- Email: [email protected]
Changes in circulating plasma levels of cortisol in lactating and non-lactating dairy cattle during the estrous cycle
THERIOGENOLOGY CHANGES IN CIRCULATING PLASMA LEVELS OF CORTISOL IN LACTATING AND NON-LACTATING DAIRY CATTLE DURING THE ESTROUS CYCLE J.D. Roussel, T.J. Clementa, T.J. Aranas and S.H. Seybt Department of Dairy Science Louisiana Agricultural Experiment Station Baton Rouge 70803 Received for Publication: October 6. 1982 Accepted: December 21, 1982
ABSTRACT
Plasma cortisol levels were determined by radioimmunoassay for five lactating cows and five open heifers from blood samples collected daily during the course of a complete estrous cycle. Each animal was fitted with an indwelling jugular catheter to minimize stress from bleeding. Mean plasma cortisol levels were 5.67 ng/ml for the lactating cows and 5.87 ng/ml for the open heifers. Mean values for individuals ranged from 3.79 ng/ml to 6.94 ng/ml in the lactating group, and 3.37 ng/ml to 11.69 ng/ml in the open group. These differences, both between and within groups, were not significant. Mean cortisol values during the estrous cycle ranged from 2.26 + 0.93 nglml on day one to 9.49 + 2.11 nglml on day six for the lactating group, and 2.74 + 0.52 ng/ml-on day six to 14.66 + 10.78 ng/ml on day twelve for the open group. Group by day interactions were not significant. Attempts to correlate plasma cortisol with lactation or day of estrus were not significant.
INTRODUCTION The estrous cycle is known to be controlled to a large extent by ovarian hormones. Those hormones, being steroids, are chemically similar to hormones from the adrenal. While glucocorticoids have been found to have little effect on stage of lactation or reproductive status (l-6), their characterization may help identify any differences in the physiological state of fertile and infertile cows. In the present study, plasma cortisol levels were characterized during the estrous cycle for lactating cows and open heifers. Rather than using the familiar competitive-protein binding assay, a radioimmunoassay (RIA) method was used to quantitate cortisol levels.
1 The authors are grateful to K.L. Koonce for his technical assistance in the statistical analysis of the data.
% epartment of Animal Science, University of Southwest Louisiana, Lafayette, LA
70504
APRIL 1983VOL. 19 NO. 4
535
THERIOGENOLOGY
MATERIALS AND METHODS Five lactating Holstein cows and five contemporary but virgin Holstein heifers were used for blood collection. The lactating cows were in their third month of lactation while the heifers had all experienced at least one normal estrous cycle. All animals were fed 100% of the National Research Council estimated net energy requirements. Both water and trace minerals were available ad libitum. Blood Collection: Daily blood samples were taken from each animal starting on the first day post-estrus and continuing until the next standing heat. To minimize stress from bleeding, each animal was fitted with an indwelling jugular catheter by the procedure outlined by McGilliard (7) and modified by Roussel and Gomila (8). Samples were collected between 0700 and 0800 hours with a 30 ml plastic disposable syringe and transferred to a 40 ml collection tube containing sodium heparin. Following centrifugation the plasma was harvested and stored at O°C until subsequent cortisol analysis. Cortisol Assay: A modified radioimmunoassay procedure was used which combined the procedures described by Endocrine Sciences (Tarzana, California, March, 1982) and Satterlee (9). For each sample, duplicate 200 nl aliquots of plasma were extracted with 2.0 ml of nanograde dichloromethane (DCM) (Mallinchkrot, St. Louis, MO). Samples were vortexed (Kraft Model VB-1 Multitube Vortex Mixer, Mineola, NY) for 1 minute, centrifuged at room temperature at 3000 g for 10 minutes, and frozen on a slant at -4O'C for 5 minutes. Freezing on a slant caused the upper plasma layer to freeze to one side of the test tube, thereby allowing the lower DCM extraction layer to be decanted quantitatively into a second test tube. Following drying under forced air in a 40°C water bath, the extract was reconstituted with 1 ml of 10% methanol in borate buffer (O.O5M, pH = 8.0) and vortexed for 10 seconds. Triplicates of standards containing 0, 12.5, 25, 50, 100, 250, and 500 pg cortisol were prepaired from a stock solution of 500 pg cortisol/ml methanol. The standards were dried under forced air in a 40°C water bath and reconstituted with 50 j.11 of 10% methanol in borate buffer. In addition to the triplicate standards, a sample aliquot of 50 pl was incubated overnight at room temperature with a 200 ~1 aliquot of anti-cortisol serum (Ab)(F3-314, Endocrine Sciences) diluted in a borate buffer containing bovine gamma globulin (Cohn Fraction II, Sigma Chemical Co., St. Louis, MO), boyine serum albumin (BSA) (Sigma Chemical Co.), and 16,600 dpm of 1,2,6,7- HN-cortisol (New England Nuclear, Boston, MA). The radioactivity not bound to the Ab was removed by precipitation with 250 pl of a saturated ammonium sulfate solution (J.T. Baker Chemical Co., NJ) and centrifugation at 4'C at 2500 g for 15 minutes. Non-specific binding for the buffer and pooled plasma was also assayed.
