Nomenclature of the proteins of cows' milk--sixth revision.

This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.

Association of kappa-casein glycosylation with milk production and composition in Holsteins.

A total of 545 milk samples were collected from 53 Holstein cows for 1 yr. On test day, morning milk yields were recorded; milk samples were analyzed for N-acetylneuraminic acid, protein, fat, casein, kappa-casein, and SCC. the relationship between the degree of glycosylation of kappa-casein and morning milk yield and composition was investigated. Data were analyzed using least squares procedures with a model that included test day, parity, stage of lactation, SCC, and phenotype for kappa-casein as fixed effects and N-acetylneuraminic acid content of kappa-casein as covariate. After adjustments were made for effects of environmental and genetic factors, the degree of glycosylation of kappa-casein was associated with morning milk yield, protein, and casein content. Milk yield increased linearly with an increase of N-acetylneuraminic content up to approximately 70 micrograms/mg of kappa-casein.

Identification of kappa-casein genotype in Holstein sires: a comparison between analysis of milk samples from daughters and direct analysis of semen samples from sires by polymerase chain reaction.

Two different methods were used to determine kappa-casein genotypes of Holstein sires. In the earlier procedure, genotypes of sires were deduced by analyzing frequency distribution data of kappa-casein variants obtained through typing of milk samples from daughters by electrophoresis. The second method involved direct analysis of DNA obtained from semen samples of the sires. The polymerase chain reaction was used to amplify a 99-bp region from the kappa-casein gene that contains nucleotide substitutions that are diagnostic of variants A and B. Identity of the amplified product was confirmed by sequencing. Results obtained by both methods of genotyping were similar. For the 42 sires that were analyzed by both methods, the distribution of kappa-casein genotypes were 31 AA, 11 AB, and 0 BB. The frequency of B allele for kappa-casein in the sire population studied was lower than in larger Holstein cow populations. Due to the high demand for kappa-casein B milk by the dairy industry, it might be advantageous to increase this allele in the dairy cattle population by identifying sires with homozygous kappa-casein B and using them more frequently as service sires.

Relationships of milk protein types to lifetime performance.

Data from 889 cows at five research stations of Agriculture Canada were used to study the effects of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin loci on herdlife and total yield over fixed parities (one, two, and three parity) and to a fixed age (36, 48, and 61 mo). Actual yields of all cows were utilized to compute total milk regardless of lactational length. The model consisted of station, breed, year of birth, season of birth, and milk protein types with age at first calving as a covariate. Of the four milk protein types studied, only the kappa-casein locus had significant effects on fixed parity and fixed age total milk and herdlife. Cows with BB kappa-casein type outproduced those with AB or AA kappa-casein types in three parity total milk by 963 and 1657 kg, respectively. Considering total milk accumulated up to 61 mo of age in life, cows with BB kappa-casein type outperformed their counterparts with AB or AA kappa-casein types by 1050 and 1923 kg, respectively. Complete replacement of A by B allele at kappa-casein locus would result in an increase of 1657 kg in three parity total milk and an increase of 1923 kg in 61-mo total milk. The moderate gene frequency of kappa-casein B allele in the current dairy population can be increased to improve lifetime total milk to the benefit of the dairy industry.

Environmental factors affecting plasmin activity in milk.

A total of 367 milk samples were collected from 43 individual Holstein cows during 1 yr. Samples were analyzed for plasmin activity, total casein, alpha s-casein (alpha s1-casein + alpha s2-casein), beta-casein, kappa-casein, and SCC. Least squares analyses showed that SCC in milk were positively related (r = .62) to plasmin activity. An increase of SCC from 100,000 to 1,300,000/ml was associated with a 2.3-fold increase in plasmin activity (100 vs. 230 x 10(-6) units/ml). Increased plasmin activity was associated with advancing stage of lactation and older cows after appropriate adjustments were made for the effects of milk yield and SCC. Milk samples obtained in fall and winter were higher, but not significantly, in plasmin activity. Plasmin activity was also associated with major casein components and milk pH. Correlations coefficients between plasmin and alpha s-casein, beta-casein, and pH were -.14, -.27, and .19.

