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Milk Protein Polymorphisms in the Kerry Breed of Cattle
T E C H N I C A L NOTES
(11)
(12)
(13) (14) (15)
in milk throughout the stages of lactation of individual cows. J. Dairy Sei., 42: 1619. Kelly, P. L. 1945. Milk lipase activity: A method for its determination and its relationship to the estrual cycle. J. Dairy S¢i., 28 : 803. Kern, F., Jr., L. Steinmann, az~d B. B. Sanders. 1961. Measurement of lipoprotein lipase activity in post heparin plasma: Description of technique. J. Lipid Res., 2:51. Korn, E. D. 1962. The lipoprotein lipase of cow's milk. J. Lipid Res., 3: 246. Lynn, W. S., Jr., and ~T. C. Perryman. 1960. Properties and purifications of adipose tissue lipase. J. Biol. Chem., 235: 1912. McBride, O. W., and E. D. Korn. 1963. The lipoprotein lipase of mammary gland and the correlation of its activity of lactation. J. Lipid Res., 4: 17.
1113
(16) Mickle, J. B., and L. J. Bush. 1965. Relationships among tri- and diglyceride fatty acids, certain protein fractions, and the acid degree of milk from individual cows. J. Dairy Sci., 48:832. (17) Pawar, S. S., and H. C. Tidwell. 1968. Effect of ingestion of unsaturated fat on lipolytie activity of rat tissues. J. Lipid Res., 9: 334. (18) Rizack, M. A. 1961. An epinephrine-sensitive lipolytic activity in adipose tissue. J. Biol. Chem., 236: 657. (19) Robinson, D. S. 1963. Changes in the lipolytic activity of the guinea pig mammary gland at parturition. J. Lipid Res. 4: 21. (20) Stadhouders, J-, and It. Mulder. 1964. Some observations on milk lipase. I I I . The effect of pH on milk ]ipase activity. Netherlands Milk Dairy J., 18:30.
Milk Protein Polymorphisms in the Kerry Breed of Cattle Abstract
Polymorphisms of fl-lactoglobulin, B-casein, %l-casein, and ~-casein were investigated in milk from the K e r r y breed using starch-gel electrophoresis. Incidences of protein phenotypes and their controlling genes were compared with those from other European breeds. Milk from the K e r r y breed contained only Variant A of B-casein and only Variant A of hemoglobin, thus resembling Northern European breeds, especially the Ayrshire and Shorthorn breeds. Electrophoretic methods have been used extensively to demonstrate the occurrence of genetically controlled polymorphisms in the principal proteins of bovine milk (2, 11, 16). The frequencies of genes which determine protein variants in cattle may be used to indicate the ancestral origins and affinities between breeds (3, 7, 15). I n the present investigation polymorphisms of fl-laetoglobulin, B-casein, asl-Casein, and K-casein were examined in milks from the K e r r y breed, which is indigenous to Southwest Ireland. Methods and Materla|s
Phenotyping of the milk proteins was achieved by the starch gel electrophoretic technique described by Asehaffenburg and Thymann (5). Slight modifications in procedure were introduced to improve the resolution of a-lactalbumin from as~- and as~-caseins (Fig. 1) and thus obviate the need for separation o£ the caseins from whey proteins, before electrophoresis. Starch gels (0.1 b y 10 by 18.5 cm)
were composed of a mixture of starch (18 g), urea (40 g), tris-borate-EDTA buffer p H 8.6 (80 ml), and mercaptoethanol (0.8 ml). Urea (0.65 g) and mercaptoethanol (0.08 ml) were added to milk samples (1 ml) and the mixtures stored at 4 C for one hour before electrophoresis. Strips of Whatman 0.33-ram chromatography p a p e r (0.1 by 0.7 cm), previously soaked in test samples, were inserted in slots (0.8-cm) cut in the gel. Prolonged storage (72 hr at 4 C) of urea-treated samples before electrophoresis was avoided, as it resulted in the conversion of homozygous B-lact0giobulin into two artifact forms which had electrophoretic mobilities approximately half that of fl-lactoglobulin. F u r thermore, electrophoresis of dried s£rips impregnated with test solution (8) proved unsatisfactory for whole milks. The gel sltrfaces were covered with polythene sheeting (0.02-cm thick), care being taken to exclude air bubbles. A constant current of 12 mamp, corresponding to an initial voltage of 160, was applied in a Shandon electrophoresis apparatus (Shandon Scientific Co. Ltd., London) at 4 C. Protein bands were located on the gels with amido black stain (Fig. 1) and quantified using a Chromoscan densitometer (Joyce Lobel and Co. Ltd., London) with the following instrument settings: aperture, l-ram diameter; specimen/drive gear ratio, 1:3, cam, no. 5 - - 0 7 7 / D ; filter, 620. The content of individual proteins in a milk was expressed as the ratio (R) of the optical density of the protein band to the sum of the densities of all the protein bands. By this procedure results obtained with different gels were comparable, as any inaccuracies due to slight differences in the degree of staining were minimized. J. DAIRY SCLEI~OE YOL. 52. NO. 7
111~
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OF DAIRY
SCIENCE
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/ REFLECTANCE DENSITOMETRY--CHROMOSCAN UNITS FIG. ]. Separation of individual milk proteins by urea-starch gel electrophoresis. Discussion of Results
The incidence of the various protein phenotypes in the 123 milks examined is shown in Table 1. The frequencies of their controlling genes are compared with those calculated for other European breeds (Table 2). I n general, the K e r r y breed resembles Northern European breeds but especially the Ayrshires and Shorthorns. I n common with the K e r r y breed, milk from the Ayrshires and Shorthorns contained only the A variant of fl-casein (Table 2) and also only the A variant of hemoglobin (6, 9). Consequently, the origin of the K e r r y breed would appear to be similar to that of Northern British breeds. I n this context it is of interest J . DAIRY SCIENCE VOL. 52, NO. 7
to note that the fat content of milk of the Kezwy breed (10) (average, 3.78%) is of the same order as that reported for the Ayrshires (14) (average, 3.81%) and Shorthorns (14) (average, 3.70%). However, the gene frequency of K-casein A (0.93) is exceptionally high in the K e r r y breed. Similar to results of Aschaffenburg and Drewry (4), it was found that fl-lactoglohulin constituted a greater proportion of the total protein in milks containing fl-lactoglobu!in A A (R ----- 0.24) than those containing either the AB (R = 0.19) or the BB (R -----0.145) types. No such relationship was observed for the other milk proteins.
TECHNZCXL NOTES
1115
TABLE 1. Incidence of genetically determined proteins in the milks of 123 K e r r y cows. Proteins
Genetic types
fl-Laetoglobulin %wCasein fl-Casein K-Casein
AA
BB
CC
AB
11 0 123 105
74 108 0 0
0 1 0
38 0 0 18
AC
BC
0 0 0
0 14 0
TABLE 2. Comparison of the frequencies of genes controlling milk protein polymorphisms in European breeds of cattle. Breeds
fl-Casein
A K e rr y 1.0 Ayrshire 1.0 Shorthorn 1.0 Friesian 0.98 Red Danish 0.95 Guernsey 0.988 Jersey 0.65 Brown Swiss 0.79
B 0 0 0 0.2 0.04 0.004 0.35 0.19
C 0 0 0 0 0.02 0.016 0 0.02
asl-Casein A 0 0 0 0.08 0.005 0 0 0
B 0.94 1.0 1.0 0.87 0.98 0.07 0.95 0.97
Gene C 0.06 0 0 0.05 0.1 0.3 0.05 0.03
R. F. MURPHY and W. K. DOWNEY, Department of Dairy Chemistry, National Dairy Research Centre, The Agricultural [nstitute, Moorepark, Fermoy, County Cork, Ireland Acknowledgments We thank R. Kearney for skillea technical assistance, D. Kelleher, The Office of Public Works, Killarney, for assistance in collecting milk samples, and the producers who supplied the samples. References (1) Aschaffenburg, R. 1963. Breed differences in the occurrence of fl-casein variants. J. Dairy Res., 30: 251. (2) Aschaffenburg, R. 1965. Variants of milk proteins and their pattern of inheritance. J. Dairy Sci., 48: ]28. (3) Aschaffenburg, R. 1968. Genetic variants of milk proteins: their breed distribution. J. Dairy Res., 35: 447. (4) Aschaffenburg, t~., and J. Drewry. ]957. Genetics of fl-lactoglobulins of cow's milk. Nature, 180: 376. (5) Aschaffenburg, R., and M. Thymann. 1965. Simultaneous phenotyping procedure for the principal proteins of cows' milk. J. Dairy Sci., 48:1524. (6) Ba~gham, A. D. 1957. Distribution of electrophoretically different haemoglobins among cattle breeds of Great Britain. Nature, 179: 467. (7) Bell, K. 1967. The detection and occurrence of fl-lactoglobulin. Biochim. et Biophys. Acta, 147: 100.