536
APRIL 1983VOL. 19 NO. 4
THERIOCENOLOCY
---
lactating
cows
open heifers
5
0
Figure plasma cortisol
1
I
I
I
5
10
15
20
DAY OF ESTRUS 1. Relationship between day of estrus and levels in lactating cows and open heifers.
APRIL 1983 VOL. 19 NO. 4
537
THERIOGENOLOGY
The radioactivity precipitated was measured in a liquid scintillation spectrometer (Model LS 8000, Beckman Instruments, Fullerton, CA). Measurements were corrected for non-specific binding. A standard curve was constructed by plotting the percentage of radioactive cortisol bound (% B) vs. loglo of the mass of the cortisol standards. For dose interpxation, a linearized curve was constructed by plotting the lqgit of % B vs. the log of pg cortisol. Coefficient of determination (r > calculazon for the curve generated was 0.92. The extraction efficiency was determined to be 87.25%. Analysis of inter-assay coefficient of variation (CV) for % B from pooled plasma samples was 5.40%. The intra-assay CV for duplicate samples of pooled plasma cortisol was 1.58%. Ten steroids were tested for cross reactivity (Table 1). The cross reactivity of those steroids with the Ab was less than 0.3% for all but corticosterone (4.9%) and cortisone (14.8%). The addition of 12.5, 25, 50, 100, and 250 pg cortisol in triplicate samples to 200 1.11 pooled plasma yielded efficiency recoveries of 99.95, 102.8, 103.8, 102.4, and 90.15X, respectively. The above indicates that the assay was specific, sensitive, accurate, and precise.
TABLE I.
Steriod cross-reaction with antiserum
3-314.
% Cross-Reaction Compound Tested Cortisol Corticosterone Cortisone 17-hydroxy progesterone 20 -hydroxy progesterone Progesterone 20 -hydroxy progesterone Aldosterone Deoxycorticosterone Testosterone Estriol
Endocrine Sciences
Dairy Physiology Laboratory
100.0 3.5 15.3 0.2 0.02 0.2 0.01 0.09 0.3 0.01 0.01
100.0 4.9 14.8 0.2 0.01 0.05 0.01 0.01 0.2 0.02 0.01
RESULTS AND DISCUSSION Individual mean cortisol levels ranged from 3.78 + 0.78 ngfml to 5.97 + 1.11 ng/ml for the five lactating cows, and 3.51 + 0.36 ng/ml to 11.69 + 3.26 ng/ml for the five open heifers. Analysis of variance revealed that the mean plasma cortisol level for the lactating group (5.67 + 0.61 ng/ml) was not significantly different from that for the open group (5.87 + 0.59 ng/ml). While somewhat higher values have been reported by others (4, 6, lo), statistical analysis yielded similar results. Daily fluctuations in plasma cortisol during the estrous cycle were not significant. Mean plasma levels ranged from 2.26 + 0.93 ngfml on day one to 9.49 2 2.11 ng/ml on day six for the lactating group, and
538
APRIL
1983 VOL. 19 NO. 4
THERIOGENOLOGY from 2.74 + 0.53 ng/ml on day six to 14.66 + 10.78 ng/ml on day twelve There were no significant group by day for the open group (Figure 1). interactions and attempts to correlate cortisol with lactation or day of estrus were not significant. The degree of intermittent variation exhibited in plasma cortisol levels for lactating cows and contemporary but open heifers during the estrous cycle indicates that cortisol would be an unreliable predictor of breeding soundness. References 1.
