Specific Gravity of Bovine Colostrum Immunoglobulins as Affected by Temperature and Colostrum Components G. D. MECHOR1 and Y. T. GROHN2 Department of Clinical Sciences New York State Col/8ge of Veterinary Medicine Comell University Ithaca. NY 14853
L. R. McDOWELL Department of Animal Science University of Florida Gainesville, 32611
R. J. VAN SAUN Department of Animal Science College of Agriculture and Ufe Sciences COmell University 11haca. NY 14853 ABSTRACT
The effects of temperature and coloscomponents on specific gravity in bovme colostrum were investigated. Thirty-nine first milking colostrum samples were collected from Holstein cows. The samples were assayed for atocopherol, fat, protein, total solids, and IgG. The concentrations of total solids. total protein, total IgO, and fat in colostrum were 26.6, 12.5, 3.7, and 9.4 g/100 g, respectively. A range of 1.8 to 24.7 Jlg/ml for a-tocopherol was measured in the colostrum samples. Specific gravity of the colostrum was measured using a hydrometer in increments of 5'C from 0 to 40·C. Specific gravity explained 76% of the variation in colostral total IgG at a colostrum temperature of 20·C. The regression model was improved only slightly with the addition of protein, fat, and total solids. The model for samples at 20'C was IgG (milligrams per milliliter) = 958 x (specific gravity) - 969. Measurement of specific gravity at variable temperatures necessitated inclusion of temperature in the model for estima-
tion of IgG. Inclusion of the other components of colostrum into the model slightly improved the fit. The regression model for samples at variable temperatures was as follows: IgG (milligrams per milliliter) 853 x (specific gravity) + .4 x temperature (Celsius degrees) 866. (Key words: colostrum, specific gravity, immunoglobulin)
tru~
Received April 7. 1992. Accepted July 8, 1992. ISection of Medicine. 2Section of Epidemiology. 1992 J Dairy Sci 75:3131-3135
=
Abbreviation key: SG = specific gravity, SRID
= single radial
immunodiffusion.
INTRODUCTION
The measurement of specific gravity (SG) of colostrum has been a management practice utilized by many dairy farmers to collect colostrum that has adequate Ig for feeding the newborn dairy calf. Fleenor and Stott (6) first developed a regression equation to estimate Ig concentration in colostrum from the SG of fresh whole colostrum. A significant effect of colostrum temperature on SG of colostrum has been demonstrated (9). The effect of temperature was not consistent through different quality strata of colostrum, suggesting a possible confounding effect of different constituents of colostrum on SG at various temperatures (9). In that study (9), we used colostrometer readings to provide estimates of globulin concentration in colos3131
3132
MECHOR ET AL.
trum. No attempt was made to investigate how accurately a colostrometer measures colostral globulin concentrations from the SG. Thus, the objectives of the current study were 1) to evaluate the relationship of colostral SG and IgG content, 2) to study how different constituents of colostrum (total solids, total protein, and fat) affect this relationship, 3) to develop a model to estimate IgG concentration of colostrum using the SG when the temperature effect is considered, and 4) to measure (X-tocopherol in the colostrum of dairy cows maintained on dry cow rations with recommended vitamin E supplementation. MATERIALS AND METHODS Collection and Measurement of Samples