K-Casein
fi-Lactoglobulin
frequencies A B A 0.93 0.07 0.24 0.31 0.11 0.68 0.32 0.5 0.74 0.26 0.1 0.73 0.27 0.22 0.52 0.48 0.26 0.56 0.44
B 0.76 0.69 0.98 0.5 0.9 0.78 0.64
C 0 0 0 0 0 0 0.01
References
Present investigation 1, 4, 12, 17 1, 4 4, 7, 13, 18 13 4, 12, 17, 18 13, 18 12, 17, 18
(8) E1-Negoumy, A. M. 1966. Rapid recovery preservation and phenotyping of milk proteins by a modified starch gel technique of superior reso]ving power. Anal. Biochem., 15 : 437. (9) Evans, J. A., and J. 2. Crowley. 1966. Haemoglobin typing of the Kerry breed of cattle. Nature, 209: 309. (10) Kelleher, D. 1968. Personal communication. (11) Kiddy, C. A. 1964. Inherited differences in specific blood and milk proteins in cattle. A review. J. Dairy Sci., 47: 510. (12) Kiddy, C. A., J. O. Johnston, and M. P. Thompson. ]964. Genetic po]ymorphism in casein of cows' milk. ][. Genetic control of as-casein variation. J. Dairy Sci., 47: 147. (13) Y-arsen, B., and M. Thymann. 1966. Studies on milk protein polymorphism in Danish cattle and the interaction of the controlling genes. Acta Vet. Scand., 7: 189. (14) Report of Interdepartmental Committee. 1960. Milk Composition in the United Kingdom. p. 53, Her Majesty's Stationery Office, London. (15) Roberts on, A. 1961. Genetics of dairy cattle. J. Dairy Res., 28: 195. (16) Thompson, M. P. 1964. Phenotyping of caseins of cows' milk: Collaborative experiment. 5. Dairy Sci., 47: 1261. (17) Thompson, )5. P., C. A. Kiddy, J. 0. Johnston, and R. M. Weinberg. 1964. Genetic polymorphism in caseins of cows' milk. II. Confirmation of the genetic control of fl-casein variation. J. Dairy Sci., 47: 378. (18) Woychlk, J. H. 1965. Phenotyping of K-casein. J. Dairy Sci., 48:496. J . ]:)AIRY SCIEI~C~ VOL. 52, NO. 7
(11)
(12)
(13) (14) (15)
in milk throughout the stages of lactation of individual cows. J. Dairy Sei., 42: 1619. Kelly, P. L. 1945. Milk lipase activity: A method for its determination and its relationship to the estrual cycle. J. Dairy S¢i., 28 : 803. Kern, F., Jr., L. Steinmann, az~d B. B. Sanders. 1961. Measurement of lipoprotein lipase activity in post heparin plasma: Description of technique. J. Lipid Res., 2:51. Korn, E. D. 1962. The lipoprotein lipase of cow's milk. J. Lipid Res., 3: 246. Lynn, W. S., Jr., and ~T. C. Perryman. 1960. Properties and purifications of adipose tissue lipase. J. Biol. Chem., 235: 1912. McBride, O. W., and E. D. Korn. 1963. The lipoprotein lipase of mammary gland and the correlation of its activity of lactation. J. Lipid Res., 4: 17.
1113
(16) Mickle, J. B., and L. J. Bush. 1965. Relationships among tri- and diglyceride fatty acids, certain protein fractions, and the acid degree of milk from individual cows. J. Dairy Sci., 48:832. (17) Pawar, S. S., and H. C. Tidwell. 1968. Effect of ingestion of unsaturated fat on lipolytie activity of rat tissues. J. Lipid Res., 9: 334. (18) Rizack, M. A. 1961. An epinephrine-sensitive lipolytic activity in adipose tissue. J. Biol. Chem., 236: 657. (19) Robinson, D. S. 1963. Changes in the lipolytic activity of the guinea pig mammary gland at parturition. J. Lipid Res. 4: 21. (20) Stadhouders, J-, and It. Mulder. 1964. Some observations on milk lipase. I I I . The effect of pH on milk ]ipase activity. Netherlands Milk Dairy J., 18:30.