Role of the adrenal cortex in intermediary Egdahl, R.L. Amer. J. Med. E:556-566 (1951). metabolism.
2.
Adrenal function related to Stott, G.H. and Thomas, J. reproduction in heifers subjected to submaintenance rations. Dairy Sci. -54:787 (1971).
3.
Stott, G.H. and Wiersma, F. Climatic hormonal depression and low fertility Biometeor, _l7_:115-122 (1973).
4.
Glucocorticoids in mammary Schwalm, J.W., and Tucker, H.A. secretions and blood serum during reproduction and lactation and distributions of glucocorticoids, progesterone, and estrogens in J. Dairy Sci. %:550-560 (1978). fractions of milk.
5.
Shayanfar, F., Head, H.H., Wilcox, Adrenal responsiveness in lactating -58~870-878 (1975).
6.
Shaw, K.E., Dutta, S., and Nichols, R.E.. Quantities of 17-hydroxycorticosteroids in the plasma of healthy cattle Amer. J. Vet. Res. -21:52-53 various physiologic states.
7.
Permanent implantation McGuilliard, A.D. J. Dairy catheters and lymphatic shunts.
8.
Roussel,
9
Satterlee,
10.
J.D.
and Gomila,
D.G.
Personal
L.F.
a cause of Int. J.
C.J., and Thatcher, W.W. Holstein cows. J. Dairy
Personal
Sci.
during (1960).
of arterial and venous Sci. %:1192-1199 (1972). communication.
communication.
S.L. Wagner, W.C., and Oxenrieder, Diurnal changes and the effects of J. Anim. Sci. 2:630-635 hormones.
APRIL 1983VOL. 19 NO. 4
thermal stress, in the bovine.
J.
Adrenal lactation (1972).
(1977).
(1978). function in the cow. and neurohypophysial
539
ABSTRACT
Plasma cortisol levels were determined by radioimmunoassay for five lactating cows and five open heifers from blood samples collected daily during the course of a complete estrous cycle. Each animal was fitted with an indwelling jugular catheter to minimize stress from bleeding. Mean plasma cortisol levels were 5.67 ng/ml for the lactating cows and 5.87 ng/ml for the open heifers. Mean values for individuals ranged from 3.79 ng/ml to 6.94 ng/ml in the lactating group, and 3.37 ng/ml to 11.69 ng/ml in the open group. These differences, both between and within groups, were not significant. Mean cortisol values during the estrous cycle ranged from 2.26 + 0.93 nglml on day one to 9.49 + 2.11 nglml on day six for the lactating group, and 2.74 + 0.52 ng/ml-on day six to 14.66 + 10.78 ng/ml on day twelve for the open group. Group by day interactions were not significant. Attempts to correlate plasma cortisol with lactation or day of estrus were not significant.
INTRODUCTION The estrous cycle is known to be controlled to a large extent by ovarian hormones. Those hormones, being steroids, are chemically similar to hormones from the adrenal. While glucocorticoids have been found to have little effect on stage of lactation or reproductive status (l-6), their characterization may help identify any differences in the physiological state of fertile and infertile cows. In the present study, plasma cortisol levels were characterized during the estrous cycle for lactating cows and open heifers. Rather than using the familiar competitive-protein binding assay, a radioimmunoassay (RIA) method was used to quantitate cortisol levels.
