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Protein Production in the Bovine. Breed and Individual Variations in the Specific Protein Constituents of Milk1
PROTEIN ~'RODUCTION IN THE BOVINE. BREED AND INDIVIDUAL VARIATIONS IN THE SPECIFIC PROTEIN CONSTITUENTS OF MILK ~ G. D. ROLLERI, B. L. LARSON, AND R. W. TOUCHBERRY
Laboratory of Biocltemistry, Department of Dairy Sc~e~ce, University of Illinois, Urbana
For biological as well as technological reasons a greater knowledge o f the relation of breed of cattle to the production of the major proteins o f milk is important. The milk from five to seven cows from each of five major breeds was used for electrophoretic analysis. The amounts of a-, /?- and ,/casein, a-lactalbumin, B-lactoglobulin, the immune globulins, and serum albumin present in each milk were determined. Editor.
The various breeds of d a i r y cattle produce significantly different amounts of protein in their milk, and individuals within the breeds show considerable variation (9,10, 11, 12, 14). I n general the breeds producing less total milk but having a higher f a t content also produce milk containing more protein. Studies o n the proteins of skimmilk have been confined to an evaluation of the variations in the total protein or of the total casein and serum (whey) protein fractions. I n some studies the serum proteins have been f u r t h e r s e p a r a t e d into " l a c t o g l o b u l i n , " " l a c t a l b u m i n , " and " p r o t e o s c - p e p t o n e " fractions on the basis of their heat stability and solubility in concentrated salt solutions (10, 11, 12, 14). These fractionation procedures and terminology have become outmoded b y the recent isolations in purified or crystalline f o r m of the m a j o r specific protein c o n s t i t u e n t s of milk (3, 8, 13). The evaluation of the specific milk proteins m a y be accomplished most easily b y an electrophoretic analysis of the isolated casein and the isolated serum proteins even though the n u m b e r of samples t h a t can be analyzed by this procedure is limited. Casein on eleetrophoresis at p H 8.6 separates into three components (a-, fi-, and 7-casein) (8). On electrophoresis at p H 8.6 the serum proteins separate into eight or more fractions; however, f o u r of these, the immune globulins, ~-lactalbumin, fl-laetoglobulin, and blood serum albumin, compose up to 85% of the normM milk serum protein (3, 8). The inmmne globulins (euglobulin and pseudoglobulin) compose most of the classical " l a e t o g l o b u l i n " fraction and are p r o b a b l y identical to some of the immune globulins of blood (1, 5,8,13). a-Laetalbumin, fl-laetoglobulin, and serum albumin have been isolated in erystalline f o r m f r o m milk and compose essentially the classical " ' l a e t a l b u m i n " fraction (3, 8). The serum albumin of milk is identical with the Received for publication April 12, 1956. 1 Supported in part by aid from the Rockefeller Foundation. 1683
1684
a.D.
R O L L E R I ET _4.L
serum albumin of blood (2). In addition there are enzymes, unidentified minor proteins, and unspecific " p r o t e o s e - p e p t o n e " materials present (3, 8, 12). The biological and technological importance of each of the major specific proteins prompted a study to determine if there are differences between the various breeds of dairy cattle in their production of these proteins. The amounts of a-, fl-, and ~/-easein, a-lactalbumin, fi-laetoglobulin, the immune globulins, and serum albumin in milk were determined in this study. ~WlgTtIODS AND PROCEDURES
Studies in this laboratory have indicated that the stage of lactation has an effect oll the total protein production per day and the ratio of the amounts of the specific proteins in the milk relative to each other (~, 5'). The relative amounts of these proteins in the milk v a r y greatly at the start and near the end of the lactation period; thus the milk samples for thi.~ study were taken from cows that had been in lactation 2 to 6 months. Samples were taken from 28 healthy cows ill the University herd including five to seven cows from each of the five major dairy breeds. Two from each breed were in their first lactations. The samples of milk were collected at five weekly intervals during J u l y and August from groups of five to seven cows, including at least one cow from each breed. Samples of milk were taken from each cow at the morning and evening milkings of two consecutive days; the four samples were composited proportional to the weight of each milking. The composited sample of milk was centrifuged for 20 minutes at 2000 x gravity in 300-ml. polyethylene bottles and the skimmilk was pipetted from beneath the layer of fat. An aliquot of 200 ml. of skimmilk was used for the preparation of the serum proteins for the electrophoretic analysis according to previously described procedures (3). Casein was p r e p a r e d for the electrophoretic analysis by precipitating it from 25 ml. of skimmilk with 10% acetic acid at p H 4.65, redispersing in sodium-potassium phosphate buffer ( p H 6.9, ionic strength of 0.1), reprecipitaring with 10% acetic acid, and redispersing in sodium veronal buffer ( p H 8.6, ionic strength of 0.1) to give a concentration of 2 to 4'~. Centrifugation in 