Effect of Adrenocorticotropin Injection and Stress on Milk Cortisol Content 1
ROBERT D. BREMEL and M I C H A E L I. G A N G W E R Department of Dairy Science University of Wisconsin Madison 53706
ABSTRACT
INTRODUCTION
The purpose of this study was to determine whether cortisol content of milk might objectively measure stress in lactating dairy cows. A procedure has been developed for measuring cortisol in milk samples by a combination of solvent extraction, chromatography, and competitive protein-binding procedures. Cortisol is normally in cow's milk, and injection of 200 IU adrenocorticotropin into five cows caused an increase in concentration of cortisol in milk from 2.5 ng/ml to 8.7 ng/ml. The stress of shipping 39 cows by truck from one dairy to another also increased the concentration of cortisol in milk and decreased milk production. We correlated the concentration of cortisol in milk with the average concentratin in blood during the interval of milk secretion. Under control conditions and after injection of adrenocorticotropin to increase circulating cortisol, the regression equations describing the transport of cortisol from blood to milk are nearly identical. These results are interpreted to indicate that the mammary gland is integrating the circulating concentration of cortisol, and, therefore, the cortisol concentration in milk reflects the average concentration in blood during the interval of milk synthesis. Because the secretion of cortisol into milk is one example of a more general phenomenon, a model is presented to describe the diffusion of materials across the mammary secretory cell and into milk.
Estrogens and progesterone are in bovine milk in concentrations generally exceeding those in blood. Although milk is not a major excretory route of the hormones, their concentrations reflect the concentration in blood, and those in milk follow concentrations of hormones in blood during the estrous cycles and pregnancy. There is limited information concerning the glucocorticoid content of bovine milk and factors which might alter it. The mammary gland has specific glucocorticoid receptors (6, 14) which are related to the physiological effect of corticoids on the mammary gland. The receptors bind corticoids and may be responsible for significant differences in arteriovenous concentrations across the mammary gland (10, 11). Cortisol concentration in milk was correlated with the post-milking concentrations in blood in (7). Because these measurements were after the milk had been secreted, they tell nothing of the relationship between the concentration in the blood and milk during the period when the milk was secreted. Cortisol in blood is considered a reliable physiological endpoint for determining animal response to stress, and studies have examined changes in blood cortisol or glucocorticoid concentrations in cows under a variety of conditions (1, 13, 14). Because of the variability in the concentration of cortisol in blood, perhaps due to the blood collection, large amounts of data are needed to determine statistical differences due to treatment (2). Nevertheless, circulating corticoid concentrations may be an indication of management problems in as much as elevated corticoids have been associated with decreased milk production (3). We felt, therefore, that sampling milk would have an advantage over sampling blood because sampling could be at the normal milking time without additional stress on the animal. Such a sample
Received December 2, 1977. 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison. 1978 J Dairy Sci 61:1103-1108
1103
l 104
BREMEL AND GANGWER
would be less apt to be influenced by shortterm variation in blood concentration of cortisol. The objective of this study was to develop a reliable assay for cortisol in milk and to determine the relationship between the concentration of cortisol in blood and in milk. Our studies focused on the relationship between the average blood concentration during the period that milk was secreted and that in the milk collected at the end of the interval. This seemed a more relevant approach than what had been done previously where a sample was taken after the milk had been collected (7). Theoretical considerations suggested that there should be a relationship between the average concentration in blood and the amount secreted in milk. Therefore, in our analysis we correlated the average blood concentration during the interval that milk was being secreted with the content in milk at the end of the interval when the cow was milked. The procedure then was tested on a group of animals subjected to the acute stress of shipping by truck to see if this stress elevated cortisol in the milk of these cows. MATERIALS AND METHODS Preparation of Samples
To extract corticoids we used duplicate 3ml samples of either fresh or previously frozen milk. Ethylenediaminetetracetic acid (EDTA) was added to the milk sample to a final concentration of .05 M by addition of .5 M EDTA at pH 7.5. A cortisol tracer (which was chromatographically purified) was mixed with each sample (2,200 dpm [1,23 H] cortisol, 42 Ci/ mmole). This sample was extracted twice with 12 ml diethyl ether (freshly opened, peroxide free) and the organic phase collected either by centrifugation or after freezing the aqueous phase in dry-ice and acetone bath. After evaporating the ether, 6 ml of methanol to water (3:7 vol/vol) were added and the less polar lipid removed by extraction twice with 6 ml of n-pentane. After the methanol to water phase was evaporated, the samples were dissolved in 1 ml methylene chloride to methanol (98:2 vol/ vol) and chromatographed on a .6 x 11.5 cm Sephadex LH-20 column in the same solvent system. The cortisol fraction was evaporated and the residue dissolved in I ml buffer (.15 M NaC1, .1 M Na-phosphate pH 7.4, .1% gelatin). Journal of Dairy Science Vol. 61, No. 8, 1978
An aliquot of 200 ul was used to determine the recovery of the 2,200 dpm of tracer initially added. Of the remaining material, aliquots of 200 and 400 ul were used in the assay for cortiSO1.
