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2012

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English Editor

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2012

Volume 4, Number 2June 2012

Online Version ISSN: 1314-412X

Genetic relationship among Nigerian indigenous sheep populations using blood protein polymorphism

M.O. Akinyemi*, A.E. Salako

Department of Animal Science, Faculty of Agriculture and Forestry, University of Ibadan, Nigeria.

Abstract. This investigetion was carried out to study the genetic relationship among three Nigerian indigenous sheep breeds, namely Balami, Uda and Yankassa using blood protein markers. Blood samples were obtained from a total of one hundred and twenty adult animals (40 per breed) of both sexes, and their protein polymorphism was investigated using cellulose acetate electrophoresis. Three loci (haemoglobin, transferrin and carbonic anhydrase) were examined. Based on the estimated allele frequencies, the populations were characterized for their genetic variability using heterozygosity (H), number of alleles (Na) and genetic distance (D) among the breeds. The estimates of heterozygosity were 0.383, 0.283 and 0.383 for Balami, Uda and Yankassa, respectively. While nine alleles were each reported for Balami and Uda, ten alleles were obtained for their Yankassa counterparts. The closest genetic relationship was found between the Balami and Yankassa sheep (D=0.008), while Uda sheep and their Balami counterparts were more distant apart (D=0.175). The relationship among the three genetic groups was consolidated by the dendrogram that emanated from their genetic distances. The present information may be exploited in formulating appropriate management and conservation strategies for Nigerian indigenous sheep.

Keywords: protein polymorphism, genetic distance, heterozygosity, Nigerian sheep

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 4, No 2, pp 107 - 112, 2012

in animal genetic diversity studies should not be underplayed, Introductionmoreso that genetic research in Africa is less fully developed than in Europe (Gifford-Gonzalez and Hanotte, 2011) and other first world Domestic animal biodiversity is the complex result of countries. Nwacharo et al. (2002) reported that for populations environmental and genetic effects leading to the differentiation of whose genetic status is unknown, protein polymorphism may be morphological, physiological and productive traits which are vital to used first to verify the degree of genetic relationship and to prioritize all production systems as it provides the raw materials for breed breeds to be analysed using microsatellites. Various electrophoresis improvement, and for adaptation to changing circumstances techniques have made rapid progress since Smithies successful (Ceriotti et al., 2003). Characterization of Farm Animal Genetic segregation in 1955 of serum protein using starch gel Resources (FAGR) encompasses all activities associated with the electrophoresis. Protein polymorphism indicates that the analogous identification, quantitative and qualitative description, and protein has two or more genetic variations. It is caused by nucleotide documentation of breed populations and the natural habitats and alternation in the DNA chain that results in the substitution of amino production systems to which they are or are not adapted (Gizaw, acid of polypeptide chain ((Lu et al., 2006). Analysis of genetic 2011). Genetic resources of livestock are threatened by erosion. The markers based on protein variants detected by electrophoretic situation is most alarming for developing countries, where industrial method has been a tool for studying genetic differentiation among breeds often replace local breeds that have unique disease breeds and in phylogenetic studies (Imumorin et al., 1999; Menrad et resistance and adaptations to the local environment (Ajmone-al., 2002; Nyasamba et al., 2003; Ibeagha-Awemu and Erhardt, Marson, 2010). In the absence of information about the genetic 2004a; Camoglu and Elmaci, 2005). Several studies in sheep have attributes of each breed available for a breeding programme, linked different blood protein variants to production traits and development of local breeds is often ignored in favour of the environmental adaptation (Vicovan and Rascu, 1989; Charon et al., introduction of germplasm from exotic breeds, about which more 1996; Darcan and Guney, 2001; Akinyemi and Salako, 2010; Al-information is generally available. One of the strategic priorities of Samarrae and Younis, 2011). Information on blood proteins has also the “Global Plan of Action for Animal Genetic Resources”, adopted in been used to study genetic relationship among sheep breeds Interlaken in 2007, is to inventory and characterize animal genetic (Ibeagha-Awemu and Erhardt, 2004b; Esharatkhah et al., 2007; Sun resources, and to monitor trends and risks associated with them et al., 2009; Shahrbabak et al., 2010).(FAO, 2007). Therefore, characterization of breeds both at the level