50ml. plastic centrifuge tubes and homogenization in a glass tissue grinder were used for the precipitation and redispersion steps, respectively. The casein solutions were centrifuged at 2000 × gravity for 30 minutes to remove traces of fat and other opaque material. An aliquot of 17.6 ml. was dialyzed against 250 nil. of veronal buffer ( p H 8.6, ionic strength of 0.1) for 12 to 24 hours at 4 ° C. and then against 500 ml. of the same strength buffer for an additional 24-hour period. At the time of the eleetrophoretie analysis the casein solutions were diluted with equilibrated buffer to the desired concentration. The concentration of the solution actually used in the e]ectrophoretic analysis was determined by a semi-micro Kjeldahl analysis. The electrophoretic analyses were all conducted in sodium veronal buffer (pII 8.6, ionic strength of 0.1, 0.12 M) for 5,580 seconds at 1.2 ° C. with a field
1685
PROTEIN PRODUCTION IN THE BOVINE
strength of 7.8 volts per square centimeter as previously described (3, 7). The casein solutions were analyzed at a concentration of about 1.5% and the serum protein solutions at 2-3% protein. Difficulties encountered with the isolation procedure and the electrophoretic analyses resulted in the loss of some samples. I n some instances, only the casein or the serum protein fraction for a given cow was carried through the complete analysis without loss. The electrophoretic p a t t e r n s of the serum proteins were divided into four areas r e p r e s e n t i n g the immune globulins, a-lactalb~min, fi-lactoglobulin, and serum albumin (3). Since in addition to the usual electrophoretic anomalies other nfinor protein components are present, the actual amounts of each constituent are p r o b a b l y 5 to 15% lower t h a n the figures r e p o r t e d (3). TABLE 1
Within and between breeds components of variance for the protein eonstituents of ~niUc Components of variance Protein component
W i t h i n breed
Between breeds
On a basis of grams per 100 ml. sl~immilk Casein : a-casein fl-easein 3,~casein Total casein
0.0831 0.0204 0.0017 0.1563
0.0166 0.0091 ~ 0.0019 ~'~ 0.0131
Serum p r o t e i n s : I m m u n e globulins adactalbumin ¢/-laetoglobulin Serum albumin Total serum p r o t e i n s
0.0044 0.0008 0.0063 0.0001 0.0143
trace" trace ~ 0.0001 trace" 0.0004
Derived f r o m a m e a n square t h a t was significant at the 0.05 level of probability. ~ Derived f r o m a m e a n square t h a t w a s significant a t the 0.01 level of p r o b a b i l i t y . Components were positive b u t the first five decimal places were zeros. RESULTS
The components of variance in Table 1 indicate t h a t there was considerably more variation between cows within the same breed t h a n between the breeds. TABLE
2
Analysis of variance of [3-casein Source Total
Mean squares
Expected mean squares
24
Between breeds 1, 2. 3. 4.
Degrees of freedom
Hol. vs (Ayr. + Guer. + J e r . + B. Sw.) Ayr. vs (Guer. + J e t . + B. Sw.) B. Sw. vs (Guer. + J e t . ) Guer. vs Jer.
W i t h i n breeds
4
0.0654 ~'
1 1 1 1
0.2413 ~ 0.0066 0.0051 0.0091
20
0.0204
( a ~ + 4.94 ~ ) a
(~)
F o r the between-breed mean squares involving the serum p r o t e i n s the coefficient of fl~ w a s 5.17.
1686
G.D. I~OLLERI ET AL
The components of variance were derived from analyses of variance tables similar to that freedom
shown
in Table
between
breeds
2.
For
every protein
were broken
down
constituent
the four
into individual
degrees of
comparisons
as is
s h o w n i n T a b l e 2, a n d t h e r e s u l t s o f t h e s e c o m p a r i s o n s a r e s h o w n i n T a b l e s 3 a n d 4. TABLE 3 The average a~ounts of proteins prese~tt in the sl~i~neailk of the various breeds
Protein component
Ayrshire
Brcwn Swiss
Guernsey Holstein
Jersey
Within breed stan(]ard deviations
Casein: (No. samples) a-casein fl-easein "/-casein Total
(g. per 100 v~l.) (5) (4) (5) 1.71" 1.83 1.92 0.85 0.84 0.82 0.08" 0.11 0.14 2.64 2.78 2.88
(7) 1.58" 0.60" 0.20" 2.38"
(4) 1.83 0.76 0.13 2.72
(25) 0.29 0.14 0.04 0.39
Serum proteins: (No. samples) Immune globulins a-laetalbumi n fl-laetoglobulin Serum albumin Total
(5) 0.06 0.11 0.31 0.03 0.51
(6) 0.09 0.13 0.30 0.04 0.56
(5) 0.08 0.15 0.39 0.04 0.66
(26) 0.07 0.03 0.08 0.01 0.12
(4) 0.97 0.11 0.31 0.04 0.53
(6) 0.08 0.1 ] 0.35 0.04 0.58
Comparison 1, Table 2, was significant for total casein and highly significant for a-, fl-, and 7-casein. b Comparison 2, Table 2, was highly significant for a- and 7-casein. TABLE 4 The ratio of ?he specific proteins present in casein and the ser~t~t proteins isolated frown the vario'~s breeds
Protein component
Ayrshire
Brown Swiss
Guernsey
ltolstein
Jersey
Within breed standard deviations
(c/c of total casein or serbian proteins)
Casein : a-casein fl-casein 7-casein
64.6 32.2" 3.1
65.8 30.1 4.1
66.4 28.3 5.2
66.3 25.2 '~ 8.5 ~
67.2 27.9 4.8
2.92 3.04 1.56
Serunl proteins : Immune globulins a-lactalbumin fl-laetoglobulin Serum albumin
11.5 21.7 61.3 5.5"
13.0 21.3 58.9 6.7
13.5 19.7 59.8 6.8
15.8 23.3 54.4" 6.6
12.8 22.2 59.6 5.6
2.71 2.18 3.55 .65
" Comparison 1, Table 2, was significant for fl- and "r-casein and for fl-laetoglobulin. Comparison 2, Table 2, was significant for fl-easein and for serum albumin. A. Amounts
o f t h e p r o t e i n s p r e s e n t i n the m i l k .