Cortisol Assay
The cortisol content in the aliquots was determined by competitive protein binding with dextran-coated charcoal and bovine transcortin. Data were accepted only if at least 20% and not more than 80% of the hormone blank (only isotopic hormone added) counts were displaced. By this criterion, analysis of variance showed that the dose response of the aliquots was not significantly different in any of the experiments. To determine the validity of the entire procedure, different amounts of unlabeled cortisol were added to bulk tank milk sample which contained 4.09 ng/ml cortisol. Cortisol additions of 5 to 20 ng/ml were chosen to maximize precision of the test. With this method two essentially independent estimates of the cortisol in the bulk tank sample are obtained - one fiom the direct measurement and the other from the intercept of the least squares line for the standard additions. The intercept gave a value of 4.02 ng/ml as compared to 4.09 by direct assay. The slope of the least squares regression line was 1.02, indicating a quantitative recovery of the added cortisol. Blood Samples
Five cows from the university herd were fitted with indwelling jugular catheters. Blood samples were taken at regular intervals, and milk samples were collected from the composite samples in the weigh jar at the normal morning and evening milkings. The interval between blood samples was 8 h during the preand post-experimental period and 30 min for the first 3 h after ACTH injection. All other intervals were approximately 4 h between sampies. Following a 40-h control, 200 IU of repository form ACTH were injected intramuscularly 12 h prior to milking. This corresponds to day zero in the figures. Blood and milk were sampled for an additional 6 days. The experimental data were divided into pre-experimental control, experimental, and post-experimental period. The post-experimental period began after 2 days when the cortisol concentrations in
MILK CORTISOL RESPONSE TO ACTH AND STRESS blood had declined to a point that was not significantly different from the concentration in the pre-experimental period as determined by analysis of variance.
boundary. The equations are a modification of the law of diffusion of Fick. The vectorial flow, Jx, of a substance is given by [1] : Jx = Lgradux
Management Stress
Cows routinely are moved by truck between two University of Wisconsin dairy farms. To determine the effects on cortisol in milk of such a stress, milk samples were collected from 39 cows for two afternoon milkings prior to moving and for two afternoon milkings after moving. The cows were moved about 6 h before the afternoon milking on the 3rd day. No blood samples were collected in this experiment. THEORY
Nonelectrolytes which are in blood and are permeable to cell membranes should be in milk at a concentration directly related to that in blood. A pregnancy test which uses the progesterone content of milk as an index takes advantage o f this principle (12), For electrolytes the situation is more complicated than that presented below due to the vectorial transport systems in the cell membranes; nevertheless, the same general principles apply. Figure 1 dramatically shows how a substance is transported through a secretory cell and interacts with a binding protein in the cell. An intercellular binding protein is not essential but is included because mammary cells contain such a protein (14). The mammary secretory cell can be considered as a penetrable boundary, and the transport of any substance, X, across it will obey the equations describing the flow across such a
BLOOD
1105
SECRETORY CELL
X
BP
BPX ~
BP "X
FLOW
[1]
Where L is a phenomenological coefficient replacing the diffusion constant in Fick's law. L = Cxcox
[21
The coefficient L is a function of the concentration, Cx, and the rate of transfer of the substance through the cell, co x. The term gradux is the gradient in chemical potential (concentration) across the cell. Thus, Jx is the amount of flow per unit time and is dependent on the blood concentration, the rate of transfer (i.e. the time it takes for the material to pass through the cell including any interaction with it within the cell), and the concentration difference between blood and milk. The flow can be in either direction; for example, a substance injected into the gland would flow toward blood because the gradient, grad/ax, would be in that direction. With a constant rate of milk secretion, J H : O = constant, the total amount transferred, Tx, in a particular time interval t i to t2 will be [3] : t2
Tx = f t~
Lgrad,xdt
[3]
Therefore, a measurement of the amount of any material that can penetrate the secretory cell will be the value o f the integral in equation [31. It will be a function of the blood concentration averaged over time. Actually, the concentration will be a ratio of two flow, JH2O and JCORT, but JH20 is assumed to be constant. The concentration of the material actually might be higher in milk as in the case of progesterone; this is because a partition coefficient between the aqueous and lipid phases must also be taken into account. A higher concentration in milk might arise if binding proteins were secreted from the cell. A more complete discussion of these principles can be found in Katchalsky and Curran (8).
