethiopia sheep, HHL sheep could not be differentiated as a breed because of high heterogeneity in most traits of observed. This could be supported by the fact that HHL sheep shared some common phenotypic character with Afar sheep and has relatively short genetic distance with BHS sheep. This might be due to gene flow as a result of the proximity of the area to the breeding tract of BHS, Afar and Arisi-Bale sheep breeds. This result is in agreement with the information given by elders during group discussion in different districts. Even though the elders could not be sure about the origin of their sheep, they believed that they might have come from both BHS and Afar sheep or other sheep type because of the farmers’ activity in purchasing sheep from low land and fatten them at highland areas (Nigussie et al 2015). Therefore, marketing system, breeding practice and geographical location of the area could be responsible factors to cause genetic admixture between breeds and sheep type. On the other hand, Shibabaw et al, (2014) indicated that HHL sheep have common attribute with Afar/Adal sheep in their coat color, ear orientation and tail type. The variation in coat color pattern, tail type, body size, shape and conformation observed among sheep breeds in the current result might be as a result of intermixing between sheep breeds or it might be associated with their adaptation to the different agro ecological conditions. A significant association between ecological variation and morphological diversity, particularly variation in quantitative traits and coat color were found among the traditional sheep breeds of Ethiopia (Gizaw et al2007).

Hemoglobin is a blood protein which composed of four subunits, two α-globin subunits and two β-globin subunits, and the interaction between these subunits dictates many oxygen binding characteristics of the protein. Change in blood–O₂ affinity is mediated by structural changes in Hb sub units. Besides, the sub units of the Hb were found in different locations, and the α-globin cluster is located on chromosome 25 while the β-globin cluster is positioned on chromosome 15 (Pieragostini et al 2010). Furthermore, the same authors’ review indicated that the ovine β-globin gene cluster is differently arranged depending on the A or B haplotypes. In the A sheep, the β-globin locus consists of 12 genes, organized as a triplicates, developmentally expressed four-gene set. Sheep with the B haplotype have a locus arrangement consisting of a duplicated four gene set as the consequence of a recent deletion from a triplicates locus. At birth, the A sheep synthesize a juvenile hemoglobin C (HbC), which is produced at birth and exclusively during severe anemia in adults. The B sheep do not synthesize HbC and continue to produce their adult Hb during anemia. This is because B sheep lack the beta C gene as well as three other genes present in A sheep (Pieragostini et al 2010).

High frequency of HbA (0.77) observed in HHL sheep were consistent with the finding of Akinyemi and Salako (2010) who reported high frequency for Hb A in West African Dwarf sheep of Nigeria. Pieragostini et al (2006) observed that Hb A is found more frequently in sheep living above 40^o C latitude. High frequency of Hb A was reported for Ethiopian cattle by Pal and Mummed (2014) that managed under similar environment with HHL sheep. The difference in Hb allele of the sheep has been adduced due to selective advantages in different geographical regions in which the animal finds itself, and possibly has an effect on its performance. This fact has been confirmed by different authors, for instance, Tsunoda et al (2006) reported that Hb A has a relatively high affinity for oxygen and is therefore very important for survival of the sheep in mountainous areas at latitude above 3000 m. On the other hand, relatively high frequency of HbB was found in Afar and BHS sheep (0.70 and 0.67, respectively) is in agreement with the report of Akinyemi and Salako (2012) who showed that Hb B was more predominant with allele frequencies of 0.75, 0.90 and 0.81 recorded for Nigeria sheep (Balami, Uda and Yankasa sheep, respectively). The predominance of Hb B over Hb A in sheep has also been reported by other authors (Mwacharo et al 2002; Boujenane et al 2008; Shahrbabake et al 2010). Sun et al (2007) also showed that sheep with Hb B were better able to withstand the stress associated with acute hypoxia compared to those with Hb A. Besides, Akinyemi and Salako (2012) indicated that high frequency of HbB type sheep have adaptive significance in arid regions which is due to the decreased hematocrit values, low body viscosity and higher availability of water associated with HbB blood type compared with HbA. According to Di Stasio (1997) the variation observed in Hb type which resulted in performance and adaptation difference among individual animals could be due to better functional properties of the Hb molecule concern as a result of greater affinity for oxygen and higher Hb concentration and packed cell volume. The frequency Hb variant observed in the three breeds considered in the current study might be associated with the fact that HHL sheep (high frequency of HbA) were managed under highland/cool environment whereas Afar and BHS sheep (high frequency of HbB) were managed under arid and semiarid lowland areas. Therefore, Hb type of the sheep will contribute to their selective advantage to the agro ecology where they are adapted. Besides, this result confirmed by relatively similar average H_E value and close genetic distance between Afar and BHS compared to HHL sheep.

Protein polymorphism indicates that the analogous protein has two or more genetic variations. It is caused by nucleotide alteration in the DNA chain that results in the substitution of amino acid of polypeptide chain (Lu et al 2006). Analysis of genetic markers based on protein variants detected by electrophoretic methods has been a tool to study genetic differentiation and relationship among breeds and phylogenetic studies (Pieragostini et al 2010; Tsunoda et al 2010). Information on blood protein has also been used to study the genetic relationship among sheep breeds (Tsunoda et al 2006; Shahrabak et al 2010; Akinyemi and Salako 2012).

Both alleles of Hb and Alb loci were polymorphic in all the three sheep breeds. Alleles of polymorphic loci can be used as diagnostic markers to discriminate between breeds though there was no significant hetrozygosity within the three breeds. Estimates of mean H_E obtained in this study were within the recommended range. An average heterozygosity should be between 0.3 and 0.8 in the population to be used in measuring genetic variation (Takezaki and Nei 1996). The present values were higher than the previous results reported for African sheep, indigenous sheep breeds of Kenya (ranged from 0.168 to 0.219) (Mwacharo et al 2002) and Nigerian sheep (ranged from 0.283 to 0.383) (Akinyemi and Salako 2012). Relatively higher number of H _E with two protein loci indicated that the higher number of alleles per locus, the higher the heterozygosity estimates that may be. So that increasing number of protein loci might increase heterozigosity and the usefulness of protein polymorphism in genetic diversity study.

Conclusion

• Generally the results showed that the genetic relationship among three indigenous sheep breeds was due to their genetic adaptation to the environment where the sheep are managed.

• Sampling area has its own limitation (restricted to eastern Hararghe) to give final conclusion about the origin and genetic relationship of HHL sheep. Including more sampling area will help to ascertain the origin of HHL sheep in the future.

• Better diversity indices from two protein loci and its consistency with phenotypic variation showed that protein loci will be a promising tool for genetic diversity study along with phenotypic data in areas where DNA technology is not feasible.

• The current information from phenotypic and genetic relationships among indigenous sheep will  provide baseline information to design cost effective and sustainable genetic improvement programs for eastern Ethiopia sheep breeds. 

Acknowledgments

The authors would like to thank Haramaya University and Swedish International Development Agency (Sida) cooperation for funding this research work. We would like to express our heartfelt gratitude to those smallholder farmers, pastoralists and agro-pastoralists for providing their animal free for blood sample collection and all experts and development agents in the study area for their cooperation for data collection.

Ethical approval

Permission was obtained from the departmental head of the Animal and Range Sciences to carry out the present work. Standard protocol for animal care and welfare was employed during sample collection.

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Appendix tables

Received 19 April 2016; Accepted 16 July 2016; Published 1 August 2016

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