1 The authors are grateful to K.L. Koonce for his technical assistance in the statistical analysis of the data.
% epartment of Animal Science, University of Southwest Louisiana, Lafayette, LA
70504
APRIL 1983VOL. 19 NO. 4
535
THERIOGENOLOGY
MATERIALS AND METHODS Five lactating Holstein cows and five contemporary but virgin Holstein heifers were used for blood collection. The lactating cows were in their third month of lactation while the heifers had all experienced at least one normal estrous cycle. All animals were fed 100% of the National Research Council estimated net energy requirements. Both water and trace minerals were available ad libitum. Blood Collection: Daily blood samples were taken from each animal starting on the first day post-estrus and continuing until the next standing heat. To minimize stress from bleeding, each animal was fitted with an indwelling jugular catheter by the procedure outlined by McGilliard (7) and modified by Roussel and Gomila (8). Samples were collected between 0700 and 0800 hours with a 30 ml plastic disposable syringe and transferred to a 40 ml collection tube containing sodium heparin. Following centrifugation the plasma was harvested and stored at O°C until subsequent cortisol analysis. Cortisol Assay: A modified radioimmunoassay procedure was used which combined the procedures described by Endocrine Sciences (Tarzana, California, March, 1982) and Satterlee (9). For each sample, duplicate 200 nl aliquots of plasma were extracted with 2.0 ml of nanograde dichloromethane (DCM) (Mallinchkrot, St. Louis, MO). Samples were vortexed (Kraft Model VB-1 Multitube Vortex Mixer, Mineola, NY) for 1 minute, centrifuged at room temperature at 3000 g for 10 minutes, and frozen on a slant at -4O'C for 5 minutes. Freezing on a slant caused the upper plasma layer to freeze to one side of the test tube, thereby allowing the lower DCM extraction layer to be decanted quantitatively into a second test tube. Following drying under forced air in a 40°C water bath, the extract was reconstituted with 1 ml of 10% methanol in borate buffer (O.O5M, pH = 8.0) and vortexed for 10 seconds. Triplicates of standards containing 0, 12.5, 25, 50, 100, 250, and 500 pg cortisol were prepaired from a stock solution of 500 pg cortisol/ml methanol. The standards were dried under forced air in a 40°C water bath and reconstituted with 50 j.11 of 10% methanol in borate buffer. In addition to the triplicate standards, a sample aliquot of 50 pl was incubated overnight at room temperature with a 200 ~1 aliquot of anti-cortisol serum (Ab)(F3-314, Endocrine Sciences) diluted in a borate buffer containing bovine gamma globulin (Cohn Fraction II, Sigma Chemical Co., St. Louis, MO), boyine serum albumin (BSA) (Sigma Chemical Co.), and 16,600 dpm of 1,2,6,7- HN-cortisol (New England Nuclear, Boston, MA). The radioactivity not bound to the Ab was removed by precipitation with 250 pl of a saturated ammonium sulfate solution (J.T. Baker Chemical Co., NJ) and centrifugation at 4'C at 2500 g for 15 minutes. Non-specific binding for the buffer and pooled plasma was also assayed.
536
APRIL 1983VOL. 19 NO. 4
THERIOCENOLOCY
---
lactating
cows
open heifers
5
0
Figure plasma cortisol
1
I
I
I
5
10
15
20
DAY OF ESTRUS 1. Relationship between day of estrus and levels in lactating cows and open heifers.
APRIL 1983 VOL. 19 NO. 4
537
THERIOGENOLOGY
The radioactivity precipitated was measured in a liquid scintillation spectrometer (Model LS 8000, Beckman Instruments, Fullerton, CA). Measurements were corrected for non-specific binding. A standard curve was constructed by plotting the percentage of radioactive cortisol bound (% B) vs. loglo of the mass of the cortisol standards. For dose interpxation, a linearized curve was constructed by plotting the lqgit of % B vs. the log of pg cortisol. Coefficient of determination (r > calculazon for the curve generated was 0.92. The extraction efficiency was determined to be 87.25%. Analysis of inter-assay coefficient of variation (CV) for % B from pooled plasma samples was 5.40%. The intra-assay CV for duplicate samples of pooled plasma cortisol was 1.58%. Ten steroids were tested for cross reactivity (Table 1). The cross reactivity of those steroids with the Ab was less than 0.3% for all but corticosterone (4.9%) and cortisone (14.8%). The addition of 12.5, 25, 50, 100, and 250 pg cortisol in triplicate samples to 200 1.11 pooled plasma yielded efficiency recoveries of 99.95, 102.8, 103.8, 102.4, and 90.15X, respectively. The above indicates that the assay was specific, sensitive, accurate, and precise.
