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International Journal of Genetics 7(2): 46-52, 2017ISSN 2222-1301 © IDOSI Publications, 2017DOI: 10.5829/idosi.ijg.2017.46.52

Corresponding Author: Manal M. El-Bramony, Buffalo Breeding Research Department, Animal Production Research Institute,Agricultural Research Center, 12618, Dokki, Giza, Egypt. E-mail: [email protected].

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Variation in Milk Composition Traits Associated withPolymorphisms in Kappa-Casein and -Lactoglobulin Genes and

Mastitis Incidence of the Egyptian Buffalo

Manal M. El-Bramony and M.A. Abo-Farw

Buffalo Breeding Research Department, Animal Production Research Institute,Agricultural Research Center, 12618, Dokki, Giza, Egypt

Abstarct: Determining the genetic polymorphisms of milk protein profile in Egyptian buffalo population wouldbe of great importance in animal breeding schemes and dairy industry. Identification of polymorphisms inkappa-casein ( -CN) and -lactoglobulin ( -LG) genes in the presence of infection status and analysis theirassociation with milk composition traits are the objectives of this study. Composite milk samples were collectedmonthly from buffalo cows in experimental herds and analysed including percentages of fat, protein, casein,whey protein and lactose and cultured for bacteria presence. PCR-RFLP method was performed for digestionthe amplified DNA fragments of -CN and -LG genes with restriction endonucleases HinfI and HaeIII in thepopulation studied. The restriction digestion of these fragments showed the existence of only one allele A: forboth -CN and -LG genes, with all buffalo cows studied being homozygous for these genes. Moreover, amonomorphic banding pattern demonstrated the presence of allele B of -CN though -LG was not detectedamong the tested buffaloes. No association between infection status and polymorphisms at the -CN and -LGgenes was found in studied buffalo cows. Statistical analysis of dataset was performed using mixed linearmodel. Fixed effects of test-day, herd-season of calving, infection status and parity were included, in addition,days in milk was analysed as a covariate. Within buffaloes, significant differences were detected in milk, protein,casein, whey protein, lactose yield and casein content.

Key words: Egyptian Buffalo Kappa-Casein -Lactoglobulin Infection Status Milk Composition Traits PCR-RFLP

INTRODUCTION 80% and divided into main four groups: -CN, -CN, -

Milk is one of the most important food products for as micelles [6]. -lactoglobulin is the major whey proteinhuman consumption in several countries. Thus, the of ruminants [7]. Several studies [2, 8-11] on dairy cattlepossibilities of modifying milk constituents by breeding stated that an association between genotypes of -CNtechniques and managerial practices have been done in and -LG genotypes with individual variations in milkdairy populations [1]. In the last decades, the use of protein composition and properties of dairy products. Inmarker data to improve breeding schemes for quantitative general, results on the effect of -CN and -LG genotypestraits such milk composition traits has been of great on milk yield traits have been conflicting, probably due tointerest. Genetic markers, implies the most likely use of breed, genetic differences within species or populations,molecular data as additional tool could be used to early the different managerial and protocols of analysis. In dairypredict future animal performance. Protein profile has been cattle, -LG and -CN genes have two common geneticfound, in particular, milk casein content to be associated variants, A and B [12-14]. Because of the economicwith many aspects of dairy industry and one of the major important of mastitis, the search for associations betweendeterminants of income to dairy farmers [2-5]. Caseins are mastitis resistance and susceptibility with a variety ofmajor content of milk protein comprise approximately 78- polymorphisms often in candidate genes or genetic

s1 s2

CN and -CN, forming supra-molecular structures known

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regions has received considerably more attention. Subclinical mastitis is one of the most common and costlydiseases in dairy farms worldwide. Although buffalo hasbeen traditionally considered less susceptible to mastitisthan cows, there was a slight increase in incidence of subclinical mastitis i.e. 13.75 and 25.58 % on animal level andquarter of Murrah and Nili-Ravi populations [15], leadingto decreased milk yield. In addition, the milk losses andthe disease results in changes in levels of specific milkcomponents [16, 17]. In Egypt, buffalo is mainly reared formilk production. The chemical composition of buffalo milkvaries from that of the milk of different species. Buffalomilk is usually consumed fresh according to the demandof the Egyptian domestic market ranging between 55 and80%. The objectives of this study were to identify thegenetic polymorphisms in -CN and -LG genes in thepresence of infection status and analysis their associationwith milk composition traits.