Thirty-nine samples of colostrum were collected from four commercial dairy herds in the Ithaca, New York area. One sample from each of 39 Holstein cows was taken at the fIrst milking within 24 h of parturition. Forty-one percent of the cows were in their fIrst lactation, 32% in their second lactation, and the remaining 27% in their third lactation or greater. Colostrum samples were collected before calves had the opportunity to nurse. Cows were machine-milked, and colostrum was mixed thoroughly before a 5OO-ml sample was taken for analysis. All samples were placed into a cooler immediately after collection, and the samples were picked up from the dairies within 12 h of collection. Colostrum was cooled, and its temperature was modifIed in a water bath. SpecifIc gravity of colostrum was measured in increments of S·C from 0 to 40·C using an SG hydrometer. A colostrometer (Biogenics, Oakland, CA) was used to measure IgG concentration indirectly at a colostrum temperature of 20·C. The hydrometer and colostrometer readings were taken on the fresh colostrum. Approximately 35 ml of colostrum were placed into a polypropylene sample bottle containing a preservative for later measurement of fat, protein, and total solids. Samples of colostrum were frozen for later evaluation of IgG and a-tocopherol. Quantitative Analysis of Constituents
Colostrum IgG (total IgG) concentrations were determined by single radial immunodiffuJournal of Dairy Science Vol. 75, No. 11, 1992
sion (SRID) methods using a commercial test kit (VMRD, Pullman, WA). Colostrum was thawed at 20·C and mixed thoroughly before plating. All samples were plated in duplicate at dilutions of 1: 1 (vol/vol) with saline. Total solids were determined by a forced-air oven method for raw milk without a steam bath predry. Samples were heated in a water bath to 38·C and then oven-dried at 100·C ± 1 for 4 h. The samples were cooled to room temperature (20·C) in a desiccator and weighed to the nearest. .01 mg. The fat content was determined with the modifIed Mojonnier ether extraction method as outlined by the AOAC (1). Duplicate samples were measured when fat content exceeded the expected range of fat in colostrum. True protein content of the colostrum was determined by Kjeldahl N determination by a new method recognized by the AOAC (1, 2). The new technique corrects for the overestimation of true protein by earlier techniques related to the presence of NPN (2). a-Tocopherol in colostrum was quantifIed using HPLC. Colostrum samples were prepared as serum samples described by Hatam and Kayden (7) without saponifIcation, and extraction was carried out in hexane. The HPLC method was based on the separation system described by Cort et al. (4). Statistical Analysis
Regression analysis was carried out using the general linear models technique of SAS (16). SignifIcance was noted at P < .05. RESULTS AND DISCUSSION
Colostrum IgO concentration on.7 ± 1.3 gJ 100 ml was slightly lower than the value of 4.8 ± 2.2 g/IOO ml reported by Pritchett et al. (13) and clearly lower than the value of 5.7 ± 3.7 g/ 100 ml for colostrum globulin reported by Fleenor and Stott (6). Lactation number did not have a significant effect on colostrum IgG content. Total solids and total protein were positively correlated, and fat was negatively correlated, with IgG (Tables 1 and 2). The correlations among all colostral constituents, except total solids, and SG were significant (Table 1). The fat content in the colostrum samples of 9.4 ± 4.4 g/loo ml was higher than
3133
IMMUNOOLOBULIN CONCENTRATION IN COLOSTRUM TABLE 1. Means and coefficients of variation for constituents in colostrum. l
CV (%)
- - (gll00 g) X SD 26.6 5.1 12.5 3.4 3.7 1.3 9.4 4.4
Total solids Total protein IgG Fat
Correlation Correlation to specific to IgG gravity
19.3 27.2
35.7 46.5
1Thirty-nine first milking colostrum samples collected from Holstein cows within
.36* .86* 1.0 -.36*
.23 .87* .87· -.54*
24 h of parturition.
·Constituent related to IgG or specific gravity (P < .05).