Sheep is an important source of meat (Salako, 2006), and plays of animal phenotypes and their interaction with production systems other socio-economic roles especially in the lives of rural dwellers in and at the genetic level is most essential (FAO, 2011). Nigeria. The four well defined sheep breeds in the country are Although DNA-based technologies are now the methods of Balami, Uda, Yankassa and West African dwarf. These differ choice for genetic characterization of livestock (Arora et al., 2011), considerably in size, coat colour and other characteristics. However, several alternative assays, such as protein/ allozyme polymor-only the first three are well adapted to the arid conditions which phisms, remain tremendously useful, especially in developing characterize the northern part of the country (Adu and Ngere, 1979). countries, because of their utility, ease, cost and amount of genetic Their broad genetic variability enables them to survive under information accessed or simplicity of data interpretation (Rege and stressful environmental conditions including high disease incidence, Okeyo, 2006). The role or potential of these alternative approaches poor nutrition which may increase susceptibility to disease, high

* e-mail: larakinyemi@gmail.com

107

108

temperature and traditional husbandry system. Previous studies on Statistical analysisthe variation among these breeds were based mainly on Allele frequencies for protein and enzyme polymorphisms were morphological differences (Salako and Ngere, 2002; Yakubu and determined by direct counting from phenotypes. Heterozygosity (H) Akinyemi, 2010; Yakubu and Ibrahim, 2011). These traits are was calculated as described by Jiskrova et al. (2002). The values of however complex in their mode of transmission and are influenced genetic distances among the breeds were estimated (Euclidean and by the environment thereby underestimating the true levels of Nei, 1973). Cluster analysis dendrogram illustrating the relationship genetic variation. Additionally, morphological characteristics do not between the three genetic groups was obtained using PAST necessarily correspond to the genetic characteristics of blood (Hammer et al., 2001) software. protein and non-protein polymorphisms (Tsunoda et al., 2010). However, biochemical variants of different proteins may present higher accuracy procedures for a better measurement of genetic Results and discussionvariation in sheep breeds because of their polymorphism and simple mode of inheritance. The distribution of the genotypes and the observed alleles and

There is dearth of information on the genetic variability of their frequencies are presented in Table 1 and 2, respectively. A total Nigerian breeds of sheep based on blood protein polymorphism. of 11 alleles were observed in the investigated loci. All loci sampled Therefore, the present study aimed at determining the allele were found to be polymorphic. The highest number of alleles frequencies at haemoglobin, transferrin and carbonic anhydrase loci occurred at Tf locus (7 alleles) and the lowest number at the in order to investigate genetic diversity among the Balami, Uda and haemoglobin and carbonic anhydrase loci (2 alleles each). At the Tf Yankassa sheep breeds in Nigeria. This may provide useful genetic locus A, B, C and E alleles were present in all the breeds with alleles

Binformation essential for developing effective management plans for G and P occurred only in Yankassa and Balami, respectively. Hb S the conservation and improvement of these genetic resources. and CA occurred at high frequency among the three sheep breeds.

TfA was the most frequent in Uda and Yankassa sheep. Mean heterozygosities were similar in the three breeds with

Material and methods 0.383, 0.283 and 0.383 in Balami, Uda and Yankassa, respectively.

AnimalsBlood samples were collected from a total of one hundred and

twenty (120) adult sheep of both sexes by jugular venipuncture. They represented 40 each of Balami, Uda and Yankassa sheep. Samples were collected from unrelated animals reared at the National Animal Production Research Institute, Zaria, Nigeria and a private herd. Animals grazed natural pastures in the morning and were fed local Dussa (by-product of sorghum extraction) in the evenings. They were housed in cross-ventilated pens and had free access to water and mineral salt licks. The routine health management system involved regular de-worming and de-ticking (dip bath). Only pure-bred animals as judged by their phenotypes were considered for the study.