A study of the stability of Record of Performance milk samples for infrared milk analysis.

The effect of switching individual cow Record of Performance milk samples from on-farm Babcock analytical system to centralized infrared analysis was investigated. The effects of lipolytic activity on the signals of the fat (5.73/5.58 and 3.48/3.56 microns) and protein (6.46/6.68 microns) wavelengths of filter instruments showed that lipolysis decreased the infrared fat signal and concurrently increased the protein signal. The chain length (3.48/3.56 microns) signal was unaffected by lipolysis due to the concordant movement of the reference wavelength baseline with the changes in the sample wavelength signal. Storage trials (3 d) of preserved milk samples indicated no direct relation between fat signal depression and chemically determined free fatty acid levels. Microbial growth and agitation also contributed to changes observed in samples studied under controlled conditions. Based on concurrent infrared, Babcock, and Mojonnier analyses of approximately 900 samples on d 1 and 3, there was a consistent but marginal drop in the carbonyl (5.73/5.56 microns) fat signal and an increase in the amide (6.46/6.68 microns) protein signal, but these changes were of limited significance. A study of 3-d and older samples indicated that significant levels of acid were produced, indicative of microbial growth. A comparison of Babcock results carried out on-farm, Mojonnier analyses in the laboratory, and their corresponding infrared results did not show statistically significant differences. Other than the inherent variability present in the individual cow samples, there were minor quality changes occurring under the present sample handling system due to a combination of lipolysis, microbial growth, and acid production. Refrigeration of Record of Performance samples during transport and at the central laboratory is recommended to minimize these changes.

Variation in milk protein concentrations associated with genetic polymorphism and environmental factors.

Concentrations of alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, alpha-lactalbumin, serum albumin, and immunoglobulin in milk from 1888 Holstein cows were determined monthly over the lactation period. Cows were phenotyped for genetic variants of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Least squares analyses showed variations in individual proteins due to parity number, month of test, stage of lactation, somatic cell count, fat content, milk yield, and phenotypes of cows for milk proteins. beta-Casein declined and serum proteins increased with advancing age of cows. Concentration of individual proteins decreased during the first 2 to 3 mo in lactation and then increased as lactation progressed. alpha s1-Casein variants significantly affected concentrations of alpha s-casein (BC greater than BB greater than AB) and beta-lactoglobulin (AB greater than BB greater than BC). Variant B for beta-casein is associated with lower alpha s-casein, beta-lactoglobulin, immunoglobulins, and higher beta-casein and alpha-lactalbumin concentrations than variant A1, A2, or A3. Milk from BB kappa-casein, and BB beta-lactoglobulin cows contained more alpha s-casein, kappa-casein, and less beta-lactoglobulin than milk from AA cows for the two proteins. Concentrations of all proteins were negatively correlated with milk production. Increased somatic cell counts were associated with lower beta-casein and higher concentrations of other proteins. Fat content of milk was positively correlated with the three casein fractions and beta-lactoglobulin.

Association of milk protein types with growth and reproductive performance of dairy heifers.

A total of 890 heifers was used to study the effects of four milk protein loci (alpha S1-casein, beta-casein, kappa-casein, and beta-lactoglobulin) on heifer growth and reproduction. The additive effects of gene substitutions at the four milk protein loci were significant only in 4 of 56 cases for all traits studied. Dominance effects at alpha S1-casein, beta-casein, and kappa-casein loci were not significant for any traits except beta-casein locus on body weight at first calving. Heifers with AB type of beta-lactoglobulin showed greater body weights and measurements and gestation length than the AA or BB type, indicating an overdominance effect. Heifers with AB type of beta-lactoglobulin were significantly younger at first conception and at first freshening and had fewer number of days from first service to conception than the AA or BB type, indicating underdominance effect. Thus, beta-lactoglobulin locus shows overdominance, underdominance, or no dominance, depending upon the traits considered. The four milk protein loci contributed more dominance variance than additive variance to total phenotypic variance. This might account for the existence of milk protein polymorphism in the cattle population. The combined genotypes of the four milk protein loci showed significant effects on 2 of 14 traits studied.