The breed averages
shown
in Table 3 indicate that the milk of the IIolstein cows contained significantly less t o t a l c a s e i n , a - c a s e i n , a n d f i - c a s e i n b u t s i g n i f i c a n t l y m o r e ~ - c a s e i n t h a n t h e m i l k o f t h e cows o f t h e f o u r o t h e r b r e e d s . T h e A y r s h i r e m i l k c o n t a i n e d s i g n i f i cantly less a-casein than the milk of the breeds other than Holstein, and sign i f i c a n t l y less 7 - c a s e i n t h a n t h e m i l k o f t h e f o u r o t h e r b r e e d s .
PROTEIN PRODUCTION IN THE BOVINE
1687
No significant differences were shown between the breeds in the total or in any of the specific serum proteins; however, the power of these tests of significance is low. It should be noted that the milk from the Holstein cows contained on the average less fl-lactoglobulin and more immune globulins than the milk of the other four breeds.
B. Relative amounts of the specific proteins present in the casein and the serum proteins. Significant differences were found between the breeds in respect to the ratio of the specific proteins present in the casein and the serum proteins. The data in Tab.le 4 show that the casein from Holstein cows contained significantly less fl-casein but significantly more v-casein than the casein from the other four breeds. The casein from the Ayrshire cows contained significantly more fl-casein than the casein from the other four breeds. The a-casein content of the total casein was practically the same (64.6 to 67.2%) for all of the breeds. Practically all of the variation shown was in the relative amounts of fl-casein and ~/-casein. The serum proteins from the Holstein cows contained significantly less fl-lactoglobulin than did the serum protein of the other four breeds, and, though not significant, they contained a higher average content of immune globulins and a-lactalbumin. The serum proteins of the Ayrshire and Jersey breeds contained significantly less serum albumin than the serum proteins of the three other breeds. DISCUSSION
Since it was a p p a r e n t that the relative and actual amounts of the specific milk proteins of the individual animals varied, the within-breed correlation coefficients between the specific constituents were calculated from the data for all animals and are shown in Table 5. The highly significant correlation between the total casein and the total serum proteins indicates that cows that produced milk high in casein composition also produced milk high in total serum proteins. The high correlations between a-casein, fl-casein, fl-lactoglobulin and ~-lactalbumin thus would be expected since these proteins compose most of the total casein and the total serum proteins. This suggests that the synthesis of these four major constituents in the m a m m a r y gland might be closely related. The amount of ~/-casein or milk serum albumin present bears no significant relationship to any of the other constituents. This agrees with the observations (6) that the p a t t e r n of production of these constituents during the lactation period is more erratic and bears little relationship to the production of the other protein constituents. I t is of interest to note that, although not significant, there is a negative correlation between the amount of 7-casein present and the amount of immune globulins, fi-lactoglobulin, and milk serum albumin. These studies indicate that the amounts of the specific proteins present in the milk of the Holstein breed appear to differ most from the other breeds, whereas the Brown Swiss, Guernsey, and Jersey breeds are remarkably alike. The a-casein content of the total casein was relatively constant for all of the individual cows. Other than the Holstein breed, which contained a significantly
1688
G.D. ROLLERI ET AL TABLE 5 Within-breed correlations between tl~e protein constituents of mill~
Correlations between grams in 100 ml. of skimmilk Total casein and total serum proteins a-casein and fl-easein a-casein and y-easeln a-casein and imnlune globulins a-casein and a-lacta]bumin a-easmn and fl-lactoglobulin a-casein and serum albumin fl-easein and "/-casein fl-easein and immune globulins fl-easein and a-lactalbumin fl-casein and fl-lactoglobulin fi-casein and serum albumin "/-casein and immune globulins "/-casein and a-lactalbumin "y-casein and fl-lactoglobulin 7-casein and serum albumin immune globulins and a-laetalbumin immune globulins and fl-laetoglobulin immune globulins and serum albumin a-laetalbumin nnd fl-laetoglotm]in a-laetalbumin and serum albumin fl-laetoglobulin and serum albumin
