Diurnal Variations in Blood Cortisol in the Dairy Cow SUSAN HUDSON, M. MULLORD, W. G. WHITI'LESTONE, and E. PAYNE Ruakura Animal Research Centrea Private Bag, Hamilton, N e w Zealand Abstract

Concentrations of adrenal corticostefolds in plasma were determined by a competitive protein-binding technique in blood samples taken hourly over 24 h from four dairy cows, each sampled for three 24 h periods. Mean blood cortisol was 7.9 ng/ml. Statistical analysis revealed no diurnal variation. Reasons for this are postulated. Introduction

A diurnal variation in peripheral plasma concentration of adrenal eortieosteroids has been demonstrated in several species including man (19), pigs and horses (5), oats (11), and rats (7). Reports on variation in dairy cattle a r e imprecise and conflicting. In this species MacAdam and Eberhart (12) obtained an early morning peak but no following trough whereas VCaguer and Oxenreider (26) obtained a trough between 1800 h and 0200 h but no early morning peak. Conversely, Paape et al. (16) and Abilay and John,son (1), in studies not primarily concerned with diurnal variation, noted a lack of daily rhythmicity in circulating cortisol. Irfformation regarding a diurnal rhythm in glucocorticoid concentration in the cow is essential because of the role of these hormones in pregnancy (2, 23), parturition (10), and lactation (17), and must be considered in the design of all experiments involving these hormones. This experiment was planned to determine the inherent daily variation of glueoeortieoid concentration in the dairy cow with environmental influences minimized. Materials and Methods

Animals. Two pairs of mature, monozygotie twin Jersey cows were used, one pair 6 mo pregnant, the other pair nonpregnant. All cows were nonlactating. Housing. The cows were restrained in pairs Received March 4, 1974. Ministry of Agriculture and Fisheries, Ruahtra Agricultural Research Centre, Hamilton, New Zealand.

in an enclosed pen with free access to w a t e r and were fed 2.7 kg maize meal and crushed barley mix at 0800 h and .125 bale of medium pasture hay at 0800 h and 1630 h. Pairs of twins were used together as they have the most stable social relationship. Animals were held in the stall for 24 h every 2 or 3 days over 6 wk prior to the experiment to accustom them to this new environment. The pen was automatically lit between 0600 ,and 1800 h by a 40 W fluorescent tube. Cannulation and sampling. To eliminate any possibility of sampling stress, blood was obtained by remote sampling from indwelling cannulae. Polyvinyl tubing, 1.5 m ~ (inner diameter) was inserted into ,a jugular vein under a tranquilizer and local anaesthesia. The canntda was sewn to the cow's skin in front of the shoulder, continued v~a a tap to a counter-balanced, pivoting rod, and then through the wall of the pen. Sampling was from outside the pen. A circulating water bath provided masking noise. Ten milliliters of blood were withdrawn every hour into a heparinized syringe and immediately cooled to + 4 C in a circulating water bath, spun, and the plasma frozen at --20 C. Heparinized saline was back-injected to keep the cannula patent. Three 24 h sets of samples were taken from each cow within a month. The sampling period was never begun less than 48 h after eannulation although later work has shown that cortisol has returned to normal 8 h after cannulation. The first set of samples, taken from cow 1108, was discarded as the mean cortisol was 100% higher than subsequent series from the same cow and all other cows. It may be that the cow's adjustment to the situation was not complete on this t~rst session. Assay. Plasma cortisol was analyzed by an adaptation of the competitive protein-binding method of Murphy (14) with tritiated cortisol and fullers earth for separation of bound and Lee eortisol. Ethyl acetate was used to extraet cortisol from plasma. Recovery of labeled eortisol was 90 ___ 3%. The coefficient of variation within assays and between assays was 10%. The values are uncorrected and could include other corticoids to a small extent. 30

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No set showed a relationship with time. A 24 h sinusoidal regression was nonsignificant for all sets, the greatest reduction in residual variation being 21%. No sigr~cant correlations between sets from the same cow or between twin cows were significant (P < .01): repeatability of measurements on the same cow was too low in respect to sampling time.