Laboratory analysisBlood was collected from the jugular vein into tubes containing

heparin as the anticoagulant and kept refrigerated during transportation. Plasma and erythrocyte samples were separated from the heparinized whole blood by centrifugation. After centrifugation, red cells were washed three times in saline solution (0.155M NaCl), and lysed with a fourfold volume of distilled H O to 2

release haemoglobin. Separate aliquots of plasma and erythrocytes were stored at 4°C until they were analysed. Gel electrophoresis was carried out on cellulose acetate strips to analyse inherited biochemical differences in the red blood cells and plasma. Three blood protein loci were analysed – Haemoglobin (Hb), Transferrin (Tf) and Carbonic anhydrase (Ca). This involved the use of Tris EDTA Borate pH8.6, Tris Glycine pH 8.4 and Sodium Acetate Trihydrate pH 5.6 for Haemoglobin, Transferrin and Carbonic anhydrase, respectively as described by RIKEN (2006). Resultant gel was stained with Red Ponceau stain to visualize the protein bands. The analysis was done in the Animal Breeding and Genetics Laboratory of the Department of Animal Science, University of Ibadan, Nigeria.

Table 1. Distribution of the genotypes in Balami, Uda and Yankassa sheep breeds

BreedLoci

UdaBalami YankassaGenotype

Haemoglobin

Carbonic

Anhydrase

Transferrin

AA

AB

BB

SS

FS

FF

AA

AB

AC

AD

AE

AG

BB

BC

BD

BE

BP

CC

CD

CE

CP

EE

GD

-

20

20

33

5

2

1

3

-

-

4

-

10

5

-

5

2

4

-

1

2

3

-

-

4

36

26

11

3

14

3

3

3

2

-

5

3

3

-

-

4

-

-

-

-

-

2

11

27

22

12

6

10

6

3

3

1

1

9

2

1

1

-

1

1

-

-

1

1

SCasati et al. (1990) that CA is generally the most occurring allele sheep breeds. However, Ibeagha Awemu and Erhardt (2004)

Sreported a fixation of CA in Yankassa, Uda, Mbororo and West African Dwarf sheep breeds and in Merinoland, EastFriesian Milk

Sand German Grey Heath sheep breeds. The fixation of CA was M Scontradicted by the report of CA and CA on similar breeds

(Missohou, 1999). Data on the polymorphism of this blood enzyme and the advantages it confer is however scarce.

The result of this study showed that all the breeds are highly differentiated at the transferrin locus. From twelve transferrin alleles

Aknown in sheep breeds (Erhardt, 1986), only seven were found: Tf , B C D E G PTf , Tf , Tf , Tf , Tf and Tf controlling 17 genotypes. Six alleles were

found at the transferrin locus in Yankassa and five alleles in Balami and Yankassa. While A, B, C and E alleles were present in all the breeds sampled, P allele was found only in Balami. D allele was found in Uda and Yankassa while G allele was found in Yankassa breed only. The A allele was the most frequent in Uda and Yankassa and B allele in Balami breed. Observation at Transferrin locus are generally difficult to compare with the result obtained in other studies because of the different electrophoresis media used by other researchers and subsequently different resolution power i.e. starch gel and polyacrylamide gel electrophoresis. Missohou et al. (1999)

Genetic distance estimates (derived from allele frequencies) using starch gel electrophoresis could only demonstrate four between each pair of breeds are shown in Table 3. The dendrogram variants of Tf for the Djallonke sheep breed while Ibeagha-Awemu generated based on the genetic distances showed that Balami was and Erhardt (2004) using PAG-IEF clearly resolved six variants for more closely related to Yankassa compared to Uda sheep (Figure 1). the same breed. Results obtained in this study on Uda breeds

This paper provides the first description of the genetic variability agreed with that of Ibeagha Awemu and Erhardt (2004) on the A Pusing blood biochemical polymorphism in Nigerian indigenous frequency of Tf in this breed. The rare Tf observed in only Balami

sheep breeds. The predominance of allele B at the haemoglobin was also reported in Sardi and Beni Ashen sheep breeds in Morocco locus in this study agrees with the report of earlier workers (Clarke et (Boujenane et al., 2008), and is more widely distributed in European al., 1989; Zanoti Cassati et al., 1990, Bunge et al., 1990; Mwacharo sheep breeds (Buis and Tucker 1983; Zanotti Casati et al., 1990).