the mean of 2.0 g/100 ml reported by Fleenor and Stott (6). Specific gravity explained 76% of the variation in IgG concentration of colostrum (fable 3). Separate addition of fat, total solids, and total protein to the model improved the fit of prediction equations to 77, 78, and 80%, respectively. Using the combination of the constituents of colostrum did not further improve the models, and the coefficients were not significant (Table 3). If the temperature effect was not considered in the conversion equation when the SG of the samples was measured at variable temperatures, the model explained only 51 % of the total variation (Table 4). Inclusion of temperature in the model improved the fit of the model to 63%. Addition of fat, total solids, and total protein further improved the fit to 64, 67, and 76%, respectively. Inclusion of all of the components in the model gave the highest R2, .77. The slight improvement, however, is irrelevant in the field setting, because the values of colostrum constituents likely are not readily available for the approximation of IgG concen-
tration. Although the best model that recognizes the temperature effect improved the fit from 63 to 77%, 23% of the variation still remained unexplained by the SG and the constituents of colostrum. In our previous report (9) describing a temperature effect on colostrometer readings, the regression model developed explained 92.5% of the total variation of gamma globulin when colostrometer reading and temperature were used as predictors. The discrepancy between this model and the present one can be explained by the fact that, in the earlier model, we relied on the ability of the colostrometer to predict Ig concentrations in colostrum accurately. The original equation described by Fleenor and Stott (6) to predict globulin concentration from SG explained 70% of the variation, which compares favorably with our equation developed at colostrum temperatures of 20·C, which explained 76% of the variation of IgG in colostrum. The equation used by Fleenor and Stott (6) to develop the colostrometer was based on samples of serial dilution
TABLE 2. Regression equations to convert measured colostrum constituents to colostrum IgGI Parameter Intercept Total protein Total solids Fat R2 P
Regression coefficient
SE
-2.4 3.4
4.2 .3
Regression coefficient
SE
Regression coefficient
SE
13.6
11.3
48.9
4.9
.9
.4 -1.0 .13 .03
.5
.74
.13
.0001
.03
lThirty-nine first milking colostrum samples collected from Holstein cows within 24 h of parturition. Journal of Dairy Science Vol. 75, No. 11, 1992
3134
MECHOR ET AL.
TABLE 3. Regression equations to convert colostral specific gravity and colostrum constituents to colostrum IgG. Specific gravity was measured using a hydrometer at a colostral temperature of 20'C.l.2 Intercept
Specific gravity
a
SE
PI
SE
-969 -544 -924 -1053 -597 --{i12 -848 -571
93 169 93 112 187 217 190 233
958 535 905 1035 584 599 830 559
88 167 89 105 182 211 186 228
Total protein
P2
5E
Fat
Total solids
P3
5E
5E
P4
.6
1.6
.7 .6
1.4· 1.5 1.4·
.7
.4
.2
.2·
.2
.6· .3·
.5 .5
.4
.3
.I. -.3· .I.
.3 .6 .6
R2
P
L. R. McDOWELL Department of Animal Science University of Florida Gainesville, 32611
R. J. VAN SAUN Department of Animal Science College of Agriculture and Ufe Sciences COmell University 11haca. NY 14853 ABSTRACT
The effects of temperature and coloscomponents on specific gravity in bovme colostrum were investigated. Thirty-nine first milking colostrum samples were collected from Holstein cows. The samples were assayed for atocopherol, fat, protein, total solids, and IgG. The concentrations of total solids. total protein, total IgO, and fat in colostrum were 26.6, 12.5, 3.7, and 9.4 g/100 g, respectively. A range of 1.8 to 24.7 Jlg/ml for a-tocopherol was measured in the colostrum samples. Specific gravity of the colostrum was measured using a hydrometer in increments of 5'C from 0 to 40·C. Specific gravity explained 76% of the variation in colostral total IgG at a colostrum temperature of 20·C. The regression model was improved only slightly with the addition of protein, fat, and total solids. The model for samples at 20'C was IgG (milligrams per milliliter) = 958 x (specific gravity) - 969. Measurement of specific gravity at variable temperatures necessitated inclusion of temperature in the model for estima-
tion of IgG. Inclusion of the other components of colostrum into the model slightly improved the fit. The regression model for samples at variable temperatures was as follows: IgG (milligrams per milliliter) 853 x (specific gravity) + .4 x temperature (Celsius degrees) 866. (Key words: colostrum, specific gravity, immunoglobulin)
tru~
Received April 7. 1992. Accepted July 8, 1992. ISection of Medicine. 2Section of Epidemiology. 1992 J Dairy Sci 75:3131-3135
=
Abbreviation key: SG = specific gravity, SRID
= single radial
immunodiffusion.