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Means of all 11 sets for 24 h are in Fig. 1 while ~eans of results from each cow are separated in Fig. 2. Average values for eaclh cow were 7.9 _ 3.1 n g / r n l (Cow 0069), 6.4 ___ 2.9 ng/ml (Cow 0070) (Fig. 2a); 8.2 :___ 2.2 ng/ml (Cow 1107), and 8.7 ~ 3.0 ng/ml (Cow 1108) (Fig. 2b). 13

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The mean cortisol of 7.9 ng/ml compares well with other values reported for cattle; 7.3 ng/ml (25), 9.7 ng/ml (17), and 6.7 ng/ml

(26). Results of this experiment fail to demonstrate a diurnal rhyth~n of cortisol in the dairy cow. These results do not agree with reports of Wagner and Oxenreider (26) and MacAdam and Eberh,art (12), both of whom find a statistically valid eh,ange in cortisoI over particular periods of time, Wagner and Oxenreider found that the value for 1800 to 0200 h was significandy lower than the other values but obtained no early morning peak. Conversely, MacAdam and Eberhart deduced the existence of a diurn,al rhythm from a statistically valid

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early morning peak with no following trough. However, our results confirm brief reports (1, 16) which note a lack of cortiso] rhythmicity. The lack of a definite diurnal rhythm in the cow is more deafly deanonstrated when these results are compared with the marked rhythms in other species. In man, (19), and in pigs and mares (5), there is over 100% deviation from the mean in the early morning, and a trough of corresponding amplitude in the evening. Work with rats (7) has produced similar results with the time scale shifted so that the peak occurs in the early evening when this nocturnal animal becomes active. In these species there is a regular and rapid "anticipatory" rise in cortisol with the peak activity at the time of awakening. Studies by Hiroshige et al. (9), Takebe et al. (24), and Selden and Brodish (21) have indicated that in the rat the daily rhythm of adrenocorticoid activity is the result of a diurnal rhythm of corticotropin-releasing factor secretion from the hypothalamus. Perkoff et al. (18) have shown that the timing of this cycle is dependent on

ET AL

the sleep-wake activity of the subject rather than on daily environmental variations. This finding is supported by the studies of Orth et al. (15), S h ~ 3 et al. (22), Weitzman et al. (27), and Franks (6). The dependence of cortisol on the sleepwake schedule of the subject may explain the lack of a dittrnal rhythm in dairy cattle. Ruminants do not enter the deep state of sleep of man and domestic animals (3, 4, 8). Ruckebusch (20), using electroencephalograph),, proved that ruminants have the physiologic capacity for sleep but that in cows sleep was transient and polyphasie, averaging 4 rain per episode, with f~equencies averaging 11 episodes in 24 h. Balch (3) states that sleep periods in the ruminant are linked dixecfly to the digestive needs of the animal since rumination requires both time and cor~sciousn~s. Support comes from Morag (13), who found that sheep fed only finely chopped grass entered states of deep sleep but that when their diet was changed to unlimited amounts of hay, they did

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DIURNAL CORTISO,LIN THE COW not sleep at all. He concluded that sleep in ruminants occurs only when their diet contains a small percentage of roughage. Since diurnal variation in corticoid activity is related to the sleep-wake schedule of the subject, we suggest that absence of deep sleep periods in the cow may explain this maimal's lack of a well defined diurnal rhythm.