Correlation coefficient 0.676~* 0.599"* 0.191 0.364 0.584~ 0.538~ 0.125 0.067 0.357 0.680*~ 0.550 ~ 0.248 -0.259 0.113 --0.243 -0.052 0.487 ~ 0.504 ~ 0.489~ 0.845*~ 0.310 0.327
Degrees of freedom 17 19 19 17 17 17 17 19 17 17 17 17 17 17 17 17 20 20 20 20 20 20
Significant at the 0.05 level of probqbility. ** Significant at the 0.01 level of prol)ability. lower p e r c e n t a g e of fl-lactoglobulin ill the total s e r u m p r o t e i n s , the f o u r other b r e e d averages for f l - l a c t o g l o b u l i n were r e m a r k a b l y close. I t also has b e e n n o t e d t h a t the f l - l a c t o g l o b u l i n c o n t e n t of the s e r u m p r o t e i n s , a l t h o u g h v a r y i n g b e t w e e n cows, does n o t v a r y m u c h for tile i n d i v i d n a l cow i n the m i d d l e r a n g e s of the lactation period (6). SUMMARY A n e v a l u a t i o n of the specific milk p r o t e i n s of the nfilk of 28 d a i r y cows i n c l u d i n g the five m a j o r breeds was made b y a q u a n t i t a t i v e e l e c t r o p h o r e t i c procedure. L a r g e i n d i v i d u a l v a r i a t i o n s a n d also s i g n i f i c a n t b r e e d differences were f o u n d . The nfilk of H o l s t e i n cows c o n t a i n e d s i g n i f i c a n t l y less t o t a l casein, a-casein a n d fl-easein, b u t s i g n i f i c a n t l y more y-casein t h a n did the m i l k of the f o u r o t h e r breeds. A y r s h i r e milk c o n t a i n e d s i g n i f i c a n t l y less a-casein t h a n d i d the m i l k of the B r o w n Swiss, G u e r n s % ' , a n d J e r s e y breeds a n d less y-casein t h a n did the m i l k of the breeds other t h a n the H o l s t e i n . A l l of the b r e e d s p r o d u c e d casein c o n t a i n i n g a p p r o x i m a t e l y the same a m o u n t of a-casein; however, the casein of the H o l s t e i n breed c o n t a i n e d s i g n i f i c a n t l y less fl-casein a n d more y-casein, whereas casein f r o m the A y r s h i r e b r e e d c o n t a i n e d s i g n i f i c a n t l y more fl-casein. T h e s e r u m p r o t e i n s of H o l s t e i n m i l k also c o n t a i n e d s i g n i f i c a n t l y less fi-laetoglobulin. H i g h c o r r e l a t i o n s were f o u n d b e t w e e n the a m o u n t s i n s k i m m i l k of a-casein, fl-easein, a - l a e t a l b u m i n , f l - l a e t o g l o b u l i n a n d to a lesser e x t e n t the i m m u n e g l o b u l i n s , b u t l i t t l e r e l a t i o n s h i p was f o u n d b e t w e e n the above c o n s t i t u e n t s a n d the a m o u n t of y-casein or s e r u m a l b u m i n .
PROTEIN PRODUCTION IN THE BOVINE
1689
REFERENCES (1) ASKO~'AS, B. A., CAMPBELL, P. N., HU~[r~EY, J. H., AI~D WORK, T. S. The Source of Antibody Globulin in Rabbit Milk and Goat Colostrum. Biochem. J., 56: 597. 1954. (2) COULS01',-, E. J., AND STEVENS, 11. The Serologic'~| ~clationship of Bovine Whey Albumin to Sermn Albumin. J. Biol. Chem., 187: 355. 1.950. (3) LARSOR~, B. L., AND ROLLERI, G. D. Hcat Denatm'ation of the Specific Serum Proteins in Milk. J. Dairy Sci., 38: 351. 1955. (4) LA~SOU', B. L., ROLLERI, G. D., AND ]t][ISNDALL, I~. A. Protein Production in the Bovine. Comtmrison of Daily Protein, ]!'at, and Milk Production During the Entire Lactation Period. J. Dairy Sci., 39: 204. ]956. (5) LAESON, B. L., ROLL~RI, G. D., :~ND KENDALL, K. A. Protein Production in the Bovine. Changes in the Specific Blood Serum Proteins Associated with Pregnancy and Parturition. (Abs.) J. Dairy Sci., 37: 662. 1954. (Paper in preparation.) (6) LA~S0~~, B. L., ROLDE,[~I, G. D., AND KENDALL, K. A. Protein Production in the Bovine. Daily Production of the Specific Milk Proteins During the Lactation Period. (Abs.) J. Dairy Sci., 38: 619. 1955. ( P a p e r in preparation). (7) LARSON, lB. L., AND SALISBURY, G. W. The Proteins of Bovine Seminal Plasma. I. Preliminary and Electrophoretic Studies. J. Biol. Chem., 205: 741. 1954. (8) McMEEKI~r, T. L. Milk Proteins. Chaptcr 16 in The Proteins by Neurath. Academic Press. 1954. (9) OVERSL~N, O. R. Monthly Variations in the Composition of Milk. J. Dairy Sci., 28: 305. 1945. (10) OVEI~BIAN, O. R., KEIRS, ]~. J,. AND CRAINE, E. M. Composition of Herd Milk of the Brown Swiss Breed. Ill. Agr. Expt. Sta., Bull. 567. 1953. (11) OVER~[_~:~, O. ]~., S~XNMAN~, F. P., AND WEIGH% K. E. Studies on the Composition of Milk. Ill. Agr. Expt. Sta., Bull. 325. 1929. (12) ROWLAND, S. J. The Protein Distribution in Normal and Abnormal Milk. J. Dairy Rssearch, 9: 47. 1938. (13) SMZTH, E. L. The Isolation and Properties of the Immune Proteins of Bovine Milk and Colostrum and Their Role in Immunity. A Review. J. Dairy Sci., 31: 127. 1948. (14) TUaNEa, C. W. Factors Affecting the Composition of Milk. Mo. Agr. Expt. Sta., Bull. 365. 1936.
