ASSOCIATON OF KAPPA-CASEIN GENOTYPE AND THE LINEAR PARAMETER IN TWO INDIGENIOUS BOS INDICUS AND BOS TAURUS CATTLE IN NIGERIA

Department of Animal Production and Health, The Federal University of Technology Akure (FUTA), Ondo State, Nigeria. Phone: 08033612523 Email: lanremore@gmail.com and or obmorenikeji@futa.edu.ng Phone: 08102620874 Email: ebruphiyoroghate@gmail.com Phone: 08150741301 Email: joshuaadetunbi@gmail.com Phone: 08032321183 Email: olawaleogunshola@gmail.com and or ojogunshola@futa.edu.ng Phone: 08035855410 Email: cliffchineke@yahoo.com and or cachineke@futa.edu.ng


INTRODUCTION
Casein is one of the members of milk proteins family that exists in different molecular forms and is the main protein present in the cow's milk (Alipanah et al., 2005). It is also a member of phosphoproteins family (αS1, αS2, β, κ). Each of the first four caseins (αs1, αs2, β and κ) manifests variability at the level of Phosphorylation and glycosylation.
Phosphorylation is a key factor responsible for the stabilization of calcium phosphate nanoclusters in casein micelles (De Kruif and Holt, 2003;Huppertz, 2013). In the intestine, the protein ingested is divided into a non-dissolved peptide (Para kappa-casein) and a soluble hydrophilic glycopeptide (caseinomacropeptide) (Ageitos et al., 2006).
Caseinomacropeptide has been identified to be responsible for higher digestion efficiency, prevention of newborn hypersensitivity to ingested proteins and inhibition of gastric pathogens (Ageitos et al., 2006). Kappa-casein is responsible for the formation, stabilization and aggregation of the casein colloidal aggregate thereby changing the manufacturing properties and digestibility of milk (Jann, 2004).
Kappa casein is different from other caseins in structure and other properties (Azevedo et al., 2008). Two allelic variants, A and B is seen on exon IV of the bovine kappa casein gene on point mutation (Alipanah et al., 2007).
These variants can be differentiated by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis (Rachagani and Gupta, 2008). Also, CSN3 gene has been seen to be highly polymorphic in bovine and according to recent review of milk protein nomenclature (Caroli et al., 2009). The allele A and B have different amino acid and the positions of these amino acids differs (Alexander et al., 1988). The variation has been associated with processing properties like cheese production (Alipanah et al., 2007) and physiological processes such as antibacterial and cytotoxic effects (Hamza et al., 2010). PCR-RFLP is one of the most frequently used methods for genetic polymorphism studies because it's simple, (Yahyaoui, 2003) and it involves the amplification of a target DNA region including the polymorphic restriction enzyme sites by PCR afterward digesting the amplicon with the respective restriction enzymes. One of the downsides of PCR-RFLP when compared with RFLP analysis is that it's difficult in finding polymorphisms within limited size of the PCR amplified fragments (Agarwal et al., 2008). Over the years, body measurements have been used to interpret growth and production factors, to describe size inheritance and types of breeds or strains, and to estimate weight in beef cattle (Lasisi et al., 2002 andNdumu et al., 2008).
Morphological descriptions have also been used to evaluate breeding goals, to assess type and function and to estimate the animals' value as potential breeding stock (Mwacharo et al., 2006). Linear body measurements (LBM) can be used in assessing growth rate, feed utilization and carcass characteristics in farm animals (Adeyinka and Mohammed, 2006;Yunusa et al., 2013). Linear body measurements are divided into skeletal and tissue measurements (Essien andAdesope, 2003 andHamito, 2009).