INTRODUCTION
The measurement of specific gravity (SG) of colostrum has been a management practice utilized by many dairy farmers to collect colostrum that has adequate Ig for feeding the newborn dairy calf. Fleenor and Stott (6) first developed a regression equation to estimate Ig concentration in colostrum from the SG of fresh whole colostrum. A significant effect of colostrum temperature on SG of colostrum has been demonstrated (9). The effect of temperature was not consistent through different quality strata of colostrum, suggesting a possible confounding effect of different constituents of colostrum on SG at various temperatures (9). In that study (9), we used colostrometer readings to provide estimates of globulin concentration in colos3131
3132
MECHOR ET AL.
trum. No attempt was made to investigate how accurately a colostrometer measures colostral globulin concentrations from the SG. Thus, the objectives of the current study were 1) to evaluate the relationship of colostral SG and IgG content, 2) to study how different constituents of colostrum (total solids, total protein, and fat) affect this relationship, 3) to develop a model to estimate IgG concentration of colostrum using the SG when the temperature effect is considered, and 4) to measure (X-tocopherol in the colostrum of dairy cows maintained on dry cow rations with recommended vitamin E supplementation. MATERIALS AND METHODS Collection and Measurement of Samples
Thirty-nine samples of colostrum were collected from four commercial dairy herds in the Ithaca, New York area. One sample from each of 39 Holstein cows was taken at the fIrst milking within 24 h of parturition. Forty-one percent of the cows were in their fIrst lactation, 32% in their second lactation, and the remaining 27% in their third lactation or greater. Colostrum samples were collected before calves had the opportunity to nurse. Cows were machine-milked, and colostrum was mixed thoroughly before a 5OO-ml sample was taken for analysis. All samples were placed into a cooler immediately after collection, and the samples were picked up from the dairies within 12 h of collection. Colostrum was cooled, and its temperature was modifIed in a water bath. SpecifIc gravity of colostrum was measured in increments of S·C from 0 to 40·C using an SG hydrometer. A colostrometer (Biogenics, Oakland, CA) was used to measure IgG concentration indirectly at a colostrum temperature of 20·C. The hydrometer and colostrometer readings were taken on the fresh colostrum. Approximately 35 ml of colostrum were placed into a polypropylene sample bottle containing a preservative for later measurement of fat, protein, and total solids. Samples of colostrum were frozen for later evaluation of IgG and a-tocopherol. Quantitative Analysis of Constituents
Colostrum IgG (total IgG) concentrations were determined by single radial immunodiffuJournal of Dairy Science Vol. 75, No. 11, 1992
sion (SRID) methods using a commercial test kit (VMRD, Pullman, WA). Colostrum was thawed at 20·C and mixed thoroughly before plating. All samples were plated in duplicate at dilutions of 1: 1 (vol/vol) with saline. Total solids were determined by a forced-air oven method for raw milk without a steam bath predry. Samples were heated in a water bath to 38·C and then oven-dried at 100·C ± 1 for 4 h. The samples were cooled to room temperature (20·C) in a desiccator and weighed to the nearest. .01 mg. The fat content was determined with the modifIed Mojonnier ether extraction method as outlined by the AOAC (1). Duplicate samples were measured when fat content exceeded the expected range of fat in colostrum. True protein content of the colostrum was determined by Kjeldahl N determination by a new method recognized by the AOAC (1, 2). The new technique corrects for the overestimation of true protein by earlier techniques related to the presence of NPN (2). a-Tocopherol in colostrum was quantifIed using HPLC. Colostrum samples were prepared as serum samples described by Hatam and Kayden (7) without saponifIcation, and extraction was carried out in hexane. The HPLC method was based on the separation system described by Cort et al. (4). Statistical Analysis
Regression analysis was carried out using the general linear models technique of SAS (16). SignifIcance was noted at P < .05. RESULTS AND DISCUSSION
Colostrum IgO concentration on.7 ± 1.3 gJ 100 ml was slightly lower than the value of 4.8 ± 2.2 g/IOO ml reported by Pritchett et al. (13) and clearly lower than the value of 5.7 ± 3.7 g/ 100 ml for colostrum globulin reported by Fleenor and Stott (6). Lactation number did not have a significant effect on colostrum IgG content. Total solids and total protein were positively correlated, and fat was negatively correlated, with IgG (Tables 1 and 2). The correlations among all colostral constituents, except total solids, and SG were significant (Table 1). The fat content in the colostrum samples of 9.4 ± 4.4 g/loo ml was higher than
3133
IMMUNOOLOBULIN CONCENTRATION IN COLOSTRUM TABLE 1. Means and coefficients of variation for constituents in colostrum. l
CV (%)
- - (gll00 g) X SD 26.6 5.1 12.5 3.4 3.7 1.3 9.4 4.4
Total solids Total protein IgG Fat
Correlation Correlation to specific to IgG gravity
19.3 27.2
35.7 46.5
1Thirty-nine first milking colostrum samples collected from Holstein cows within
.36* .86* 1.0 -.36*
.23 .87* .87· -.54*
24 h of parturition.