Laboratory of Biocltemistry, Department of Dairy Sc~e~ce, University of Illinois, Urbana
For biological as well as technological reasons a greater knowledge o f the relation of breed of cattle to the production of the major proteins o f milk is important. The milk from five to seven cows from each of five major breeds was used for electrophoretic analysis. The amounts of a-, /?- and ,/casein, a-lactalbumin, B-lactoglobulin, the immune globulins, and serum albumin present in each milk were determined. Editor.
The various breeds of d a i r y cattle produce significantly different amounts of protein in their milk, and individuals within the breeds show considerable variation (9,10, 11, 12, 14). I n general the breeds producing less total milk but having a higher f a t content also produce milk containing more protein. Studies o n the proteins of skimmilk have been confined to an evaluation of the variations in the total protein or of the total casein and serum (whey) protein fractions. I n some studies the serum proteins have been f u r t h e r s e p a r a t e d into " l a c t o g l o b u l i n , " " l a c t a l b u m i n , " and " p r o t e o s c - p e p t o n e " fractions on the basis of their heat stability and solubility in concentrated salt solutions (10, 11, 12, 14). These fractionation procedures and terminology have become outmoded b y the recent isolations in purified or crystalline f o r m of the m a j o r specific protein c o n s t i t u e n t s of milk (3, 8, 13). The evaluation of the specific milk proteins m a y be accomplished most easily b y an electrophoretic analysis of the isolated casein and the isolated serum proteins even though the n u m b e r of samples t h a t can be analyzed by this procedure is limited. Casein on eleetrophoresis at p H 8.6 separates into three components (a-, fi-, and 7-casein) (8). On electrophoresis at p H 8.6 the serum proteins separate into eight or more fractions; however, f o u r of these, the immune globulins, ~-lactalbumin, fl-laetoglobulin, and blood serum albumin, compose up to 85% of the normM milk serum protein (3, 8). The inmmne globulins (euglobulin and pseudoglobulin) compose most of the classical " l a e t o g l o b u l i n " fraction and are p r o b a b l y identical to some of the immune globulins of blood (1, 5,8,13). a-Laetalbumin, fl-laetoglobulin, and serum albumin have been isolated in erystalline f o r m f r o m milk and compose essentially the classical " ' l a e t a l b u m i n " fraction (3, 8). The serum albumin of milk is identical with the Received for publication April 12, 1956. 1 Supported in part by aid from the Rockefeller Foundation. 1683
1684
a.D.
R O L L E R I ET _4.L
serum albumin of blood (2). In addition there are enzymes, unidentified minor proteins, and unspecific " p r o t e o s e - p e p t o n e " materials present (3, 8, 12). The biological and technological importance of each of the major specific proteins prompted a study to determine if there are differences between the various breeds of dairy cattle in their production of these proteins. The amounts of a-, fl-, and ~/-easein, a-lactalbumin, fi-laetoglobulin, the immune globulins, and serum albumin in milk were determined in this study. ~WlgTtIODS AND PROCEDURES
Studies in this laboratory have indicated that the stage of lactation has an effect oll the total protein production per day and the ratio of the amounts of the specific proteins in the milk relative to each other (~, 5'). The relative amounts of these proteins in the milk v a r y greatly at the start and near the end of the lactation period; thus the milk samples for thi.~ study were taken from cows that had been in lactation 2 to 6 months. Samples were taken from 28 healthy cows ill the University herd including five to seven cows from each of the five major dairy breeds. Two from each breed were in their first lactations. The samples of milk were collected at five weekly intervals during J u l y and August from groups of five to seven cows, including at least one cow from each breed. Samples of milk were taken from each cow at the morning and evening milkings of two consecutive days; the four samples were composited proportional to the weight of each milking. The composited sample of milk was centrifuged for 20 minutes at 2000 x gravity in 300-ml. polyethylene bottles and the skimmilk was pipetted from beneath the layer of fat. An aliquot of 200 ml. of skimmilk was used for the preparation of the serum proteins for the electrophoretic analysis according to previously described procedures (3). Casein was p r e p a r e d for the electrophoretic analysis by precipitating it from 25 ml. of skimmilk with 10% acetic acid at p H 4.65, redispersing in sodium-potassium phosphate buffer ( p H 6.9, ionic strength of 0.1), reprecipitaring with 10% acetic acid, and redispersing in sodium veronal buffer ( p H 8.6, ionic strength of 0.1) to give a concentration of 2 to 4'~. Centrifugation in 50ml. plastic centrifuge tubes and homogenization in a glass tissue grinder were used for the precipitation and redispersion steps, respectively. The casein solutions were centrifuged at 2000 × gravity for 30 minutes to remove traces of fat and other opaque material. An aliquot of 17.6 ml. was dialyzed against 250 nil. of veronal buffer ( p H 8.6, ionic strength of 0.1) for 12 to 24 hours at 4 ° C. and then against 500 ml. of the same strength buffer for an additional 24-hour period. At the time of the eleetrophoretie analysis the casein solutions were diluted with equilibrated buffer to the desired concentration. The concentration of the solution actually used in the e]ectrophoretic analysis was determined by a semi-micro Kjeldahl analysis. The electrophoretic analyses were all conducted in sodium veronal buffer (pII 8.6, ionic strength of 0.1, 0.12 M) for 5,580 seconds at 1.2 ° C. with a field