Milk Protein Polymorphisms in the Kerry Breed of Cattle Abstract
Polymorphisms of fl-lactoglobulin, B-casein, %l-casein, and ~-casein were investigated in milk from the K e r r y breed using starch-gel electrophoresis. Incidences of protein phenotypes and their controlling genes were compared with those from other European breeds. Milk from the K e r r y breed contained only Variant A of B-casein and only Variant A of hemoglobin, thus resembling Northern European breeds, especially the Ayrshire and Shorthorn breeds. Electrophoretic methods have been used extensively to demonstrate the occurrence of genetically controlled polymorphisms in the principal proteins of bovine milk (2, 11, 16). The frequencies of genes which determine protein variants in cattle may be used to indicate the ancestral origins and affinities between breeds (3, 7, 15). I n the present investigation polymorphisms of fl-laetoglobulin, B-casein, asl-Casein, and K-casein were examined in milks from the K e r r y breed, which is indigenous to Southwest Ireland. Methods and Materla|s
Phenotyping of the milk proteins was achieved by the starch gel electrophoretic technique described by Asehaffenburg and Thymann (5). Slight modifications in procedure were introduced to improve the resolution of a-lactalbumin from as~- and as~-caseins (Fig. 1) and thus obviate the need for separation o£ the caseins from whey proteins, before electrophoresis. Starch gels (0.1 b y 10 by 18.5 cm)
were composed of a mixture of starch (18 g), urea (40 g), tris-borate-EDTA buffer p H 8.6 (80 ml), and mercaptoethanol (0.8 ml). Urea (0.65 g) and mercaptoethanol (0.08 ml) were added to milk samples (1 ml) and the mixtures stored at 4 C for one hour before electrophoresis. Strips of Whatman 0.33-ram chromatography p a p e r (0.1 by 0.7 cm), previously soaked in test samples, were inserted in slots (0.8-cm) cut in the gel. Prolonged storage (72 hr at 4 C) of urea-treated samples before electrophoresis was avoided, as it resulted in the conversion of homozygous B-lact0giobulin into two artifact forms which had electrophoretic mobilities approximately half that of fl-lactoglobulin. F u r thermore, electrophoresis of dried s£rips impregnated with test solution (8) proved unsatisfactory for whole milks. The gel sltrfaces were covered with polythene sheeting (0.02-cm thick), care being taken to exclude air bubbles. A constant current of 12 mamp, corresponding to an initial voltage of 160, was applied in a Shandon electrophoresis apparatus (Shandon Scientific Co. Ltd., London) at 4 C. Protein bands were located on the gels with amido black stain (Fig. 1) and quantified using a Chromoscan densitometer (Joyce Lobel and Co. Ltd., London) with the following instrument settings: aperture, l-ram diameter; specimen/drive gear ratio, 1:3, cam, no. 5 - - 0 7 7 / D ; filter, 620. The content of individual proteins in a milk was expressed as the ratio (R) of the optical density of the protein band to the sum of the densities of all the protein bands. By this procedure results obtained with different gels were comparable, as any inaccuracies due to slight differences in the degree of staining were minimized. J. DAIRY SCLEI~OE YOL. 52. NO. 7
111~
JOURNAL
OF DAIRY
SCIENCE
U i,-
I
C
m
100 u
eo~
u
o
80
o
i
u ~s ..I
I e~
f:O
~u
•
Y
c
E 40
"20
/ REFLECTANCE DENSITOMETRY--CHROMOSCAN UNITS FIG. ]. Separation of individual milk proteins by urea-starch gel electrophoresis. Discussion of Results
The incidence of the various protein phenotypes in the 123 milks examined is shown in Table 1. The frequencies of their controlling genes are compared with those calculated for other European breeds (Table 2). I n general, the K e r r y breed resembles Northern European breeds but especially the Ayrshires and Shorthorns. I n common with the K e r r y breed, milk from the Ayrshires and Shorthorns contained only the A variant of fl-casein (Table 2) and also only the A variant of hemoglobin (6, 9). Consequently, the origin of the K e r r y breed would appear to be similar to that of Northern British breeds. I n this context it is of interest J . DAIRY SCIENCE VOL. 52, NO. 7
to note that the fat content of milk of the Kezwy breed (10) (average, 3.78%) is of the same order as that reported for the Ayrshires (14) (average, 3.81%) and Shorthorns (14) (average, 3.70%). However, the gene frequency of K-casein A (0.93) is exceptionally high in the K e r r y breed. Similar to results of Aschaffenburg and Drewry (4), it was found that fl-lactoglohulin constituted a greater proportion of the total protein in milks containing fl-lactoglobu!in A A (R ----- 0.24) than those containing either the AB (R = 0.19) or the BB (R -----0.145) types. No such relationship was observed for the other milk proteins.