BLOOD AND MORPHOMETRIC DATA COLLECTION
Blood samples were collected from a total of 100 healthy animals belonging to two indigenous Bos indicus and Bos taurus cattle. White Fulani (n=50) and N'dama (n=50). Blood was drawn from the ventral region of the tail from each animal into a 10ml EDTA bottle containing (Ethylene Di amine Tetra acetic Acid) as an anticoagulant and kept on ice until transferred to -4oC freezers. Also, data on seven metric traits (Body length, chest girth, withers height, ear length, tail length, leg length and head circumference) were taken in centimeter (cm) on individual cattle with the aid of a tape rule. The Animals were restrained by running them into the crunch and their sexes were also taken into consideration. Measurements was taken early in the morning prior to grazing.

GENOMIC DNA EXTRACTION AND PCR-RFLP ASSAY FOR KAPPA-CASEIN GENOTYPES
The Genomic DNA was extracted from whole blood using the DNA Genomic kit (Bioline) following the manufactural's protocol. DNA fragment was amplified by PCR, using Kappa casein forward primer as K-F:5'-ATAGCCAAATATATCCCAATTCAGT-3' and reverse primer as K-R:5'TTTATTAATAAGTCCATGAATCTTG-3'.
The PCR reaction volume of 50l contains 3μl of genomic DNA, 1μl of each primer, 10μl of NFW and10μl of Master Mix to a final volume of 50μl. the amplification conditions include: pre -denaturation at 94 o C for 4 minutes, annealing temperature at 54.74 o C for 30s, extension of DNA at 72 o C for 30s, final extension at 72 o C for 4minutes,ending at 4minutes to keep it cool and the cycle repeated for 35 times. For genotyping, PCR products were digested with HindIII restriction enzyme which was used for the determination of kappa-casein A and B alleles.

STATISTICAL ANALYSIS
Data analysis was performed to examine the effects of breed, sex and genotype on body weight and linear measurements of the experimental animals with Analysis of Variance (ANOVA) using the General Linear Model (GLM) from the Statistical Analysis Software (SAS vs 9.2).
The statistical model used was: Where Yijkl, is the individual observation, general mean, Bibreed effect, Sj -sex effect, Gk-genotypic effect, BSijeffect of breed and sex interaction, BGik-effect of breed and genotype interaction, SGjkeffect of sex and genotype interaction, BSGijk-effect of breed, sex and genotype interaction and eijkl, is the experimental error.

RESULTS
The genomic DNA extraction protocol used in this study gave a reasonable quality and quantity of DNA. Upon gel electrophoresis on 1.5% agarose gel, sharp high molecular weight bands suitable for PCR-RFLP analysis were observed. Also, PCR amplification using Kappa casein Forward and Reverse primers yielded a high molecular weight band of 530 bpDNA fragment of CSN3 gene from all tested animals. The amplified products from the two indigenous Bos indicus and Bos taurus cattle, after the digestion with HindIII endonuclease generated three different DNA fragments of 530bp, 370bp and 160bp. The restriction digest of CSN3 with HindIII endonuclease reveal three distinct genotypes. Genotype AA with a single undigested fragment of 530-bp, genotype AB has two digested fragments of 370-and 160-bp and genotype BB with three fragments of 530-, 370-and 160-bp. Hence, two kappa casein variants A and B were identified in this study ( Fig. 1-4).
The CSN3 variants detected in this study and their frequencies are presented in Table 1. The genotypic frequencies and gene frequencies of the CSN3 variants varied across the two breeds examined. Genotypes AA and AB were more predominant compared to genotype BB in the two breeds examined in this study. In all, genotypic variant BB was less frequent in the two breeds but appears more frequent in the N'dama breed (0.20) than in the white Fulani breed (0.18) ( Table 1). In White Fulani breed, the genotypic frequency of the BB and AB variants were found to be identical (0.18) and (0.64) for AA variant. While for N'dama cattle has AA (0.52), BB (0.22) and AB (0.26). Also, allele frequencies A and B where found on both breeds. Allele A was predominant than allele B in the two-breed population in this study. The allele frequencies for the White Fulani are A (0.73) and B (0.27) and that of N'dama, A The association of the various genotypes and the body parameters are presented in