I
FIG. 1. Diagramatic representation of a substanae, X, going across a mammary secretory cell and its interaction with an intracellular binding protein, BP.
RESULTS AND DISCUSSION
After corticotropin was injected, cortisol increased in the circulation. The concentrations Journal of Dairy Science Vol. 61, No. 8, 1978
1106
BREMEL AND GANGWER 6
12
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....
1111 Hill
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Cortisol in Blood
tit
2.67 + .007 X |
•
14
,6
ng/ml
FIG. 3. Relationship between average blood concentration and milk concentration of cortisol. (A) Preexperimental period; (B) Post-experimental period. The hashed line in panel A is the regression of the post-experimental data superimposed on the pteexperimental data. The average blood concentration =
~4
o
n
|
I -1
| 0
I I
I 2
I 3
I 4
I 5
I 6
I 7
DAY
FIG. 2. Effect of 200 IU ACTH injection on cortisol in milk and on milk production of 5 Holstein cows. The shaded area represents one SE about the mean which is denoted by (e). Average milk production of the group is shown in the inset.
reached a maximum of 60 ng/ml in 8 to 10 h and remained elevated above control for approximately 48 h. Cortisol concentration was also higher than control in milk during this time (Fig. 2). The increase of cortisol in milk follows the same course with time as that in blood, but the concentration reached in milk is lower. The concentration was highest in the first milking after ACTH injection. As in the inset of the figure, there was also a decrease in milk production for the group of animals after the ACTH injection. This decrease in milk production was concomitant with the elevation in cortisol in the milk and is similar in magnitude to that in (3).
If the mammary gland takes up a certain fraction of the cortisol from blood, then the concentration of cortisol in milk should be related directly to that in blood during the interval since the previous milking. To see whether the m a m m a r y gland was acting as an integrator of blood concentration we plotted the data from the above experiment for the differJournal of Dairy Science VoI. 61, No. 8, 1978
n
•~citi/~t i i=1 i=1 where c = concentration and t = time interval in hours. Each point in the plot is an evaluation of the integral in equation [31, and is the amount of cortisol flow which occurs with a given concentration gradient between blood and milk.
