Romanian Biotechnological Letters Copyright © 2012 University of Bucharest

Vol. 17, No.5, 2012 Printed in Romania. All rights reserved ORIGINAL PAPER

Preliminary study on milk composition and milk protein polymorphism in the Romanian local sheep breed Teleorman Black Head Tsigai Received for publication, June 25, 2011 Accepted, January 20, 2012 R. S. PELMUS1a*, G. C. PISTOL1*, C. LAZAR1, D. E. MARIN1, M. GRAS1, M. RADU2, E. GHITA1 1 INCDBNA, National Research-Development Institute of Biology and Animal Nutrition, Balotesti, Romania 2 University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, Bucharest, Romania * Joint work Present address: National Institute of Research and Development for Biology and Animal Nutrition, 1, Calea Bucuresti, 077015, Balotesti, Romania a Corresponding author: Rodica Stefania PELMUS, INCDBNA, 1, Calea Bucuresti, 077015, Balotesti, Romania, tel. 0040213512082, fax. 0040213512080, E-mail: [email protected]

Abstract There is an international interest for preservation and improvement of local animal breeds, due to their superior biological traits: rusticity, resistance and adaptability to very different local environment. The local Romanian Teleorman Black Head Tsigai sheep breed fits very well the current economic demands, such as milk production and prolificacy. The purpose of our study was to determine milk quality indices as well as milk protein polymorphism in local sheep breed, using 24 milk samples. The types of different milk proteins were identified by SDS-PAGE. The test day milk yield and chemical composition assays performed during the milking period of studied sheep showed that the yield of milk fat and protein ranged within the quality indices specific to breed (6.56% fat and 5.9% protein), described in literature. Milk samples were further analyzed for milk protein polymorphism. The electrophoretic pattern of milk samples showed the presence of four major caseins variants (αs1-, αs2β- and k-casein) and two whey proteins (β-lactoglobulin, α-lactalbumin). Our study on percentage analysis of protein fractions of interest revealed that caseins represented 74.16% of the total protein of sheep milk, followed by whey proteins. SDS-PAGE analysis showed that majority of milk samples is characterised by a medium expression level of both caseins and whey proteins (66.67-79.17%) followed by higher level of expression (12.6-29.17%). Further studies (Real Time qPCR) are requisite for certitude of the results on the polymorphic genes of proteins from sheep milk, in order to identify the genetic variants from the locus of each protein.

Keywords: sheep, local breed, milk quality parameters, milk proteins, proteins polymorphism

Introduction There is a particular worldwide interest for the conservation and breeding of the local breeds of animals due to their superiod biological traits. The high efficiency of local sheep production for example is given by their superior traits – rusticity, resistance and the capacity to acclimatize to different environmental conditions, their better productive potential in relation to the level of improvement and technology of rearing and exploitation (Carta et al., 2009 [1]). Being increasingly aware of the benefits offered (meat and milk production with special nutritional qualities) the government, organizations and associations of state and private breeders and specialists in animal husbandry have created programs to study, to conserve and ameliorate these breeds. 7582

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The Teleorman Black Head Tsigai sheep is a Romanian local breed which fits best the current economic requirements: milk prolificacy and early weaned lambs. It originates from Teleorman County, wherefrom it disseminated cross country. The Teleorman Black Head Tsigai is enlisted in the national database of the genetic resources and in the FAO database for animal genetic resources. This local breed can be competitive for milk and meat production with the best specialised breeds, making useless their importation. Genetic improvement program for production and reproduction traits of Teleorman Black Head Tsigai sheep is necessary, and also genetically characterization is important to demonstrate the importance of this local breed. Despite the limited genetic knowledge on milk protein variability in sheep, interesting relationships between genetic milk protein variants and traits of economical interest were already described (Rampilli et al., 1992 [2]; Pirisi et al., 1999 [3]). Relationships of such variation with milk quality, composition, and technological characteristics have been found for α s1-casein (α s1-cn), where D allele is less favourable for cheese making than the most common C variant. Caseins are responsible for the enzymatic coagulation of milk and their hydrolysis pattern is an important characteristic of cheese ripening. Contradictory results have been obtained regarding β-lactoglobulin (β-lg) associated with the yield milk production. Some works found significant relationships between the main variants A and B and milk production (Bolla et al., 1989 [4]; Fraghi et al., 1996 [5]; Nudda et al., 2000 [6]) fat and protein content (Garzon and Martinez, 1992 [7]; Giaccone et al., 1997 [8]; Rampilli et al., 1997 [9]; Dario et al., 2003 [10]), while other studies did not reveal any influence on milk traits (Barillet et al., 1993 [11]; Recio et al., 1997 [12]). Important relationships exist also between protein polymorphism and breed. For instance, Celuk et al. (2006) [13] observed that the Awassi individuals with genotype BB from β-lactoglobulin locus had a higher milk fat percentage, while the milk from Morkaraman sheep with genotype BB had a higher level of milk protein and non-fat dry matter. Genotype BB from β-lactoglobulin locus has a favourable effect on the dairy processing yield. This study is part of a larger project concerning the use of milk protein genetic polymorphism as potential genetic marker for the local sheep breed Teleorman Black Head Tsigai milk production trait. The purpose of this work was to determine the milk quality parameters and to study milk protein polymorphism in the Teleorman Black Head Tsigai, allowing thus a better knowledge of the breed, for sustainable genetic improvement and conservation. The two main protein groups found in sheep milk: caseins (αs1-, αs2-, β-, kcasein) and whey proteins (α-lactalbumin, β-lactoglobulin) were analysed.