Effects of milk protein loci on first lactation production in dairy cattle.

A total of 920 cows of Holstein-based H line, Ayrshire-based A line, and cross-bred C line between H and A lines was used to determine the genotypic and gene frequencies of milk protein types and to study the relationships of milk protein loci to first lactation yields. Effects of milk protein loci on first lactation performance were examined using classification and gene substitution models. Gene frequencies at the five milk protein loci studied were similar to those reported in the literature. Gene substitution at alpha s1-casein locus showed the greatest effects on first lactation yields compared to those at other milk protein loci. Unfortunately, the favorable B allele at this locus is almost fixed (the frequency of the B allele = .955), a result of long-term selection for high milk production in dairy cattle. The extremely high frequency of a favorable allele at the alpha s1-casein locus imposes a limitation for further genetic improvement at this locus unless a more favorable mutation can be induced. Although favorable alleles at beta-casein, kappa-casein, and beta-lactoglobulin loci exerted smaller effects on first lactation performance than those at the alpha s1-casein locus, their moderate frequencies in the current population can be raised to improve lactation yields through milk protein typing. The combined contribution of the four milk protein loci accounted for 8.9% of phenotypic variance in milk yield, 8.6% in protein yield, ad 5.0% in fat yield.

Percentages of protein and nonprotein nitrogen with varying fat and somatic cells in bovine milk.

Over 15 mo, 28,390 individual milk samples from 3,600 cows in 62 Quebec Holstein herds were analyzed for crude protein, true protein, nonprotein nitrogen, fat, and somatic cell counts. Unadjusted means with standard errors were 3.51% +/- .002, 3.31% +/- .002, 31.70 mg/100 ml +/- .12, 3.67% +/- .004, and 297,230 cells/ml +/- 4002. Nonprotein nitrogen as a percentage of total nitrogen was 5.57% +/- .02. Least squares analyses showed significant effects of herd, age of cow, month of test, stage of lactation, somatic cell count, and fat percentage on contents of crude protein, true protein, and ratio of nonprotein nitrogen to total nitrogen. Highly variable nonprotein nitrogen fraction content during different months of the year and various stages of lactation is responsible for changes of crude protein content whereas changes of crude protein for different ages of cows, fat, and somatic cells in milk are due to changes of true protein content. Automatic infrared instrument calibrated against crude protein standards can be used satisfactorily to measure crude protein in milk if variations are due to age of cow, fat, and somatic cell counts. However, the instrument becomes inaccurate for measuring large variation of crude protein caused by variability of nonprotein nitrogen due to season and stage of lactation.

Association of genetic variants of casein and milk serum proteins with milk, fat, and protein production by dairy cattle.

Polyacrylamide gel electrophoresis methods were used to phenotype caseins for 2045 cows and milk serum proteins for 3870 cows distributed in 63 Quebec dairy herds. Frequencies were: alpha s1-casein A .003, alpha s1-casein B .970, alpha s1-casein C .027; beta-casein A1 .561, beta-casein A2 .421, beta-casein A3 .011, beta-casein B .007; kappa-casein A .744, kappa-casein B .256; beta-lactoglobulin A .387, beta-lactoglobulin B .613; alpha-lactalbumin B 1.00. Overall unadjusted means for 305-day production in first lactation were: 5530 +/- 26.6 kg, 197 +/- 1.0 kg, 172 +/- 1.0 kg, 3.58 +/- .009, 3.12 +/- .009 for milk yield, fat yield, protein yield, fat percentage, and protein percentage for 1687 cows for casein systems. Similar data for 2906 cows phenotyped for milk serum proteins were: 5412 +/- 20.6 kg, 193 +/- .8 kg, 170 +/- .7 kg, 3.57 +/- .007, and 3.13 +/- 007. Least squares analysis of variance showed that herd location, month of calving, age of cow at first calving, and protein variants had significant effects on production traits. alpha s1-Casein B and beta-casein A phenotypes were associated with higher milk, fat, and protein yields than other variants in the two casein systems. Milk from kappa-casein BB and beta-lactoglobulin AA phenotypes contained .13 and .05% more protein than the AA and BB phenotypes with the AB phenotype intermediate.