Aet al., 2002; Boujenane et al., 2008; Shahrbabak et al., 2010) that The present finding on the high frequency of Tf in Yankassa is at BHb is generally the most occurring allele in most sheep breeds. variance with the report of Ibeagha-Awemu and Erhardt (2004) on

CHowever, Schillhorn and Folaranmi (1978) reported that different the same breed, where Tf was reported to have the highest haemoglobin allele types have selective advantages in different frequency in Yankassa. This might be due to varying sample size,

Ageographical regions, while Hb has been shown to have selective differences in management, selection and breeding patterns as well B B Dadvantage in sheep at higher altitudes. Hb occurs more commonly as geographical locations. However, the presence of Tf , Tf and TfE

Bin lowland breeds. In Nigeria, Hb type has a very high frequency in reported in this study was consistent with the findings of Ibeagha-Gsheep of the northern savannah zone, the region in which the breeds Awemu and Erhardt (2004). The occurrence of Tf in Yankassa was

studied are predominantly found. This predominance appears to be also observed in some Moroccan sheep breeds (Boujenane et al., E G Pof adaptive significance in the arid regions to which these breeds fits 2008). Tf , Tf and Tf occurred in the studied breeds, but were totally

particularly well. This is due to the decreased haematocrit values, absent in a report on Kenyan sheep breeds (Mwacharo et al., 2002). lower blood viscosity and higher availability of water associated with It is however important to note that literature on the use of cellulose

B AHb blood types compared to Hb types, which seems to be of acetate electrophoresis method for transferrin analysis is scarce, adaptive significance in habitats characterized by the aridity of the thus the discrepancies observed can only be verified if the samples climate such as the northern zone of Nigeria. This is buttressed by used in other studies are compared with the current samples on the

Athe reports of Tsunoda et al. (2006) that Hb allele has a high affinity same gel.for oxygen and is important for survival in mountain areas at altitudes Mean heterozygosities which are considered as good over 3000 m. measures of genetic diversity were similar in the breeds studied with

SA high frequency of CA in Balami, Uda and Yankassa sheep is values ranging between 0.283 and 0.383, an indication of the similar in agreement with the findings of Bius and Tucker (1983) and Zanotti number of occurring allele in each breed (both Balami and Uda had 9

alleles each while Yankassa had 10 alleles). These values are within the range obtained for sheep breeds using blood protein markers (Clarke et al., 1989; Mwacharo et al., 2002; Ibeagha and Awemu 2004). However, the present values are higher than the range of 0.229-0.259 reported for Iranian fat-tailed sheep breeds (Shahrbabak et al., 2010). The low heterozygosity points to decreasing genetic variability in the Nigerian sheep populations at the three investigated blood protein loci. This may be due to the population structure of the sheep sample (Rychlik et al., 2011) as these animals are kept in small sizes. Similarly, Zahrane et al. (2011)

109

Balami

Uda

Yankassa

***

0.175

0.11

0.015

***

0.08

0.008

0.003

***

Table 3. Genetic distance values among the three indigenous sheep breeds (above the diagonal (Nei's (1973) and Euclidean genetic distance below the diagonal)

Breed

Breed UdaBalami Yankassa

Table 2. Allele frequencies of blood protein variants in Balami, Uda and Yankassa sheep breeds.

BreedLoci

UdaBalami YankassaAlleles

Haemoglobin (Hb)