Materials and methods Animals and biological samples To determine the milk quality parameters as well as the milk protein polymorphism, milk samples were obtained from 24 local sheep breed, Teleorman Black Head Tsigai. For the determination of milk quality indices 6 test –day from two and two weeks were realized. Determination of milk quality indices The total percentage of milk solids non-fat (SNF) content, fat and protein content was determined using a Ekomilk M analyzer (Bulteh 2000 Ltd, EON Trading LLC, Delaware, USA). Preparation of milk samples for total protein determination

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50 mL milk from each sample were centrifuged at 5000 rpm at 4°C for 20 minutes, and then kept in the refrigerator for 30 minutes, in order to allow the fat to separate at the surface. The skimmed milk was preserved at -20o C until analysis. Milk protein separation The Bradford method (Krauspe, 1986) [14] was used to determine the concentration of total milk proteins before their separation. Briefly, 5 μL of milk samples (diluted 1:40) were mixed with 250 μL Bradford reagent in 96 wells plates (Corning, Sigma-Aldrich, Steinheim, Germany) and incubated at room temperature for 15 minutes. The absorbance was measured at 595 nm using a microplate reader (SunriseTM, TECAN Group Ltd, Männedorf, Switzerland). Milk proteins were further separated by SDS-PAGE electrophoresis (Mini-Protean 3 BioRad system, BioRad, Hercules CA, USA) in 15% polyacrylamide gel. 5µg of protein samples diluted in migration buffer (1.5M Tris-HCl pH 6.8, 17.4% glycerol, 8% sodium dodecylsulphate (SDS), 0.08% bromphenol blue and 3M beta-mercaptoethanol) were migrated for 1h and 30 minutes, to facilitate separation of proteins, based on their apparent molecular weight. After migration, gels were stained by immersion in staining dye (Coomassie Blue 250 R, BioRad, Hercules CA, USA) for 15 minutes. The apparent molecular weights of analyzed proteins were established in relation with a standard known protein (Precision Plus Protein Standars Kaleidoscope, BioRad, Hercules CA, USA). Gel visualisation The gels were subjected to densitometry quantification using a transluminator (UV BIO PROFIL Vilber Lourmat, Marne-la-Vallée, France) and the BIO 1D software (Vilber Lourmat, Marne-la-Vallée, France). The relative expression of milk proteins were expressed as Arbitrary Units (AU). Statistical analysis Student's t- tests were used to analyze the differences between all samples. p values < 0.05 were considered significant. All data are expressed as mean ± standard error of the mean (SEM).