ent experimental periods in Fig. 3 and 4. The results indicate that during the preinjection control period (Fig. 3A) and after ACTH injection, during a period of increased adrenal activity (Fig. 4), there is a close relationship between the average concentration of cortisol in blood and in milk. The regression equations describing the relationship between the concentration of cortisol in blood and in milk are nearly identical during these periods and are not statistically different. A peculiar aspect of the data is the lack of correlation between the blood and milk concentrations after the period of increased adrenal activity in the post-experimental period (Fig. 3B). The mean concentration of cortisol in blood was lower during the postexperimental period. It was 4.3 ng/ml as compared to 6,4 ng/ml during the pre-experimental period (P
ROBERT D. BREMEL and M I C H A E L I. G A N G W E R Department of Dairy Science University of Wisconsin Madison 53706
ABSTRACT
INTRODUCTION
The purpose of this study was to determine whether cortisol content of milk might objectively measure stress in lactating dairy cows. A procedure has been developed for measuring cortisol in milk samples by a combination of solvent extraction, chromatography, and competitive protein-binding procedures. Cortisol is normally in cow's milk, and injection of 200 IU adrenocorticotropin into five cows caused an increase in concentration of cortisol in milk from 2.5 ng/ml to 8.7 ng/ml. The stress of shipping 39 cows by truck from one dairy to another also increased the concentration of cortisol in milk and decreased milk production. We correlated the concentration of cortisol in milk with the average concentratin in blood during the interval of milk secretion. Under control conditions and after injection of adrenocorticotropin to increase circulating cortisol, the regression equations describing the transport of cortisol from blood to milk are nearly identical. These results are interpreted to indicate that the mammary gland is integrating the circulating concentration of cortisol, and, therefore, the cortisol concentration in milk reflects the average concentration in blood during the interval of milk synthesis. Because the secretion of cortisol into milk is one example of a more general phenomenon, a model is presented to describe the diffusion of materials across the mammary secretory cell and into milk.
Estrogens and progesterone are in bovine milk in concentrations generally exceeding those in blood. Although milk is not a major excretory route of the hormones, their concentrations reflect the concentration in blood, and those in milk follow concentrations of hormones in blood during the estrous cycles and pregnancy. There is limited information concerning the glucocorticoid content of bovine milk and factors which might alter it. The mammary gland has specific glucocorticoid receptors (6, 14) which are related to the physiological effect of corticoids on the mammary gland. The receptors bind corticoids and may be responsible for significant differences in arteriovenous concentrations across the mammary gland (10, 11). Cortisol concentration in milk was correlated with the post-milking concentrations in blood in (7). Because these measurements were after the milk had been secreted, they tell nothing of the relationship between the concentration in the blood and milk during the period when the milk was secreted. Cortisol in blood is considered a reliable physiological endpoint for determining animal response to stress, and studies have examined changes in blood cortisol or glucocorticoid concentrations in cows under a variety of conditions (1, 13, 14). Because of the variability in the concentration of cortisol in blood, perhaps due to the blood collection, large amounts of data are needed to determine statistical differences due to treatment (2). Nevertheless, circulating corticoid concentrations may be an indication of management problems in as much as elevated corticoids have been associated with decreased milk production (3). We felt, therefore, that sampling milk would have an advantage over sampling blood because sampling could be at the normal milking time without additional stress on the animal. Such a sample
Received December 2, 1977. 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison. 1978 J Dairy Sci 61:1103-1108
1103
l 104
BREMEL AND GANGWER
would be less apt to be influenced by shortterm variation in blood concentration of cortisol. The objective of this study was to develop a reliable assay for cortisol in milk and to determine the relationship between the concentration of cortisol in blood and in milk. Our studies focused on the relationship between the average blood concentration during the period that milk was secreted and that in the milk collected at the end of the interval. This seemed a more relevant approach than what had been done previously where a sample was taken after the milk had been collected (7). Theoretical considerations suggested that there should be a relationship between the average concentration in blood and the amount secreted in milk. Therefore, in our analysis we correlated the average blood concentration during the interval that milk was being secreted with the content in milk at the end of the interval when the cow was milked. The procedure then was tested on a group of animals subjected to the acute stress of shipping by truck to see if this stress elevated cortisol in the milk of these cows. MATERIALS AND METHODS Preparation of Samples
To extract corticoids we used duplicate 3ml samples of either fresh or previously frozen milk. Ethylenediaminetetracetic acid (EDTA) was added to the milk sample to a final concentration of .05 M by addition of .5 M EDTA at pH 7.5. A cortisol tracer (which was chromatographically purified) was mixed with each sample (2,200 dpm [1,23 H] cortisol, 42 Ci/ mmole). This sample was extracted twice with 12 ml diethyl ether (freshly opened, peroxide free) and the organic phase collected either by centrifugation or after freezing the aqueous phase in dry-ice and acetone bath. After evaporating the ether, 6 ml of methanol to water (3:7 vol/vol) were added and the less polar lipid removed by extraction twice with 6 ml of n-pentane. After the methanol to water phase was evaporated, the samples were dissolved in 1 ml methylene chloride to methanol (98:2 vol/ vol) and chromatographed on a .6 x 11.5 cm Sephadex LH-20 column in the same solvent system. The cortisol fraction was evaporated and the residue dissolved in I ml buffer (.15 M NaC1, .1 M Na-phosphate pH 7.4, .1% gelatin). Journal of Dairy Science Vol. 61, No. 8, 1978
An aliquot of 200 ul was used to determine the recovery of the 2,200 dpm of tracer initially added. Of the remaining material, aliquots of 200 and 400 ul were used in the assay for cortiSO1.