Carbonic Anhydrase

Transferrin

A

B

F

S

A

B

C

D

E

G

P

0.19

0.81

0.30

0.70

0.42

0.38

0.04

0.09

0.05

0.02

–

0.10

0.90

0.21

0.79

0.49

0.23

0.18

0.08

0.02

–

–

0.25

0.75

0.11

0.89

0.11

0.44

0.20

–

0.20

–

0.05

110

reported that D'ma sheep in Tunisia are distributed in small size 0.283). Among six Moroccan local sheep namely D'man, Beni nuclei in different locations and defect of heterozygotes may Ahsen, Sardi, Timahdite, Beni Guil and Boujaad, Boujenane et al. therefore be probably due to the Wahlund effect. However, the (2008) reported a genetic distance range of 0.006-0.026. Distances current estimates (Balami and Yankassa sheep) fall within the obtained in this study between Yankassa and Uda (0.01, Cavalli recommended average heterozygosity between 0.3 and 0.8 in a Sforza and 0.004, Nei 1973) compared well with 0.012 obtained population (Takezaki and Nei, 1996), for markers to be useful for between the same breeds by Ibeagha and Awemu (2004). The measuring genetic variation. Adebambo et al. (2004) analysed 20 present estimates are also similar to most of the ranges reported by loci and generated 285 alleles using microsatellites markers Tsunoda et al. (2010). The Balami sheep seemed closer to Yankassa however reported higher heterozygosity values (0.57-0.72) among than Uda in the present study. This was consolidated by the Nigerian sheep breeds indicating that the higher number of alleles phylogenetic tree depicting the relationship among the sheep per locus, the higher the heterozygosity estimates that may be breeds. Although Balami and Yankassa are predominantly white in obtained. Thus heterozygosity values obtained with microsatellite colour, which is a pointer to the probable closeness of both breeds, markers are generally higher due to higher levels of polymorphism there might be need to explore more loci to make a justifiable than those obtained with protein markers. statement. The present findings were however contrary to the report

Nei's standard genetic distances obtained in this study were of Adebambo et al., 2004 based on microsatellite DNA within the range reported by Nei (1976) for local breeds. Similarly, polymorphism where Uda sheep was found to be genetically closer Ordas and Primitivo (1986) estimated genetic distance between to Balami than Yankassa. The discrepancy observed may be a Spanish dairy sheep breeds in the range of 0.0094 and 0.055 using reflection of the varying efficiency of biochemical markers and DNA data from 8 loci. Zanotti Casati et al. (1990) using data from four microsatellites in determining the relationship among breeds. Theirs blood groups and six protein coding loci found a distance equal to was however based on fewer animals.0.012-0.060 in five Italian sheep breeds. Qing et al. (2009) in a study on four Chinese breeds reported a range of 0.0164-0.0455 as genetic distance among the breeds. In a similar study using data on Conclusionfive protein-coding loci Mwacharo et al., (2002) obtained a closer estimate of genetic distance between Kenyan sheep breeds (0.044 - The estimates of heterozygosity obtained using information 0.169) than between Kenyan and the exotic Merino sheep (0.044 - from haemoglobin, transferrin and carbonic anhydrase loci were

Dis

tanc

e

4

8

12

16

20

24

28

32

36

40

Figure 1. Dendrogram of the relationship among the three Nigerian sheep breeds drawn from Euclidean genetic distance matrix (pooled for the three loci studied)

UdaBalami Yankassa

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within the range required for measuring genetic variation among the characterisation by Polyacrylamide Gel electrophoresis. Animal three Nigerian sheep breeds. The shortest genetic distance was Genetics 17, 343-352found between the Balami and Yankassa sheep, while that between Esharatkhah B, Safi S, Khaki Z, Khazraei NP and Sadaghian M, Balami and Uda was the longest. However, there is the need for 2007. Study on haemoglobin polymorphism in two breeds of Iranian further characterization employing more blood protein loci in a larger sheep. Journal of Animal and Veterinary Advances, 6, 12, 1426-142.population and complement this with DNA microsatellite information FAO, 2011. Draft guidelines on molecular genetic characterization of for sustainable management and conservation of animal genetic animal genetic resources. Commission on Genetic Resources for