Results and discussions 24 milk samples from the local Teleorman Black Head Tsigai sheep breed were used in this study in order to determine several indices (quality parameters and protein polymorphism) important for their reproduction and ameliorating process. The test day milk and milk chemical composition assays performed during the milking period of the local sheep breed revealed that the milk fat content varied from one day test to another and ranged between 6.35% in test day 1 and 10.19% in test day 4. In contrast, the protein content registered a slight increase from the second day milk test which remained constant until test day 6 (Table 1). A similar evolution was observed for milk SNF content (9.51-11.01). Table 1. Indices of milk quality in the Teleorman Black Head Tsigai sheep during milking period

Test Day 1d 2d 3d 4d 5d 6d

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Fat (g %) Mean SEM 6.35 0.40 8.93 0.20 7.97 0.10 10.19 0.20 8.16 0.10 9.07 0.20

Milk parameters Protein (g %) Mean SEM 4.82 0.20 5.57 0.10 5.43 0.10 5.37 0.10 5.69 0.70 5.63 0.10

SNF (g %) Mean SEM 9.51 0.50 10.87 0.10 10.71 0.13 10.30 0.27 11.01 0.08 10.92 0.13

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Preliminary study on milk composition and milk protein polymorphism in the Romanian local sheep breed Teleorman Black Head Tsigai

Similar results were found by Hamdon et al., (2006) [15] in the Egyptian breeds Chios and Farafra in which the milk protein increased slightly from 4.12% at the first control to 6.54% at the final control, while SNF had a similar evolution throughout the lactation (between 10.19% at the first control and 10.10% at the final control). By contrast the fat content increased from 3.05% in test day 1 to 7.28% in the last milking day test (Hamdon et al., 2006 [15]). The fat variation in our study might be due to the variation in the animal diet. In dairy sheep, as in other ruminants, feeding is an important factor affecting the quality of milk, which directly influences the synthesis and rates of milk fat, total protein, minerals and vitamins secretion (Nudda et al., 2004 [16]). It is well known that milk traits performance such as milk production, composition and physical properties are influenced by genetic (breed and genotype) and physiological (age, type of lambing, body weight, number of lambs, stage and rank of the lambing) factors as well as by nutrition and milking method (Pavic et al., 2002 [17]). The breed of sheep can affect the composition of the milk mostly because there is a negative correlation between milk yield and concentration of milk components. Breeds highly selected for dairy production tend to have a lower concentration of fat, protein and total SNF (Anifantakis 1986 [18], Bedo et al., 1999 [19]). Studying the milk composition in some European and Asian sheep breeds, different authors have noticed that the milk fat can differed with the breed, being lower in Nadjii (5.33%) and Merino-Balbass (5.84%), (Alichanidis and Polychroniadou, 1996 [20]), medium in White Maritza (6.23) and Tsurcana (6.91%), (Dimov and Mihailova, 1999 [21]; Lujerdean et al., 2008 [22]) and higher in Vlahico and Lacaune (9.05% and 7.40%, respectively), (Alichanidis and Polychroniadou, 1996 [20]); milk protein was lower only in Nadjii (4.75%) and Tsurcana (5.08%), compared to 6.52% in Vlahico, 5.91 % in White Maritza, 5.63% in Lacaune and 5.29% in Merino-Balbass. The concentration of total protein determined by Bradford method and used for protein separation confirmed the results obtained with the Ekomilk M analyzer being 5.69 mg%mL milk. Milk samples were further analyzed for protein polymorphism. SDS PAGE electrophoresis was used to determine the following proteins in the analysed milk samples: αs1-casein and αs2-casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin. Migrated protein bands were obtained for each analysed milk sample, with different expressions within the same type of protein. The electrophoretic pattern of milk samples of Teleorman Black Head Tsigai sheep showed the presence of four major caseins variants αs1-casein, αs2-casein β-casein, k-casein and two whey proteins, β-lactoglobulin and α-lactalbumin. A representative selection of electrophoretic pattern of milk proteins (from sample 1 to 8) is shown in Figure 1.

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R. S. PELMUS, G. C. PISTOL, C. LAZAR, D. E. MARIN, M. GRAS, M. RADU, E. GHITA Figure 1. Representative selection of electrophoresis pattern of milk proteins (samples 1-8) in Teleorman Black Head Tsigai sheep.

ilk proteins were separated by SDS-PAGE electrophoresis, in 15% polyacrylamide gel, based on their apparent molecular weight. The standards of known molecular weight (MW) were included in each experiment, thus permit to assess the type of analyzed protein. Milk samples analysed are characterized by different expression of milk proteins, depending on individual sample. Analyses of the different types of milk protein revealed that the highest expression was observed for β-casein and followed by k-casein, αs1-casein, αs2casein, β–lactoglobulin and α-lactalbumin (Figure 1 and Figure 2).