Cortisol Assay
The cortisol content in the aliquots was determined by competitive protein binding with dextran-coated charcoal and bovine transcortin. Data were accepted only if at least 20% and not more than 80% of the hormone blank (only isotopic hormone added) counts were displaced. By this criterion, analysis of variance showed that the dose response of the aliquots was not significantly different in any of the experiments. To determine the validity of the entire procedure, different amounts of unlabeled cortisol were added to bulk tank milk sample which contained 4.09 ng/ml cortisol. Cortisol additions of 5 to 20 ng/ml were chosen to maximize precision of the test. With this method two essentially independent estimates of the cortisol in the bulk tank sample are obtained - one fiom the direct measurement and the other from the intercept of the least squares line for the standard additions. The intercept gave a value of 4.02 ng/ml as compared to 4.09 by direct assay. The slope of the least squares regression line was 1.02, indicating a quantitative recovery of the added cortisol. Blood Samples
Five cows from the university herd were fitted with indwelling jugular catheters. Blood samples were taken at regular intervals, and milk samples were collected from the composite samples in the weigh jar at the normal morning and evening milkings. The interval between blood samples was 8 h during the preand post-experimental period and 30 min for the first 3 h after ACTH injection. All other intervals were approximately 4 h between sampies. Following a 40-h control, 200 IU of repository form ACTH were injected intramuscularly 12 h prior to milking. This corresponds to day zero in the figures. Blood and milk were sampled for an additional 6 days. The experimental data were divided into pre-experimental control, experimental, and post-experimental period. The post-experimental period began after 2 days when the cortisol concentrations in
MILK CORTISOL RESPONSE TO ACTH AND STRESS blood had declined to a point that was not significantly different from the concentration in the pre-experimental period as determined by analysis of variance.
boundary. The equations are a modification of the law of diffusion of Fick. The vectorial flow, Jx, of a substance is given by [1] : Jx = Lgradux
Management Stress
Cows routinely are moved by truck between two University of Wisconsin dairy farms. To determine the effects on cortisol in milk of such a stress, milk samples were collected from 39 cows for two afternoon milkings prior to moving and for two afternoon milkings after moving. The cows were moved about 6 h before the afternoon milking on the 3rd day. No blood samples were collected in this experiment. THEORY
Nonelectrolytes which are in blood and are permeable to cell membranes should be in milk at a concentration directly related to that in blood. A pregnancy test which uses the progesterone content of milk as an index takes advantage o f this principle (12), For electrolytes the situation is more complicated than that presented below due to the vectorial transport systems in the cell membranes; nevertheless, the same general principles apply. Figure 1 dramatically shows how a substance is transported through a secretory cell and interacts with a binding protein in the cell. An intercellular binding protein is not essential but is included because mammary cells contain such a protein (14). The mammary secretory cell can be considered as a penetrable boundary, and the transport of any substance, X, across it will obey the equations describing the flow across such a
BLOOD
1105
SECRETORY CELL
X
BP
BPX ~
BP "X
FLOW
[1]
Where L is a phenomenological coefficient replacing the diffusion constant in Fick's law. L = Cxcox
[21
The coefficient L is a function of the concentration, Cx, and the rate of transfer of the substance through the cell, co x. The term gradux is the gradient in chemical potential (concentration) across the cell. Thus, Jx is the amount of flow per unit time and is dependent on the blood concentration, the rate of transfer (i.e. the time it takes for the material to pass through the cell including any interaction with it within the cell), and the concentration difference between blood and milk. The flow can be in either direction; for example, a substance injected into the gland would flow toward blood because the gradient, grad/ax, would be in that direction. With a constant rate of milk secretion, J H : O = constant, the total amount transferred, Tx, in a particular time interval t i to t2 will be [3] : t2
Tx = f t~
Lgrad,xdt
[3]
Therefore, a measurement of the amount of any material that can penetrate the secretory cell will be the value o f the integral in equation [31. It will be a function of the blood concentration averaged over time. Actually, the concentration will be a ratio of two flow, JH2O and JCORT, but JH20 is assumed to be constant. The concentration of the material actually might be higher in milk as in the case of progesterone; this is because a partition coefficient between the aqueous and lipid phases must also be taken into account. A higher concentration in milk might arise if binding proteins were secreted from the cell. A more complete discussion of these principles can be found in Katchalsky and Curran (8).