MATERIALS AND METHODS MilkoScan (Foss, Hilleröd, Denmark) milk analyzer. On

Experimental Population: The current study was carried analysed: fractions of nitrogen: total nitrogen (TN),out from twenty-one lactating buffalo daughters of 13 nitrogen soluble (SN). Then the parameters weresires and 21dams in experimental herds belonging to the calculated as follows: whey protein (SNx6.38/1000), caseinAnimal Production Research Institute (APRI), Agricultural ((TN-SN) x6.38/1000). Means and standard deviations ofResearch Center (Mehalet Mousa, Kafr El-Sheikh daily milk composition traits in composite milk samplesGovernorate, Egypt) during year of calving 2016. Buffalo from healthy and infected buffalo cows are presented incows were housed in semi-open sheds and were kept Table 1.under the regular systems of feeding and management Prior to milking, teat ends of sampled were sanitizedadopted by APRI. Ration given to the animals were using 70% ethanol. First streams of foremilk weredetermined according to their live body weight and level discarded and then 15-ml of milk was collected fromof milk production. Mineral salt and vitamins were offered aseptically each teat into sterile tubes. To minimize effectregularly. The ration was offered twice daily and clean of stage of lactation or incidence sub-clinical mastitis,water was available all the time. Buffalo cows were hand- mammary quarter foremilk samples from buffalo cows canmilked twice daily with recorded the quantity of milk be performed through mid-lactation stage. Milk samplesproduced by individual buffaloes. were tested by California Mastitis Test (CMT).

Buffalo cows were to be dried two-months before Bacteriological culturing of each milk sample weretheir expected calving dates. Dry off treatment is practiced performed according to standards of the National Mastitisfor lactating females in the herds. Drugs against diseases Council [18]. Sub-clinical mastitis was defined as theand parasites were applied twice a year. Buffalo cows presence of Staphylococcus aureus, Coagulase Negativeincluded in the current study were selected from the first Staphylococci and Corynbcateria spp in the same milkto the seventh lactations. sample.

Milk Samples and Bacteriological Assay: Test day were selected for 11 healthy and 10 infected buffalo cows.records from the first seven lactations between 5 and There was no abnormal udder or milk that indicated the270 days in milk (DIM) were used. TD records/ lactation infection with clinical mastitis among buffalo cowswere classified according to DIM into ten test-days studied.The following: daily milk composition traits of milk(TD1 to TD 9). Daily milk yield was recorded and fat yield (MY), fat (FY), protein (PY), casein (CY), wheyand protein and lactose content were determined on protein (WY) and lactose (LY) in kilogram and theircomposite milk samples of the two milkings with a contents were evaluated.

Table 1: Descriptive statistics of test-day milk composition traits fromhealthy and infected buffalo cows.

Healthy [n=11] Infected [n=10]------------------- --------------------

Trait Mean SD Mean SD3

Milk yield, kg 5.69 2.61 5.12 2.13Fat content, % 4.78 1.51 4.75 1.23Fat yield, kg 0.27 0.14 0.24 0.13Protein content, % 3.45 0.46 3.42 0.40Protein yield, kg 0.20 0.09 0.18 0.07Casein content, % 3.20 0.38 2.94 0.12Casein yield, kg 0.16 0.07 0.14 0.05Casein number 79.20 0.02 79.00 0.021

Whey protein, % 0.76 0.12 0.83 0.16Whey protein yield, kg 0.02 0.02 0.05 0.03Whey protein, number 20.80 0.02 21.00 0.022

Lactose content, % 5.34 0.43 5.30 0.40Lactose yield, kg 0.30 0.13 0.27 0.10

(Casein or Whey protein/ protein); SD: standard deviation1, 2 3

each whole milk sample, following parameters were

According to this criterion, a total of 21 buffalo cows

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Fig. 7: The electrophoretic gel pattern of the digestionamplified DNA fragment -CN gene withrestriction endonuclease HinfI, M: ladder marker,lanes: 1, 2…, 10 are homologues AA genotype ofthe infected buffalo cows.

Fig. 8: The electrophoretic gel pattern of the digestionamplified DNA fragment -LG gene with restrictionendonuclease HaeIII, M: ladder marker, lanes: 1,2…, 10 are homologues AA genotype of theinfected buffalo cows.