Figure 2. The expression level of milk proteins in Teleorman Black Head Tsigai sheep.

After electrophoresis separation of milk proteins, the gels were analysed on a transluminator and the results were scanned and analyzed by densitometry using specialised software. The results were expressed as arbitrary units per 1000 (AU/1000), to facilitate data analysis. The mean values ± standard error of the mean (SEM) were calculated for each analyzed protein and presented as histograms. Caseins are the major proteins in sheep milk, accounting for 76-83% of the total proteins, being positively correlated with cheese production (Park et al., 2007 [23]). Furthermore, the milk of the local sheep breeds is more appropriate for cheese production because of the higher content of all types of casein (Revilla et al., 2009 [24]). These authors have found that the local breeds Churra and Castellana had a higher content of all the caseins in comparison with Assaf sheep. It was demonstrated that the proportion of the four milk caseins influences the physical-chemical, nutritional and technological properties of the milk (Ramuno et al., 2000 [25]). Indeed, the analysis of the milk proteins from Teleorman Black Head Tsigai sheep, separated by SDS-PAGE electrophoresis showed that the caseins account for 74.16% of the total milk proteins, while the major proteins from the whey represent 25.84% of the total protein (Table 2a). Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7586

Preliminary study on milk composition and milk protein polymorphism in the Romanian local sheep breed Teleorman Black Head Tsigai Table 2.a. Proportion of different protein fractions in milk of local sheep breed Teleorman Black Head Tsigai *

Protein fractions

% of total protein Mean

SEM

74.15

7.05

β-casein

25.00

2.29

k-casein

17.45

1.66

Total caseins Caseins

αs1- casein

17.38

1.32

αs2- casein

14.32

1.78

25.84

3.92

β-lactoglobulin

14.08

1.68

α-lactalbumin

11.76

2.24

Total whey proteins Whey proteins

*Protein fractions were separated by SDS-PAGE and intensity of bands was analyzed by densitometry using specialised software. The results were expressed as percentage from total milk protein analysed by SDS-PAGE. The mean values ± standard error of the mean (SEM) were calculated for each analyzed protein. A higher expression for β-casein (25%) followed by k-casein (17.45%), αs1-casein (17.38%), αs2–casein (14.32%), β-lactoglobulin (14.08) and α-lactalbumin (11.76%) was observed (Table 2a). The analysis of the casein fractions shows that β-casein has the highest proportion, 33.72%, of all the caseins, followed by k-casein (23.53%), αs1-casein (23.43%) and αs2-casein (19.31%) (Table 2b). Table 2.b. Proportion of casein fractions in milk of local sheep breed Teleorman Black Head Tsigai *

Casein fractions

% of total casein Mean

SEM

β -casein

33.72

3.10

k -casein

23.54

2.24

αs1- casein

23.43

1.78

αs2- casein 19.31 2.40 *Casein fractions were separated by SDS-PAGE and intensity of bands was analyzed by densitometry using specialised software. The results were expressed as percentage from total milk casein. The mean values ± standard error of the mean (SEM) were calculated for each analyzed casein. The RP-HPLC analysis of the casein fractions from the milk of the Greek sheep breeds Karogouniko, Boutsiko, Chios and Frisarta (Moatsou et al., 2004 [26]) also showed that β-casein is the best represented casein fraction (37-42.3%) of the total caseins, but unlike the local breed investigated in our study, in which k-casein is the second fraction as proportion, in the Greek breeds investigated by Moatsou et al. (2004) [26], αs1-casein follows closely β-casein as proportion (33.9-39.9%) from the total caseins, while k-casein accounts Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7587

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for just 9.1 – 10.8% of the total caseins, which shows once again the influence of the breed on milk composition. Other authors have also shown the influence of the breed on the proportion of the milk protein fractions. Thus, Law et al. (1992) [27] and Papoff et al. (1993) [28] have found that β-casein separated by cation-exchange chromatography is the milk protein fraction best represented in the Sardinian sheep, but unlike the Greek breeds (Moatsou et al., 2004 [26]) and the Teleorman Black Head Tsigai sheep from our study, the percentage of β-casein is much higher (50%) of the total caseins, followed by αs1-casein (34.2%) and αs2-casein (26.8%). On the contrary, Pirisi et al. (1999) [3], using RP-HPLC, showed a higher proportion of the αs-caseins (αs1-casein and αs2-casein), 45.3-50.2%, from the total caseins and only 9.8% for k-casein in Sarda sheep. A distribution of milk samples related to the expression level (high, medium and low) of protein of interest is shown in Table 3. Table 3. Distribution of sheep groups depending on milk protein expression*