1685
PROTEIN PRODUCTION IN THE BOVINE
strength of 7.8 volts per square centimeter as previously described (3, 7). The casein solutions were analyzed at a concentration of about 1.5% and the serum protein solutions at 2-3% protein. Difficulties encountered with the isolation procedure and the electrophoretic analyses resulted in the loss of some samples. I n some instances, only the casein or the serum protein fraction for a given cow was carried through the complete analysis without loss. The electrophoretic p a t t e r n s of the serum proteins were divided into four areas r e p r e s e n t i n g the immune globulins, a-lactalb~min, fi-lactoglobulin, and serum albumin (3). Since in addition to the usual electrophoretic anomalies other nfinor protein components are present, the actual amounts of each constituent are p r o b a b l y 5 to 15% lower t h a n the figures r e p o r t e d (3). TABLE 1
Within and between breeds components of variance for the protein eonstituents of ~niUc Components of variance Protein component
W i t h i n breed
Between breeds
On a basis of grams per 100 ml. sl~immilk Casein : a-casein fl-easein 3,~casein Total casein
0.0831 0.0204 0.0017 0.1563
0.0166 0.0091 ~ 0.0019 ~'~ 0.0131
Serum p r o t e i n s : I m m u n e globulins adactalbumin ¢/-laetoglobulin Serum albumin Total serum p r o t e i n s
0.0044 0.0008 0.0063 0.0001 0.0143
trace" trace ~ 0.0001 trace" 0.0004
Derived f r o m a m e a n square t h a t was significant at the 0.05 level of probability. ~ Derived f r o m a m e a n square t h a t w a s significant a t the 0.01 level of p r o b a b i l i t y . Components were positive b u t the first five decimal places were zeros. RESULTS
The components of variance in Table 1 indicate t h a t there was considerably more variation between cows within the same breed t h a n between the breeds. TABLE
2
Analysis of variance of [3-casein Source Total
Mean squares
Expected mean squares
24
Between breeds 1, 2. 3. 4.
Degrees of freedom
Hol. vs (Ayr. + Guer. + J e r . + B. Sw.) Ayr. vs (Guer. + J e t . + B. Sw.) B. Sw. vs (Guer. + J e t . ) Guer. vs Jer.
W i t h i n breeds
4
0.0654 ~'
1 1 1 1
0.2413 ~ 0.0066 0.0051 0.0091
20
0.0204
( a ~ + 4.94 ~ ) a
(~)
F o r the between-breed mean squares involving the serum p r o t e i n s the coefficient of fl~ w a s 5.17.
1686
G.D. I~OLLERI ET AL
The components of variance were derived from analyses of variance tables similar to that freedom
shown
in Table
between
breeds
2.
For
every protein
were broken
down
constituent
the four
into individual
degrees of
comparisons
as is
s h o w n i n T a b l e 2, a n d t h e r e s u l t s o f t h e s e c o m p a r i s o n s a r e s h o w n i n T a b l e s 3 a n d 4. TABLE 3 The average a~ounts of proteins prese~tt in the sl~i~neailk of the various breeds
Protein component
Ayrshire
Brcwn Swiss
Guernsey Holstein
Jersey
Within breed stan(]ard deviations
Casein: (No. samples) a-casein fl-easein "/-casein Total
(g. per 100 v~l.) (5) (4) (5) 1.71" 1.83 1.92 0.85 0.84 0.82 0.08" 0.11 0.14 2.64 2.78 2.88
(7) 1.58" 0.60" 0.20" 2.38"
(4) 1.83 0.76 0.13 2.72
(25) 0.29 0.14 0.04 0.39
Serum proteins: (No. samples) Immune globulins a-laetalbumi n fl-laetoglobulin Serum albumin Total
(5) 0.06 0.11 0.31 0.03 0.51
(6) 0.09 0.13 0.30 0.04 0.56
(5) 0.08 0.15 0.39 0.04 0.66
(26) 0.07 0.03 0.08 0.01 0.12
(4) 0.97 0.11 0.31 0.04 0.53
(6) 0.08 0.1 ] 0.35 0.04 0.58
Comparison 1, Table 2, was significant for total casein and highly significant for a-, fl-, and 7-casein. b Comparison 2, Table 2, was highly significant for a- and 7-casein. TABLE 4 The ratio of ?he specific proteins present in casein and the ser~t~t proteins isolated frown the vario'~s breeds
Protein component
Ayrshire
Brown Swiss
Guernsey
ltolstein
Jersey
Within breed standard deviations
(c/c of total casein or serbian proteins)
Casein : a-casein fl-casein 7-casein
64.6 32.2" 3.1
65.8 30.1 4.1
66.4 28.3 5.2
66.3 25.2 '~ 8.5 ~
67.2 27.9 4.8
2.92 3.04 1.56
Serunl proteins : Immune globulins a-lactalbumin fl-laetoglobulin Serum albumin
11.5 21.7 61.3 5.5"
13.0 21.3 58.9 6.7
13.5 19.7 59.8 6.8
15.8 23.3 54.4" 6.6
12.8 22.2 59.6 5.6
2.71 2.18 3.55 .65
" Comparison 1, Table 2, was significant for fl- and "r-casein and for fl-laetoglobulin. Comparison 2, Table 2, was significant for fl-easein and for serum albumin. A. Amounts
o f t h e p r o t e i n s p r e s e n t i n the m i l k .