·Constituent related to IgG or specific gravity (P < .05).
the mean of 2.0 g/100 ml reported by Fleenor and Stott (6). Specific gravity explained 76% of the variation in IgG concentration of colostrum (fable 3). Separate addition of fat, total solids, and total protein to the model improved the fit of prediction equations to 77, 78, and 80%, respectively. Using the combination of the constituents of colostrum did not further improve the models, and the coefficients were not significant (Table 3). If the temperature effect was not considered in the conversion equation when the SG of the samples was measured at variable temperatures, the model explained only 51 % of the total variation (Table 4). Inclusion of temperature in the model improved the fit of the model to 63%. Addition of fat, total solids, and total protein further improved the fit to 64, 67, and 76%, respectively. Inclusion of all of the components in the model gave the highest R2, .77. The slight improvement, however, is irrelevant in the field setting, because the values of colostrum constituents likely are not readily available for the approximation of IgG concen-
tration. Although the best model that recognizes the temperature effect improved the fit from 63 to 77%, 23% of the variation still remained unexplained by the SG and the constituents of colostrum. In our previous report (9) describing a temperature effect on colostrometer readings, the regression model developed explained 92.5% of the total variation of gamma globulin when colostrometer reading and temperature were used as predictors. The discrepancy between this model and the present one can be explained by the fact that, in the earlier model, we relied on the ability of the colostrometer to predict Ig concentrations in colostrum accurately. The original equation described by Fleenor and Stott (6) to predict globulin concentration from SG explained 70% of the variation, which compares favorably with our equation developed at colostrum temperatures of 20·C, which explained 76% of the variation of IgG in colostrum. The equation used by Fleenor and Stott (6) to develop the colostrometer was based on samples of serial dilution
TABLE 2. Regression equations to convert measured colostrum constituents to colostrum IgGI Parameter Intercept Total protein Total solids Fat R2 P
Regression coefficient
SE
-2.4 3.4
4.2 .3
Regression coefficient
SE
Regression coefficient
SE
13.6
11.3
48.9
4.9
.9
.4 -1.0 .13 .03
.5
.74
.13
.0001
.03
lThirty-nine first milking colostrum samples collected from Holstein cows within 24 h of parturition. Journal of Dairy Science Vol. 75, No. 11, 1992
3134
MECHOR ET AL.
TABLE 3. Regression equations to convert colostral specific gravity and colostrum constituents to colostrum IgG. Specific gravity was measured using a hydrometer at a colostral temperature of 20'C.l.2 Intercept
Specific gravity
a
SE
PI
SE
-969 -544 -924 -1053 -597 --{i12 -848 -571
93 169 93 112 187 217 190 233
958 535 905 1035 584 599 830 559
88 167 89 105 182 211 186 228
Total protein
P2
5E
Fat
Total solids
P3
5E
5E
P4
.6
1.6
.7 .6
1.4· 1.5 1.4·
.7
.4
.2
.2·
.2
.6· .3·
.5 .5
.4
.3
.I. -.3· .I.
.3 .6 .6
R2
P