TECHNZCXL NOTES
1115
TABLE 1. Incidence of genetically determined proteins in the milks of 123 K e r r y cows. Proteins
Genetic types
fl-Laetoglobulin %wCasein fl-Casein K-Casein
AA
BB
CC
AB
11 0 123 105
74 108 0 0
0 1 0
38 0 0 18
AC
BC
0 0 0
0 14 0
TABLE 2. Comparison of the frequencies of genes controlling milk protein polymorphisms in European breeds of cattle. Breeds
fl-Casein
A K e rr y 1.0 Ayrshire 1.0 Shorthorn 1.0 Friesian 0.98 Red Danish 0.95 Guernsey 0.988 Jersey 0.65 Brown Swiss 0.79
B 0 0 0 0.2 0.04 0.004 0.35 0.19
C 0 0 0 0 0.02 0.016 0 0.02
asl-Casein A 0 0 0 0.08 0.005 0 0 0
B 0.94 1.0 1.0 0.87 0.98 0.07 0.95 0.97
Gene C 0.06 0 0 0.05 0.1 0.3 0.05 0.03
R. F. MURPHY and W. K. DOWNEY, Department of Dairy Chemistry, National Dairy Research Centre, The Agricultural [nstitute, Moorepark, Fermoy, County Cork, Ireland Acknowledgments We thank R. Kearney for skillea technical assistance, D. Kelleher, The Office of Public Works, Killarney, for assistance in collecting milk samples, and the producers who supplied the samples. References (1) Aschaffenburg, R. 1963. Breed differences in the occurrence of fl-casein variants. J. Dairy Res., 30: 251. (2) Aschaffenburg, R. 1965. Variants of milk proteins and their pattern of inheritance. J. Dairy Sci., 48: ]28. (3) Aschaffenburg, R. 1968. Genetic variants of milk proteins: their breed distribution. J. Dairy Res., 35: 447. (4) Aschaffenburg, t~., and J. Drewry. ]957. Genetics of fl-lactoglobulins of cow's milk. Nature, 180: 376. (5) Aschaffenburg, R., and M. Thymann. 1965. Simultaneous phenotyping procedure for the principal proteins of cows' milk. J. Dairy Sci., 48:1524. (6) Ba~gham, A. D. 1957. Distribution of electrophoretically different haemoglobins among cattle breeds of Great Britain. Nature, 179: 467. (7) Bell, K. 1967. The detection and occurrence of fl-lactoglobulin. Biochim. et Biophys. Acta, 147: 100.
K-Casein
fi-Lactoglobulin
frequencies A B A 0.93 0.07 0.24 0.31 0.11 0.68 0.32 0.5 0.74 0.26 0.1 0.73 0.27 0.22 0.52 0.48 0.26 0.56 0.44
B 0.76 0.69 0.98 0.5 0.9 0.78 0.64
C 0 0 0 0 0 0 0.01
References
Present investigation 1, 4, 12, 17 1, 4 4, 7, 13, 18 13 4, 12, 17, 18 13, 18 12, 17, 18
(8) E1-Negoumy, A. M. 1966. Rapid recovery preservation and phenotyping of milk proteins by a modified starch gel technique of superior reso]ving power. Anal. Biochem., 15 : 437. (9) Evans, J. A., and J. 2. Crowley. 1966. Haemoglobin typing of the Kerry breed of cattle. Nature, 209: 309. (10) Kelleher, D. 1968. Personal communication. (11) Kiddy, C. A. 1964. Inherited differences in specific blood and milk proteins in cattle. A review. J. Dairy Sci., 47: 510. (12) Kiddy, C. A., J. O. Johnston, and M. P. Thompson. ]964. Genetic po]ymorphism in casein of cows' milk. ][. Genetic control of as-casein variation. J. Dairy Sci., 47: 147. (13) Y-arsen, B., and M. Thymann. 1966. Studies on milk protein polymorphism in Danish cattle and the interaction of the controlling genes. Acta Vet. Scand., 7: 189. (14) Report of Interdepartmental Committee. 1960. Milk Composition in the United Kingdom. p. 53, Her Majesty's Stationery Office, London. (15) Roberts on, A. 1961. Genetics of dairy cattle. J. Dairy Res., 28: 195. (16) Thompson, M. P. 1964. Phenotyping of caseins of cows' milk: Collaborative experiment. 5. Dairy Sci., 47: 1261. (17) Thompson, )5. P., C. A. Kiddy, J. 0. Johnston, and R. M. Weinberg. 1964. Genetic polymorphism in caseins of cows' milk. II. Confirmation of the genetic control of fl-casein variation. J. Dairy Sci., 47: 378. (18) Woychlk, J. H. 1965. Phenotyping of K-casein. J. Dairy Sci., 48:496. J . ]:)AIRY SCIEI~C~ VOL. 52, NO. 7