I
FIG. 1. Diagramatic representation of a substanae, X, going across a mammary secretory cell and its interaction with an intracellular binding protein, BP.
RESULTS AND DISCUSSION
After corticotropin was injected, cortisol increased in the circulation. The concentrations Journal of Dairy Science Vol. 61, No. 8, 1978
1106
BREMEL AND GANGWER 6
12
A
4 10
O
....
1111 Hill
•-~
•
:
.
0
:E
16
8
Z
°
-
-1
0
:E 6
111 1
2 3 OAY
4
5
6
7
Z
•£
4
] o
tJ
• .2.~
-
:
.
2 y •
o
2'
4'
,b
Cortisol in Blood
tit
2.67 + .007 X |
•
14
,6
ng/ml
FIG. 3. Relationship between average blood concentration and milk concentration of cortisol. (A) Preexperimental period; (B) Post-experimental period. The hashed line in panel A is the regression of the post-experimental data superimposed on the pteexperimental data. The average blood concentration =
~4
o
n
|
I -1
| 0
I I
I 2
I 3
I 4
I 5
I 6
I 7
DAY
FIG. 2. Effect of 200 IU ACTH injection on cortisol in milk and on milk production of 5 Holstein cows. The shaded area represents one SE about the mean which is denoted by (e). Average milk production of the group is shown in the inset.
reached a maximum of 60 ng/ml in 8 to 10 h and remained elevated above control for approximately 48 h. Cortisol concentration was also higher than control in milk during this time (Fig. 2). The increase of cortisol in milk follows the same course with time as that in blood, but the concentration reached in milk is lower. The concentration was highest in the first milking after ACTH injection. As in the inset of the figure, there was also a decrease in milk production for the group of animals after the ACTH injection. This decrease in milk production was concomitant with the elevation in cortisol in the milk and is similar in magnitude to that in (3).
If the mammary gland takes up a certain fraction of the cortisol from blood, then the concentration of cortisol in milk should be related directly to that in blood during the interval since the previous milking. To see whether the m a m m a r y gland was acting as an integrator of blood concentration we plotted the data from the above experiment for the differJournal of Dairy Science VoI. 61, No. 8, 1978
n
•~citi/~t i i=1 i=1 where c = concentration and t = time interval in hours. Each point in the plot is an evaluation of the integral in equation [31, and is the amount of cortisol flow which occurs with a given concentration gradient between blood and milk.
ent experimental periods in Fig. 3 and 4. The results indicate that during the preinjection control period (Fig. 3A) and after ACTH injection, during a period of increased adrenal activity (Fig. 4), there is a close relationship between the average concentration of cortisol in blood and in milk. The regression equations describing the relationship between the concentration of cortisol in blood and in milk are nearly identical during these periods and are not statistically different. A peculiar aspect of the data is the lack of correlation between the blood and milk concentrations after the period of increased adrenal activity in the post-experimental period (Fig. 3B). The mean concentration of cortisol in blood was lower during the postexperimental period. It was 4.3 ng/ml as compared to 6,4 ng/ml during the pre-experimental period (P