Group

Protein

High (n=4) Medium (n=18)

16.67 β-casein

75.00

Low (n=2)

8.33

High (n=3)

12.50

Medium (n=16)

k-casein

66.67

Low (n=5)

20.83

High (n=5)

20.83

Medium (n=18)

αs1-casein

75.00

Low (n=1)

4.17

High (n=4)

16.67

Medium (n=19)

αs2-casein

79.17

Low (n=1)

4.17

High (n=3)

12.50

Medium (n=18)

β-lactoglobulin

75.00

Low (n=3)

12.50

High (n=7)

29.17

Medium (n=17) *

% of total samples

Low (n=0)

α-lactalbumin

70.83 0.00

Protein fractions were separated by SDS-PAGE and intensity of bands was analyzed by densitometry using specialised software. The results were expressed as percentage of total milk samples.

Percent analysis showed that the majority of milk samples (16-19 from 24) is characterised by a medium expression level of both caseins and whey proteins (66.6779.17%) followed by the higher level of expression (12.6-29.17%) (3-5 from 24). As shown in Figure 3, there are statistically significant differences especially between ewes groups characterized by high and medium expression levels of all analysed milk proteins. Also, for βcasein, k-casein and β–lactoglobulin expression, statistically significant differences between subgroups with high versus low expression level and medium versus low expression level were found. 7588

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Preliminary study on milk composition and milk protein polymorphism in the Romanian local sheep breed Teleorman Black Head Tsigai

Figure 3. Distribution of milk samples related to the expression level (high, medium and low) of milk protein in Teleorman Black Head Tsigai sheep.

After electrophoresis separation of milk proteins, the gels were analyzed by densitometry using specialised software. To facilitate data analysis, the results were expressed as arbitrary units per to 1000 (AU/1000). Student's t- tests were used to analyze the differences between protein expression levels of all samples. P-values < 0.05 were considered significant. All data are expressed as mean ± standard error of the mean (SEM). The whey proteins amount, according to some authors, to 17-22% (Park et al., 2007 [23]) or 18-20% (Jovanovic et al., 2007 [29]) of the total milk proteins, β-lactoglobulin accounting for 50% of the whey proteins, and α-lactalbumin 20% (Jovanovic et al., 2007 [29]). Similar proportions have been found for the whey proteins in Teleorman Black Head Tsigai sheep after processing by SDS-PAGE electrophoresis: 25.90% total whey protein of which 14.10% β-lactoglobulin and 11.80% α-lactalbumin from the total milk proteins. This protein profile associated with the variability at the casein and whey proteins locus could be used to differentiate the individual genotypes and exploited in the breeding selection strategies for the improvement of the milk protein and milk casein, when cheese production is the main purpose of selection. Similar investigations were performed by Marzia et al. (2009) [30] who determined by 2D electrophoresis the variability of milk caseins in Garganica goats in order to find the global genotype of the casein αs1-, αs2-, β- and κcasein. These investigators expressed casein profile associated with casein genotype and thus showed differences in the protein expression level due to the interaction of loci. Conclusions In summary, this study suggests that the local sheep breed Teleorman Black Head Tsigai showed good performance for milk traits performance (quality parameters and protein polymorphism). Percentage analysis of the protein fractions of interest revealed that caseins Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

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represented 74.16% of the total protein of sheep milk, followed by whey proteins. Most of the milk samples are characterised by a medium intensity level of expression of both caseins and whey proteins followed by the higher level of expression. In addition to our preliminary results, both increase the number of the animal studied and further studies (Real Time qPCR) are requisite for certitude of the results on the polymorphic genes of the milk proteins from sheep milk, in order to identify the genetic variants from the locus of each protein. Also, correlations between diet and milk protein composition will be performed to estimate the potential effect of diet composition (different energy: protein ratio in the diet and dietary crude protein concentration) on the the genetic potential of this breed.

Acknowledgement This work was financially supported from CNCSIS-UEFISCDI Project no.56/ PNII/TE, 2010-2013, granted by the Romanian Ministry of Research and Technology.

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