thresources in a hot humid tropical environment of Nigeria. Food and Agriculture, 13 Regular Session, 18-22 July, 2011, Rome. Available at http: //www.fao.org/docrep/meeting/022/am651e.pdf.FAO, 2007. Interlaken Declaration on Animal Genetic Resources. Annex 1. The First International Technical Conference on Animal ReferencesGenetic Resources for Food and Agriculture. 3–7 September, Interlaken, Switzerland.Adebambo O, Williams JL, Blott, S and Urquhart B, 2004. Gifford-Gonzalez D and Hanotte O, 2011. Domesticating Animals Genetic relationships between native sheep breeds in Nigeria based in Africa: Implications of Genetic and Archaeological Findings. on microsattelite DNA polymorphism. Animal Genetic Resources Journal of World Prehistory, 24 1, 1-23.Information, 34, 27- 40. Gizaw S, Komen H, Hanote O, van Arendonk JAM, Kemp S, Adu IF and Ngere LO, 1979. The indigenous sheep of Nigeria. Aynalem H, Mwai O and Dessie T, 2011. Characterization and World Review of Animal Production, 15, 3, 51-62.conservation of indigenous sheep genetic resources: A practical Ajmone-Marsan P and The GLOBALDIV Consortium, 2010. A framework for developing countries. ILRI Research Report 27. global view of livestock biodiversity and conservation–GLOBALDIV. Nairobi, Kenya.Animal Genetics, 41 (Suppl. 1), 1–5.Hammer O, Harper DAT and Ryan PD, 2001. PAST. Akinyemi MO and Salako AE, 2010. Hemoglobin polymorphism Paleontological Statistics Software Package for Education and Data and Morphometrical, correlates in the West African Dwarf Sheep of Analysis. Paleontological Electronica 4 (1) http://palaeo-Nigeria. International Journal of Morphology, 28, 205-208.electronica.orgAl-Samarrae SH and Younis KhH, 2011. Blood polymorphism and Ibeagha-Awemu EM and Erhardt G, 2004a. An evaluation of its relation with sheep production and reproduction. Diyala genetic diversity indices of the Red Bororo and White Fulani cattle Agricultural Sciences Journal, 3, 10-21.breeds with different molecular markers and their implications for Arora R, Bhatia S, Mishra BP and Joshi BK, 2011. Population current and future improvement options. Tropical Animal Health and structure in Indian sheep ascertained using microsatellite Production 38, 431-441.information. Animal Genetics 42, 242–250. Ibeagha-Awemu EM and Erhardt G, 2004b. Genetic variations Boujenane I, Ouragh L, Benlamlih S, Aarab B, Miftah J and between African and German sheep breeds and description of a new Oumrhar H, 2008. Variation at post-albumin, transferrin and variant of vitamin D-binding protein. Small Ruminant Research 55, haemoglobin proteins in Moroccan local sheep. Small Ruminant 33–43.Research 79, 113-117.Imumorin IG, Ologun AG and Oyeyemi MO, 1999. Preliminary Buis RC and Tucker EM, 1983. Relationships between rare breeds observations on effects of hemoglobin genotype and estimate of of sheep in the Netherlands as based on blood-typing. Animal Blood genetic distance at the Hb locus in West African Dwarf and Red Groups and Biochemical Genetics, 14, 17-26.Sokoto goats. Tropical Journal of Animal Science1, 1–9.Bunge R, Thomas DL and Stookey JM, 1990. Factors affecting the Jiskrova I, Glasnak V and Misar D, 2002. The use of blood protein productivity of Ramboillet ewes mated to rams lambs. Journal of polymorphism for determining the genetic distance between the Animal Science 68, 2253-2262.Moravian warm-blooded horse and the Czech warm-blooded and Camoglu G and Elmaci C, 2005. Carbonic Anhydrase (Ca) and X-Trakehner horses. Czech Journal of Animal Science 47, 98-105.Protein(X-p) Types in Turkish Sheep Breeds. Journal of Applied Lu SX, Chang H, Ji DJ, Tsunoda K, Ren ZJ, Ren XL, Sun W, Yang Animal Research, 28, 125-128.ZP and Chang GB, 2006. The levels of genetic differentiation of Ceriotti G, Caroli A, Rizzi R and Crimella C, 2003. Genetic small-tailed Han sheep and Tan sheep populations using structural relationships among taurine (Bos taurus) and zebu (Bos indicus) loci. Agricultural Sciences in China 5, 865-872.populations as revealed by blood groups and blood proteins. Journal Menrad M, Stier CH, Geldermann H and Gall CF, 2002. A study on of Animal Breeding and Genetics, 120, 57–67.the Changthangi Pashmina and the Bakerwali goat breeds in Clarke SW, Tucker EM and Osterhoff DR, 1989. Blood groups and Kashmir: I. Analysis of blood protein polymorphisms and genetic biochemical polymorphisms in the Namaqua sheep breed. Animal variability within and between the populations. Small Ruminant Blood Groups and Biochemical Genetics, 20, 279-286.Research 43, 1, 3-14.Charon K, Lipecka C, Siudek T, Swiderek WP and Skiba E, 1996. Missohou A, Nguyen TC, Sow R and Gueye A, 1999. Blood Relationship between transferrin and globulin antigen Polymorphisms in West African Breeds of Sheep. Tropical Animal polymorphism and sheep resistance to mastitis. Journal of Applied Health and Production, 31,175-179.Genetics 37,2, 161-172.Mwacharo JM, Otieno CJ and Okeyo AM, 2002. Genetic Darcan N and Guney O, 2001. Effects of haemoglobin and variations between indigenous fat tailed sheep populations in transferrin polymorphisms on the performance of Awassi and Kenya. Small Ruminant Research, 44,173-178.crossbred ewes under subtropic environment. Journal of Applied Mwacharo JM, Otieno CJ, Okeyo AM and Aman RA, 2002. Animal Research, 19, 187-192.Characterization of indigenous fat-tailed and fat-rumped hair sheep Di Stasio L, 1995. Biochemical Genetics In: Genetics of Sheep eds in Kenya: Diversity in blood proteins. Tropical Animal Health and Piper, L. Ruvinsky, A. Pp 133-147.Production, 34, 515-524.Erhardt G, 1986. Transferrin variants in sheep: separation and