DISCUSSION
Many candidate genes have been identified and selected for analysis based on a known relationship with productivity traits. In this study, genetic polymorphisms of the bovine Kappa casein gene were analyzed in two indigenous breed of cattle and their genotypes determination was performed using PCR-RFLP technique in association with their linear parameters. The PCR-RFLP technique has been used to study the frequency of the CSN3 genotypes (alleles) in both meat and dairy cattle (Alipanah et al., 2005;Azevedo et al., 2008). The identification of the variant genotypes of the CSN3 gene in this study was performed through the PCR-RFLP method. Earlier studies have shown some restriction sites for endonucleases HindIII and other restriction enzyme on kappa-casein AA, BB and AB variants and the use of PCR-RFLP techniques for detecting polymorphism in kappa-casein gene (Mitra et al., 1998;Otaviano et al., 2005;Ahani et al., 2006). The three genotypes AA, AB, and BB identified in this study were in accordance to other previous studies reporting the two variant A and B alleles of the CSN3 gene as the two common alleles in Bos taurus dairy breeds (Beata et al., 2008).  (Liron et al., 2002), Holstein fersain dairy cattle (Anggraeni et al., 2010) and Russian Black and Red Pied cattle breed (Alipanah et al., 2005). Significances of body weight, milk yield and body measurements in dairy cattle breeds have been studied by a number of researchers. Most of the investigators reported that larger and longer cows produced higher amount of milk (Yanar et al., 2000). This corresponds to what is observed in this study that the cows with genotype AB and BB (allele B) have longer body length and larger chest girth and as such will produce higher amount of milk which will contain higher milk protein and fat because of the CSN3 B allele that is positively correlated with milk protein and fat. Statistically significant differences were observed in kappa casein genotypes AA, AB and BB. The genotype AA appeared more in the cattle population than AB and BB. Next to genotype AA is the genotype AB and then the Genotype BB which has the least appearance but is the most closely associated with higher milk protein because of the homozygosity of the allele B. this correspond to Denisenko, 2004, work that say the B allele is correlated with higher protein milk content than allele A. Mean values of body measurement studied exhibited sexual dimorphism in favour of Female N'dama cattle, this may be due to physiological condition of the animals as it was not taken into consideration during the conduct of this research work, Females were significantly (P≤0.05) superior to males in most of the body measurement taken, this observation is in line with submission of various workers ((Adeyinka and Mohammed, 2006;Yunusa et al., 2013). Essien and Adesope (2003) and Seifemichael et al. (2014) submitted that the influence of sex on the body weight and some morphometric traits indicate the usual difference between sexes due to hormonal actions leading to different in growth rates. Whereas for the white Fulani cattle studied, the males were significantly (p≤0.05) superior to females in most of the vital body measurement taken and this observation was contrary to the work submitted by Ndumu et al. (2008) and Seifemichael et al. (2014) and this condition may also be due to the physiological condition of the male animals which are known to be more muscular.

CONCLUSION
The PCR-RFLP technique used was informative in distinguishing between the most widely reported CSN3 variants, sequencing of the PCR fragments can also be done to reveal additional CSN3 variants which were not previously identified through restriction digest analysis. The genotype frequency of AA was higher than that of AB and BB in the White Fulani and N'dama cattle population studied and the allele frequency of A was also higher than that of allele B.
However, allele B is positively correlated with milk proteins and as such, the AB and BB genotype identified in CSN3 gene is associated with higher milk protein and cows with AB and BB genotype were seen to have longer and larger and as such will produce higher amount of milk. Therefore, incorporation of these genotypes for CSN3 may help to improve the milk yield and body conformation in the two indigenous Bos indicus and Bos taurus cattle population.
Linear body measurements are important in predicting the contribution of cattle performance in improvement programs and for commercial production purpose. Linear body measurement traits in this study showed that sex is a main source of variation in the two cattle breeds.