The breed averages
shown
in Table 3 indicate that the milk of the IIolstein cows contained significantly less t o t a l c a s e i n , a - c a s e i n , a n d f i - c a s e i n b u t s i g n i f i c a n t l y m o r e ~ - c a s e i n t h a n t h e m i l k o f t h e cows o f t h e f o u r o t h e r b r e e d s . T h e A y r s h i r e m i l k c o n t a i n e d s i g n i f i cantly less a-casein than the milk of the breeds other than Holstein, and sign i f i c a n t l y less 7 - c a s e i n t h a n t h e m i l k o f t h e f o u r o t h e r b r e e d s .
PROTEIN PRODUCTION IN THE BOVINE
1687
No significant differences were shown between the breeds in the total or in any of the specific serum proteins; however, the power of these tests of significance is low. It should be noted that the milk from the Holstein cows contained on the average less fl-lactoglobulin and more immune globulins than the milk of the other four breeds.
B. Relative amounts of the specific proteins present in the casein and the serum proteins. Significant differences were found between the breeds in respect to the ratio of the specific proteins present in the casein and the serum proteins. The data in Tab.le 4 show that the casein from Holstein cows contained significantly less fl-casein but significantly more v-casein than the casein from the other four breeds. The casein from the Ayrshire cows contained significantly more fl-casein than the casein from the other four breeds. The a-casein content of the total casein was practically the same (64.6 to 67.2%) for all of the breeds. Practically all of the variation shown was in the relative amounts of fl-casein and ~/-casein. The serum proteins from the Holstein cows contained significantly less fl-lactoglobulin than did the serum protein of the other four breeds, and, though not significant, they contained a higher average content of immune globulins and a-lactalbumin. The serum proteins of the Ayrshire and Jersey breeds contained significantly less serum albumin than the serum proteins of the three other breeds. DISCUSSION
Since it was a p p a r e n t that the relative and actual amounts of the specific milk proteins of the individual animals varied, the within-breed correlation coefficients between the specific constituents were calculated from the data for all animals and are shown in Table 5. The highly significant correlation between the total casein and the total serum proteins indicates that cows that produced milk high in casein composition also produced milk high in total serum proteins. The high correlations between a-casein, fl-casein, fl-lactoglobulin and ~-lactalbumin thus would be expected since these proteins compose most of the total casein and the total serum proteins. This suggests that the synthesis of these four major constituents in the m a m m a r y gland might be closely related. The amount of ~/-casein or milk serum albumin present bears no significant relationship to any of the other constituents. This agrees with the observations (6) that the p a t t e r n of production of these constituents during the lactation period is more erratic and bears little relationship to the production of the other protein constituents. I t is of interest to note that, although not significant, there is a negative correlation between the amount of 7-casein present and the amount of immune globulins, fi-lactoglobulin, and milk serum albumin. These studies indicate that the amounts of the specific proteins present in the milk of the Holstein breed appear to differ most from the other breeds, whereas the Brown Swiss, Guernsey, and Jersey breeds are remarkably alike. The a-casein content of the total casein was relatively constant for all of the individual cows. Other than the Holstein breed, which contained a significantly
1688
G.D. ROLLERI ET AL TABLE 5 Within-breed correlations between tl~e protein constituents of mill~
Correlations between grams in 100 ml. of skimmilk Total casein and total serum proteins a-casein and fl-easein a-casein and y-easeln a-casein and imnlune globulins a-casein and a-lacta]bumin a-easmn and fl-lactoglobulin a-casein and serum albumin fl-easein and "/-casein fl-easein and immune globulins fl-easein and a-lactalbumin fl-casein and fl-lactoglobulin fi-casein and serum albumin "/-casein and immune globulins "/-casein and a-lactalbumin "y-casein and fl-lactoglobulin 7-casein and serum albumin immune globulins and a-laetalbumin immune globulins and fl-laetoglobulin immune globulins and serum albumin a-laetalbumin nnd fl-laetoglotm]in a-laetalbumin and serum albumin fl-laetoglobulin and serum albumin