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Acknowledgments We thank D. Duganzich for statistical analysis of our data and Mr. H. deLangen for the figures. References (1) Abilay, T. A., and H. D. Johnson. 1973. Plasma steroids during the ovarian cycle at 18.2 C temperature. J. Anin~ Sei. 37:298. (2) Adams, W. M., and W. C. Wagner 1970. The role of eortieeids in parturitio~L Biol. Reprod. 3:223. (3) Balch, C. C. 1955. Sleep in ruminants. Nature (Lend.). 175:940. (4) Bell, F. R. 1960. The electroencephalogram of goats during somnolence and rnrnination. Anita. Behav. 8:39. (5) Rottoms, G. D., O. F. Roessel, F. D. Rausch, and E. L. Atkins. 1972. Circadian wtriation in plasma cortisol and eorticosteroaae in pigs and mares. Amer. 1. Vet. Res. 33:785. (6) Franks, R. C. 1967. Diurnal variation of plasma 17 hydroxycortieosteroids in childrama. 1- Ctin. Enclocrinol. 27:75. (7) Guilleman, R., W. E. Dear, and L. A. Liebelt. 1959. Nyehthermal variations in plasma free cortieestero~d levels of the rat. Prec. See. Exp. Biol. Mad. 101:394. (8) Hancock, .T. 1950. Grazing habits of dairy cows in N.Z. Emp. J. Exp. Agr. 18:1~9. (9) Hireshige, T., M. Sakakura, and S. Itoh. 1969. Diurnal variation of corticc~ropinreleasing activity in the rat hypotl~alamus. Endocrinology (Japan). 16:465. (10) Hoffman, B., D. Schams, T. Ginaenez, M. L. Ender, C. Herrman, and H. Karg. 1973. Changes of progesterone, total oestrogens, corticosteroids, prolaetin and LH in bovine peripheral plasma around parturition wlth special reference to the effect of exegenous eertieeids and a prolactin inhibitor respectively. Aeta Endoerinol. 73:385. (11) Krieger, D. T., A. I. Silverberg, F. Rizzo, and M. P. Krieger. 1968. Abolition of circadian periodicity of plasma 17-OHCS levels in the cat. Amer. 1. Physiol. 215'.959. (12) MacAdam, W. R., and R. J. Eberhart. 1972. Diurnal variation in plasma cortieesteroid concenti~tion in dairy cattle. J. Dairy 8ci. 55:1792. (13) Morag, M, 1967. Influence of diet on the behaviour pattern of sheep. Nature (Lend.) 213:1t0. (14) Murphy, B. E. P. 1967. Some studies of the

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protein binding of steroids and their application to the routine micro and ultramicro measuremer~t of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Eudocrinol. Met, 27: 973. Orth, D. N., D. P. Island, and G. W. Liddlo. 1967. Experimental alteratiort of the circadian rhythm in plasma cortisol (17OHCS) concentration in man. J. Clin. Endocrinol. Met. 27:549. Paape, M. J., C. Desjardins, and R. H. Miller. 1973. Diurnal and certieetropin effects on adrenal and leukocyte responses. J. Dairy SCI. 56:1382. (Abstr.) Paape, M. J., C. D. Desjardins, W. D. Schultze, and .T.W. Smith. 1972. Cortieosteroid concentrations in jugular and mammary vein blood plasma of co~vs after overanilking. Amer. J. Vet. Res. 33:1753. Perkoff, G. T., K. Eik-Nes, C. A. Nugent, H. L. Fred, R. A. Nimer, L. Rush, L. T. Samuels, and F. H. Tyler. 1959. Studies of the diurnal variation of plasma 17-hydmxyeortieesteroids in man. J. Clin. Endoednol. 19:432. Rose, R. M., L E. Kreuz, J. W. Holaday, K. ]. Sulak, and C. E. Johnson. 1972. Diurnal variation of plasma testosterone and cortlsol. J. Endocrinol. 54:177. Ruckebuseh, Y. 1972. Th0 relevance of drowsiness in the circadian cycle of farm animals. Anim. Behav. 24:637. Selden, G., and A. Brodish. 1972. Persistence of a diurnal rhythm in hypothalamie CRF in the absence of hormone feedback. Endoerlnology 90:1401. Sharp, G. W. G., S. A. Slorach, and H. J. Vipond. 1961. Diurnal rhythms of kateand ketogenie steroid excretion and the adaptation to changes of the activity-sleep routine. J. Endoerhaol. 22:377. Smith, V. G., L. A. Edgerton, H. D. Hafs, and E. M. Convey. 1973. Bevine serum estrogens, progestins and glucoeerticoids during late pregnancy, parturition, and early lactation. J. Anita. SCI. 36:391. Takebe, K., M. Sakakura, and K. Mashimo. 1972. Continuance of diurnal rhythmicity of CRF activity in hypophysectomized rats. Endocrinology 91:1515. Venkataseshu, G. K., and V. L. Estergreen, Jr. 1970. Co~isol and cortieesterone in bo~eine plasma and the effect of adrenocorticotropin. J. Dairy SCI. 53:480. Wagner, W. C., andS. L Oxenreider. 1972. Ardenal funetio~ ha the cow. Diurnal changes and the effects of lactation and neurohypophyseal hormones. J. Anita. SCI. '34:630. Weitzman, E. D., H. Sehaumburg, and W. Fishbein. 1966. Plasma 17-hydroxyeorticosteroid levels during sleep in man. J: Clin. Endocrinol. Met. 26:121. JOURNAL OF DAIRY SCIENCE VOL. 58, NO. 1