(Figure 6 and Figure 8). These findings showed theexistence of only one allele A: for both -CN and -LGgenes, with all the studied buffalo cows beinghomozygous for these genes. In general, only onerestriction pattern could be detected among the buffalocows studied for -CN and -LG genes. Moreover, amonomorphic banding pattern which demonstrated thepresence of allele B was not detected among the buffaloesstudied. In the study of Mitra et al. [23], an only one alleleB: for -CN gene from Murrah, Nili-Ravi and Egyptianbuffaloes were reported. A similar finding was statedby Otaviano et al. [24], Riaz et al. [25] and Nair et al. [26]for different buffalo populations. In another study byLin et al. [27], alleles -CN A and B were found with allelefrequencies 17.4 and 82.6% in different buffalopopulations. In Nagpuri buffaloes, Nair et al. [26],

detected one pattern for -LG with three fragments of148, 99 and 74 bp.

Fixed Effects: The results of the analysis of varianceshowed highly significant effect of test-day on studiedtraits (p<0.01) except for MY and FY while, herd-season ofcalving had a significant effect (p<0.05) on MY, PY, LY,CY, WY and C %. Infection status had also a significanteffect (p<0.05) on these traits. Both parity and days in milkhad highly significant effect (p<0.01) on the currentlystudied traits but had insignificant effect (p>0.05) onpercentages of milk, fat and lactose. Similar results werealso observed for other buffalo populations [10, 11, 21,28].

Association Between Infection Status and Milk Traitswith -CN and -LG Genotypes AA: Table 2 presentsleast squares means (LSM) by infection status for buffalocows with -CN and -LG genotypes AA. Infection statushad clear effects on studied traits, except FY, F%, P%,W% and L%. Differences of LSM and standard errors ofdifferences by fitting linear contrasts of the solution formilk composition traits by infection status with -CN and

-LG genotypes AA are given in Table 2.Milk, protein and lactose yield were significantly

influenced by infection status. Healthy buffalo cowsproduced more milk yield (MY, 2.98 kg), protein yield (PY,0.10 kg) and lactose yield (LY, 0.17 kg). Contrary, FY, F%,P%, W% and L% were insignificantly (P>0.05) affected bythe infection status. Similarly, healthy buffaloesproducing significantly more CY by 0.72 (±0.08 kg, p<0.5)and had high C% by 0.07 (±0.03), but differences were notinsignificant. While, WY had significant association(p<0.05) by the infection status compared to W%. Buffaloinfected produced more W% (0.16 ±0.08) and WY (0.03±0.01). In dairy cattle, Petar et al.]29] found that -CN Aallele was associated with high milk and fat yield. Otherfindings, have shown no significant differences with Aand B alleles in milk, fat and lactose yield or contents [11].In the same context, Petar et al. [29] confirmed that afavorable trend with dominance of -LG B allele in milkyield, fat and content A similar trend was observed byTsiaras et al. [10]. In spite most studies, suggested afavorable effect on milk yield and quality with genotypesBB or AB for -CN and -LG. Unfavorable associationbetween the incidence of subclinical mastitis andgenotypes with BB or AB for -CN and -LG was alsodetected [30]. In another study, Hamza et al., [31] reportedthat -CN is associated with physiological processessuch as cytotoxic and antibacterial effects that enhanceimmunity.

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Table 2: Least squares means (LSM) and differences of means and standard errors of differences (±SED) for milk composition traits by infection statusTraits LSM Healthy H LSM Infected I Infection status Means of difference SED P valueMilk yield, kg 7.31 4.33 H-I 2.98 1.05 0.01*

Fat content, % 5.35 5.09 H-I 0.26 0.62 0.68Fat yield, kg 0.36 0.24 H-I 0.12 0.06 0.05Protein content, % 3.60 3.38 H-I 0.22 0.16 0.18Protein yield, kg 0.25 0.15 H-I 0.10 0.04 0.01*

Casein content, % 3.66 2.94 H-I 0.72 0.08 0.10Casein yield, kg 0.20 0.13 H-I 0.07 0.03 0.02*

Whey protein content, % 0.70 0.86 H-I -0.16 0.08 0.06Whey protein yield, kg 0.02 0.05 H-I -0.03 0.01 0.01*

Lactose content, % 5.21 5.10 H-I 0.11 0.20 0.62Lactose yield, kg 0.39 0.22 H-I 0.17 0.06 0.01*

P <0.05*

CONCLUSION 9. Lundèn, A., M. Nilsson and L. Janson, 1997.

The present study showed that only one pattern ofgenetic polymorphisms in kappa-casein and -lactoglobulin genes with all buffalo cows studied byinfection status. Buffalo cows had significant differencesin milk, protein, casein, whey protein, lactose yield andcasein content.

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