Heritability of milk casein and genetic and phenotypic correlations with production traits.

Individual milk samples were obtained monthly from November 1979 to November 1981 from approximately 2,800 Holstein cows in 63 herds enrolled in the Quebec Dairy Herd Analysis Service. These milk samples were analyzed for fat, total protein, casein, and serum protein. After editing, the data comprised 2,813 lactations representing 109 sires. By approximate maximum likelihood procedures, the model included herd (absorbed), year-month, and parity fixed effects, and sire and error random effects. Lactation casein yield increased with advancing parity to parity five and then decreased slightly. Lactation casein percent and number (percent casein in protein) declined until parities four and three, respectively, and essentially remained constant thereafter. Lactation casein yield was high for lactations initiated during winter months as opposed to summer months. No seasonal trend was apparent for casein percent or number. Heritabilities for lactation casein yield, percent, and number were .11, .26, and .08. Genetic and phenotypic correlations of casein yield with milk, fat, and protein yields were large and positive. Genetic correlation of casein percent with milk yield was negative (-.76) but positive (.96) with protein percent. Genetic correlations involving casein number tended to be small.

Variability of test-day milk production and composition and relations of somatic cell counts with yield and compositional changes of bovine milk.

Between November 1979 and November 1981, 41,783 test-day observations were obtained from 63 Holstein herds in the province of Quebec. Measured were milk yield, percentages of fat, protein, casein, and serum protein, and somatic cell counts that had unadjusted means with standard errors 20.44 +/- .04 kg, 3.684 +/- .003%, 3.314 +/- 002%, 2.694 +/- .001, .669 +/- .001%, and 313625 +/- 3238/ml of milk. Casein as a percentage of total protein (casein number) was 79.35 +/- .015. Least squares analyses showed significant effects of herd, calendar month of test, age of sample at laboratory testing, stage of lactation, age of cow, and somatic cell counts on milk yield and composition. Milk yield was higher whereas percentages of fat and protein and somatic cell counts were lower in summer than in winter months. Somatic cells, fat, protein, and casein contents were high during early stages of lactation, reached a minimum at 2 mo in lactation, and rose gradually throughout the rest of the lactation. A comparison of younger cows (less than or equal to 2 yrs) with older one (greater than or equal to 6 yr) revealed that somatic cell counts increased from 166,000 to 507,000/ml of milk and casein number decreased from 80.14 to 78.88. Increase of somatic cell counts in milk was associated with increased protein content, which was mainly from the serum protein fraction as the casein fraction was not related with somatic cells.

Plasma protein patterns in captive American kestrels.

Plasma proteins of the American kestrel (Falco sparverius) were analyzed by polyacrylamide disc electrophoresis for 3 different groups of birds: laying and non-laying females, and males. The electrophoretic patterns were homogeneous for each group and showed differences in the mobility of some proteins among the 3 groups. There was no significant difference among the 3 groups in the amount of proteins and in the standard parameter of albumin to globulin ratio.

Effects of potassium dichromate and sample storage time on fat and protein by Milko-Scan and on protein and casein by a modified Pro-Milk Mk II method.