TABLE I.
Steriod cross-reaction with antiserum
3-314.
% Cross-Reaction Compound Tested Cortisol Corticosterone Cortisone 17-hydroxy progesterone 20 -hydroxy progesterone Progesterone 20 -hydroxy progesterone Aldosterone Deoxycorticosterone Testosterone Estriol
Endocrine Sciences
Dairy Physiology Laboratory
100.0 3.5 15.3 0.2 0.02 0.2 0.01 0.09 0.3 0.01 0.01
100.0 4.9 14.8 0.2 0.01 0.05 0.01 0.01 0.2 0.02 0.01
RESULTS AND DISCUSSION Individual mean cortisol levels ranged from 3.78 + 0.78 ngfml to 5.97 + 1.11 ng/ml for the five lactating cows, and 3.51 + 0.36 ng/ml to 11.69 + 3.26 ng/ml for the five open heifers. Analysis of variance revealed that the mean plasma cortisol level for the lactating group (5.67 + 0.61 ng/ml) was not significantly different from that for the open group (5.87 + 0.59 ng/ml). While somewhat higher values have been reported by others (4, 6, lo), statistical analysis yielded similar results. Daily fluctuations in plasma cortisol during the estrous cycle were not significant. Mean plasma levels ranged from 2.26 + 0.93 ngfml on day one to 9.49 2 2.11 ng/ml on day six for the lactating group, and
538
APRIL
1983 VOL. 19 NO. 4
THERIOGENOLOGY from 2.74 + 0.53 ng/ml on day six to 14.66 + 10.78 ng/ml on day twelve There were no significant group by day for the open group (Figure 1). interactions and attempts to correlate cortisol with lactation or day of estrus were not significant. The degree of intermittent variation exhibited in plasma cortisol levels for lactating cows and contemporary but open heifers during the estrous cycle indicates that cortisol would be an unreliable predictor of breeding soundness. References 1.
Role of the adrenal cortex in intermediary Egdahl, R.L. Amer. J. Med. E:556-566 (1951). metabolism.
2.
Adrenal function related to Stott, G.H. and Thomas, J. reproduction in heifers subjected to submaintenance rations. Dairy Sci. -54:787 (1971).
3.
Stott, G.H. and Wiersma, F. Climatic hormonal depression and low fertility Biometeor, _l7_:115-122 (1973).
4.
Glucocorticoids in mammary Schwalm, J.W., and Tucker, H.A. secretions and blood serum during reproduction and lactation and distributions of glucocorticoids, progesterone, and estrogens in J. Dairy Sci. %:550-560 (1978). fractions of milk.
5.
Shayanfar, F., Head, H.H., Wilcox, Adrenal responsiveness in lactating -58~870-878 (1975).
6.
Shaw, K.E., Dutta, S., and Nichols, R.E.. Quantities of 17-hydroxycorticosteroids in the plasma of healthy cattle Amer. J. Vet. Res. -21:52-53 various physiologic states.
7.
Permanent implantation McGuilliard, A.D. J. Dairy catheters and lymphatic shunts.
8.
Roussel,
9
Satterlee,
10.
J.D.
and Gomila,
D.G.
Personal
L.F.
a cause of Int. J.
C.J., and Thatcher, W.W. Holstein cows. J. Dairy
Personal
Sci.
during (1960).
of arterial and venous Sci. %:1192-1199 (1972). communication.
communication.
S.L. Wagner, W.C., and Oxenrieder, Diurnal changes and the effects of J. Anim. Sci. 2:630-635 hormones.
APRIL 1983VOL. 19 NO. 4
thermal stress, in the bovine.
J.
Adrenal lactation (1972).
(1977).
(1978). function in the cow. and neurohypophysial
539