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Nei M, 1976. Mathematical models of speciation and genetic Veterinary Science, 25, 3, 397-398.distance. In: Karlin, S., Nevo, E. (Eds.), Population Genetics and Shahrbabak HM, Farahani AHK, Shahrbabak MM and Yeganeh Ecology. (Academic Press, New York) HM, 2010. Genetic variations between indigenous fat-tailed sheep Nyamsamba D, Nomura K, Nozawa M, Yokohama K, populations. African Journal of Biotechnology, 9, 5993-5996.Yo.Zagdsuren K and Amano T, 2003. Genetic Relationship among Sun W, Musa HH, Chang H and Tsunoda K, 2009. Comparison of Mongolian native goat populations estimated by blood protein genetic detection efficiency of different markers under the same polymorphism. Small Ruminant Research, 47, 171-181. genetic background. African Journal of Biotechnology 8, 2437-2442.Oni OO, 2002. Breeds and genetic improvement of small ruminants. Tsunoda K, Chang H, Chang G, Sun W, Dorji T, Tshering G, Small Ruminant Training Workshop, held at the National Animal Yamamoto Y and Namikawa T, 2010. Phylogeny of Local Sheep Production Research Institute, Ahmadu, Bello University, Shika, Breeds in East Asia, Focusing on the Bayanbulak Sheep in China Zaria. and the Sipsu Sheep in Bhutan. Biochemical Genetics, 48, 1-12.Rege JEO and Okeyo AM 2006. Improving our knowledge of Tsunoda K, Hong C, Wei S, Hasnath MA, Nyunt MM, tropical indigenous animal genetic resource. Version II, Module 2. In: Rajbhandary HB, Dorji T, Tumennasan, H and Sato K, 2006. Animal Genetic Training Resource Version 2, 2006. Ojango J.M., Phylogenetic relationships among indigenous sheep populations in Malmfors, B. and Okeyo A.M. (eds) International Livestock East Asia based on five informative blood protein and nonprotein Research Institute Nairobi, Kenya and Swedish University of polymorphisms. Biochemical Genetics, 44, 287-306.Agricultural Science, Uppsala, Sweden. Vicovan G and Rascu D, 1989. Types of haemoglobin in sheep RIKEN, 2006. Available at http://www.riken.go.jp/engn/r- related to environmental adaptation. Archiva Zootechnica, 1, 33-44.world/research/lab/index.html. Yakubu A and Akinyemi MO, (2010) An evaluation of sexual size Rychlik T, Pakulski T and Krawczyk A, 2011. Monitoring genetic dimorphism in Uda sheep using multifactorial discriminant analysis. variation in a conservation flock of Coloured Merino sheep based on Acta Agriculturae Scandinavica A-Animal Science 60, 74-78. blood group and protein polymorphism. Annals of Animal Science Yakubu A and Ibrahim IA, 2011. Multivariate analysis of 11, 41-52. morphostructural characteristics in Nigerian indigenous sheep. Salako AE, 2006. Application of morphological indices in the Italian Journal of Animal Science 10, 83-86. assessment of type and Function in Sheep. International Journal of Zanotti Casati M, Gandini GC and Leone P, 1990. Genetic Morphology, 24, 13-18. variation and distances of five Italian native sheep breeds. Animal Salako AE and Ngere LO, 2002. Application of Multifactorial Genetics, 21, 87-92.discriminant analysis in the morphometric structural differentiation of Zharane K, Boulbaba R, Brahim H, Lazher Z and Sami S, 2011. West African Dwarf (WAD) and Yankassa sheep in South West Blood protein polymorphism in three sheep breeds from the south of Nigeria. Nigerian Journal of Animal Production 29, 2, 163-167. Tunisia. Research Opinions in Animal and Veterinary Sciences, 1, Schillhorn van Veen TW and Folaranmi DOB, 1978. The 69-73.haemoglobin types of northern Nigeria sheep. Research in