Correlation coefficient 0.676~* 0.599"* 0.191 0.364 0.584~ 0.538~ 0.125 0.067 0.357 0.680*~ 0.550 ~ 0.248 -0.259 0.113 --0.243 -0.052 0.487 ~ 0.504 ~ 0.489~ 0.845*~ 0.310 0.327
Degrees of freedom 17 19 19 17 17 17 17 19 17 17 17 17 17 17 17 17 20 20 20 20 20 20
Significant at the 0.05 level of probqbility. ** Significant at the 0.01 level of prol)ability. lower p e r c e n t a g e of fl-lactoglobulin ill the total s e r u m p r o t e i n s , the f o u r other b r e e d averages for f l - l a c t o g l o b u l i n were r e m a r k a b l y close. I t also has b e e n n o t e d t h a t the f l - l a c t o g l o b u l i n c o n t e n t of the s e r u m p r o t e i n s , a l t h o u g h v a r y i n g b e t w e e n cows, does n o t v a r y m u c h for tile i n d i v i d n a l cow i n the m i d d l e r a n g e s of the lactation period (6). SUMMARY A n e v a l u a t i o n of the specific milk p r o t e i n s of the nfilk of 28 d a i r y cows i n c l u d i n g the five m a j o r breeds was made b y a q u a n t i t a t i v e e l e c t r o p h o r e t i c procedure. L a r g e i n d i v i d u a l v a r i a t i o n s a n d also s i g n i f i c a n t b r e e d differences were f o u n d . The nfilk of H o l s t e i n cows c o n t a i n e d s i g n i f i c a n t l y less t o t a l casein, a-casein a n d fl-easein, b u t s i g n i f i c a n t l y more y-casein t h a n did the m i l k of the f o u r o t h e r breeds. A y r s h i r e milk c o n t a i n e d s i g n i f i c a n t l y less a-casein t h a n d i d the m i l k of the B r o w n Swiss, G u e r n s % ' , a n d J e r s e y breeds a n d less y-casein t h a n did the m i l k of the breeds other t h a n the H o l s t e i n . A l l of the b r e e d s p r o d u c e d casein c o n t a i n i n g a p p r o x i m a t e l y the same a m o u n t of a-casein; however, the casein of the H o l s t e i n breed c o n t a i n e d s i g n i f i c a n t l y less fl-casein a n d more y-casein, whereas casein f r o m the A y r s h i r e b r e e d c o n t a i n e d s i g n i f i c a n t l y more fl-casein. T h e s e r u m p r o t e i n s of H o l s t e i n m i l k also c o n t a i n e d s i g n i f i c a n t l y less fi-laetoglobulin. H i g h c o r r e l a t i o n s were f o u n d b e t w e e n the a m o u n t s i n s k i m m i l k of a-casein, fl-easein, a - l a e t a l b u m i n , f l - l a e t o g l o b u l i n a n d to a lesser e x t e n t the i m m u n e g l o b u l i n s , b u t l i t t l e r e l a t i o n s h i p was f o u n d b e t w e e n the above c o n s t i t u e n t s a n d the a m o u n t of y-casein or s e r u m a l b u m i n .
PROTEIN PRODUCTION IN THE BOVINE
1689
REFERENCES (1) ASKO~'AS, B. A., CAMPBELL, P. N., HU~[r~EY, J. H., AI~D WORK, T. S. The Source of Antibody Globulin in Rabbit Milk and Goat Colostrum. Biochem. J., 56: 597. 1954. (2) COULS01',-, E. J., AND STEVENS, 11. The Serologic'~| ~clationship of Bovine Whey Albumin to Sermn Albumin. J. Biol. Chem., 187: 355. 1.950. (3) LARSOR~, B. L., AND ROLLERI, G. D. Hcat Denatm'ation of the Specific Serum Proteins in Milk. J. Dairy Sci., 38: 351. 1955. (4) LA~SOU', B. L., ROLLERI, G. D., AND ]t][ISNDALL, I~. A. Protein Production in the Bovine. Comtmrison of Daily Protein, ]!'at, and Milk Production During the Entire Lactation Period. J. Dairy Sci., 39: 204. ]956. (5) LAESON, B. L., ROLL~RI, G. D., :~ND KENDALL, K. A. Protein Production in the Bovine. Changes in the Specific Blood Serum Proteins Associated with Pregnancy and Parturition. (Abs.) J. Dairy Sci., 37: 662. 1954. (Paper in preparation.) (6) LA~S0~~, B. L., ROLDE,[~I, G. D., AND KENDALL, K. A. Protein Production in the Bovine. Daily Production of the Specific Milk Proteins During the Lactation Period. (Abs.) J. Dairy Sci., 38: 619. 1955. ( P a p e r in preparation). (7) LARSON, lB. L., AND SALISBURY, G. W. The Proteins of Bovine Seminal Plasma. I. Preliminary and Electrophoretic Studies. J. Biol. Chem., 205: 741. 1954. (8) McMEEKI~r, T. L. Milk Proteins. Chaptcr 16 in The Proteins by Neurath. Academic Press. 1954. (9) OVERSL~N, O. R. Monthly Variations in the Composition of Milk. J. Dairy Sci., 28: 305. 1945. (10) OVEI~BIAN, O. R., KEIRS, ]~. J,. AND CRAINE, E. M. Composition of Herd Milk of the Brown Swiss Breed. Ill. Agr. Expt. Sta., Bull. 567. 1953. (11) OVER~[_~:~, O. ]~., S~XNMAN~, F. P., AND WEIGH% K. E. Studies on the Composition of Milk. Ill. Agr. Expt. Sta., Bull. 325. 1929. (12) ROWLAND, S. J. The Protein Distribution in Normal and Abnormal Milk. J. Dairy Rssearch, 9: 47. 1938. (13) SMZTH, E. L. The Isolation and Properties of the Immune Proteins of Bovine Milk and Colostrum and Their Role in Immunity. A Review. J. Dairy Sci., 31: 127. 1948. (14) TUaNEa, C. W. Factors Affecting the Composition of Milk. Mo. Agr. Expt. Sta., Bull. 365. 1936.