A rapid procedure is described for measuring casein in milk by a combination of isoelectric precipitation and amido black dye-binding. Individual milk samples from 38 cows were subsampled, preserved with potassium dichromate at a concentration of 8 mg/ml of milk, and analyzed for fat and protein by the Milko-Scan 300 and for protein and casein by the dye-binding procedure on day 1, 4, 7, and 10 after milk collection. Fat tests were not affected by sample storage time, but protein tests showed variation depending on the interval between milk sample collection and analysis. In a separate study effects of potassium dichromate used as a preservative at 0, 2, 4, 6, 8, and 12 mg/ml of milk on chemical determinations were investigated. No changes were significant in fat and protein analyzed by the infrared method for concentrations of preservative. There were no significant differences in protein determination by the two methods for nonpreserved samples. For preserved samples, the dye-binding method for protein gave determinations higher than those by the infrared method. The difference increased linearly with increasing concentration of potassium dichromate at .07% in protein for every 2 mg of potassium dichromate per milliliter of milk.

Environmental influences on protein content and composition of bovine Milk.

A total of 24,405 individual milk samples from approximately 2,800 Holstein-Friesian cows in 63 dairy herds enrolled in the Quebec Dairy Herd Analysis Service program were analyzed monthly for protein, serum protein, casein, and somatic cell counts during 17 mo. Unadjusted means for protein, serum protein, and casein were 3.396% +/- .002, .687% +/- .001, and 2.708% +/- .002. Least squares analyses showed significant effects of calendar month of test, stage of lactation, age of cow, and somatic cell count on milk protein content and composition. Total protein, casein, and serum protein contents of milk showed a generally increasing trend from July to December. These components were highest during the first 10 days in lactation when means were 3.81%, 3.05%, and .76% and reached a minimum at 2 mo in lactation to give corresponding means of 3.08%, 2.46%, and .62%. The proportion of casein in milk protein decreased as cows became older. For every unit of increase in log somatic cell count there was an increase in protein content of 0.99%, mainly from change in the serum protein fraction. The same change in somatic cells would decrease ratio of casein to protein by 2.79%.

Glycosaminoglycans of tissues of the domestic fowl.

Mucopolysaccharide-peptide complexes (MPS-P) from skin, comb, wattle, liver, kidney, spleen, lung, heart, ovaries, egg yolk, infundibulum, magnum, isthmus, shell gland, and vagina of the White Leghorn laying hens were isolated after extraction with acetone, papain hydrolysis, and cetyl pyridinium chloride precipitation. They were analyzed for characteristic components of the glycosaminoglycan (GAG) and carbohydrate parts of glycoproteins and for amino acids. The infrared spectra of the MPS-P were studied for detection of the GAG present. The MPS-P were submitted to cellulose acetate electrophoresis for detection of GAG and to column chromatography on an anionic resin for quantitative determination of GAG.

Studies on the in vivo uptake and incorporation of 1-14C-labeled D-hexosamines in tissues, mucopolysaccharide-peptide complexes, and glycosaminoglycans of the domestic fowl.

The incorporation of radioactivity from D-[1-14C]glucosamine and D-[1-14C]galactosamine into the tissues of skin, comb, wattle, liver, kidney, spleen, lung, heart, ovaries, egg yolk, infundibulum, magnum, isthmus, shell gland, and vagina of the White Leghorn laying hens was studied for different time intervals up to 120 h. A total of 28 laying hens were involved in this experimentation. The radioactivity was measured in the above whole tissue, the acetone extracts, the acetone-extracted tissues, the mucopolysaccharide-peptide complex (MPS-P), and in individual glycosaminoglycan (GAG) isolated from skin, comb, liver, kidney, and egg yolk. The radioactivities of the expired CO2 of the eggs and of the excreted urine and faeces were also measured in some cases. There was an increase in 14C associated with MPS-P with increasing time of experiment in all the tissues studied except in the case of the liver. The rate of increase of radioactivity was dependent on the tissue. A useful scheme for the study of the speed of the formation of GAG is to consider the acetone extract and the acetone-extracted tissue as macrocompartments from where the GAG draw hexosamines for their formation.