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp.

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Volume 4, Number 2June 2012

CONTENTS

Genetics and Breeding

Nutrition and Physiology

Production Systems

Agriculture and Environment

Product Quality and Safety

Polymorphism of the C3 component of the complement system and its impact on serum lysozyme concentrations and complement activityTs. Koynarski, L. SotirovUse of cluster analysis for selection of quality at the early stages of wheat breedingI. Stoeva, I. Belchev, K. Kostov, I. Ivanova, E. PenchevGenetic relationship among Nigerian indigenous sheep populations using blood protein polymorphism

M. Akinyemi, A. SalakoEffect of melatonin treatment on fertility, fecundity, litter size and sex ratio in ewesG. BonevThe effects of productive status and age on blood serum parameters before oestrous synchronisation in Awassi and Awassi crosses sheep breedG. Bonev, R. Slavov, S. Georgieva, P. Badarova, S. Omar

Chemical composition and nutritive value of fungal treated acha strawA. Akinfemi Carcass traits, intestinal morphology and cooking yield of broilers fed different fermented soyabean meal based dietsM. M. Ari, B.A. Ayanwale, T. Z. Adama, E.A. Olatunji

Possibilities for application of plant promotor Immunocitofit in integrated pepper productionS. Masheva, V. Todorova, G. ToskovStudy on vegetative and generative manifestations as well as yield from glasshouse tomatoes when foliar fertilizers are appliedN. Valchev, S. MashevaPlant species screening for biofumigant activity against soil-borne pathogens and root-knot nematodesS. Masheva, V. Yankova, G. Toskov

Water quality assessment from own source at poultry farm located in rural region in South BulgariaR. Stefanova, G. Kostadinova, N. GeorgievaInfluence of different factors on tannins and flavonoids extraction of some thyme varieties representatives of thymol, geraniol and citral chemotypeG. Zhekova, D. PavlovIdentification of pathogenic races of wheat common bunt using differential lines in Lorestan provinceZ. Noruzi, V. Mahinpoo, M. Mohamedi, F. Fayazi, F. Jalali, A. Sohilinejad, M. DarvishniaReview of species of the phylum Acanthocephala from the Middle-Bulgarian RegionZ. Dimitrova, N. DimitrovaEffect of experimentally polluted water on the morphological characteristics on the leaves of two varieties of Triticum aestivum L. grown on different soil types K. Velichkova, D. Pavlov, D. NinovaEvaluation the relative resistance of 14 corn cultivars to Fusarium maize rot infection L. Ashnagar, S. Rezaei, M. Darvishnia, M. Zamani

Modeling of spectral data characteristics of healthy and Fusarium diseased corn kernelsTs. Draganova

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