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C97. 1200 1800 2400 0600 1200 1800 2400 0600 1200. Time of Day (h). Figure 2. Peripheral GH concentrations over 48 hr for the second two cows sampled ...
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C I R C A D I A N A N D U L T R A D I A N R H Y T H M S OF P E R I P H E R A L G R O W T H H O R M O N E C O N C E N T R A T I O N S IN L A C T A T I N G DAIRY COWS A.M. Lefcourt, TM J. Bitman,* D.L. Wood,* and R.M. Akers** *Agricultural Research Service, USDA, Livestock and Poultry Sciences Institute, Beltsville, MD 20705 and **Department of Dairy Science, Virginia Tech, Blacksburg, VA 24061 Received June 23, 1994

ABSTRACT To investigate possible circadian and ultradian periodicities for plasma growth hormone in lactating dairy cows, integrated 15-min blood samples taken sequentially over 48 hr from six cows were analyzed by radioimmunoassay. The cows were housed in an environmental chamber at 19 --0.5 ° C, 50% relative humidity, and 16 hr of light and 8 hr of darkness (lights on at 0700 hr); fed daily at 0900 hr; and milked at 0800 and 2000 hr. Peripheral concentrations of growth hormone for all six cows exhibited sinusoidal circadian rhythms with average minima of 4.1 ng/ml at 1820 hr and maxima of 5.3 ng/ml at 0630 hr. Estimated periods of ultradian rhythms for individual cows by spectral analysis, peak identification, and fitting cosine functions using least squares were 71 to 83 min for all cows. No direct relationship between ultradian peaks and milking or feeding was apparent. In conclusion, a circadian rhythm and an ultradian rhythm with a period around 80 min are probably intrinsic to mechanisms regulating peripheral growth hormone concentrations in the lactating dairy cow. INTRODUCTION In every mammalian species studied to date, growth hormone (GH) is secreted in a pulsatile manner with multiple pulses per day. The secretion of GH is primarily governed by the interplay of two hypothalamic peptidergic systems: GH-releasing hormone, which stimulates, and somatostatin, which inhibits, release (1). However, controversy remains as to whether the genesis of the pulses is episodic or periodic (2). Numerous studies have measured peripheral GH in cows; however, for most of these studies, sampling duration, frequency, or both were insufficient to characterize secretory patterns. To reliably discern the existence of an ultradian rhythm, or a circadian rhythm when an ultradian rhythm is also present, it is necessary to sample frequently enough to fully characterize the ultradian rhythm (3-5). Ultradian circa 1.5 hr rhythms are c o m m o n in biologic systems (6,7). To characterize such rhythms requires sampling every 30 to 40 min. The episodic secretion of GH for lactating cows was first reported by Vasilatos and Wangsness (8); however, because visual observation of the data indicated to the authors that GH secretory patterns for individual cows were unique, no attempt was made to uncover any potential circadian rhythms. Mollett and Malven (9) obtained sequential blood samples at 30-min intervals over 72 hr. Using spectral analysis of average concentrations, they found no tendency toward rhythmicity for any of the frequencies tested. The inability to detect repeatable ultradian or circadian rhythms in these two studies may be a consequence of how the studies were conducted and the methodology used to analyze Domestic Animal Endocrinology 12:247-256, 1995 © Elsevier Science Inc. 1995 655 Avenue of the Americas, New York, NY 10010

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the data. For both studies, blood was collected manually and cows were housed in stanchions. It has been demonstrated that handling can alter GH concentrations (10) and that controlled environmental conditions are sometimes necessary to allow endogenous rhythms to be expressed (1,4,11). Furthermore, when attempting to determine the frequency content of time-series data, it is better to determine the frequency content of data for individual time series and then to average the frequency data, as opposed to analyzing the time-averaged data (4,5). This study was designed to investigate hormone rhythms in lactating cows that were completely acclimated to their environment and housed under rigidly controlled conditions. An automated blood sampling system was developed to reduce the effect of human presence and to comply with stringent sampling constraints (12). It was hypothesized that under these conditions, any rhythms inherent to mechanisms regulating peripheral GH concentrations could be observed. Related studies on cortisol (4), prolactin (13), and thyroid hormones (14) have been published. M A T E R I A L S AND M E T H O D S M a n a g e m e n t . Six Holstein cows (4 to 7 mo into lactation) were housed in 1.22- × 2.59-m stanchion stalls in a 6.40- × 9.14-m environmental chamber (19 -+ 0.5 ° C; 50 +10% relative humidity; lights on from 0700 to 2300 hr) for at least 6 wk before blood sampling. Milking began at 0800 and 2000 hr daily. Cows were fed a TMR (total mixed ration) once daily at 0900 hr at rates based on milk production and were allowed to exercise outdoors in an adjacent lot for 20 to 30 min after milkings except on the day before and during periods of blood sampling. Water was available for ad libitum intake. Outdoor temperatures during exercise periods were similar to chamber temperatures. Blood sampling. An automated blood sampling system (12) was used to collect continuous, integrated blood samples (18 ml) over 15-min intervals for 48 hr (192 blood samples). At weekly intervals, samples of blood diluted with heparin saline were collected from two cows into test tubes containing sodium fluoride (to inhibit enzymes). Plasma was obtained by centrifugation and stored at - 2 0 ° C until analysis. G H determination. GH was quantified by a double-antibody radioimmunoassay according to a procedure described by Barnes et al. (15). The GH standard was NIH B18, and this preparation was used for radioiodinations as described by Akers et al. (16). GH intra- and interassay coefficients of variation averaged 7.9 and 8.2%, respectively. Samples were assayed in duplicate. Analyses of r h y t h m s . The circadian rhythm underlying a data set is often difficult to observe visually when the data include higher frequency rhythms. To address this problem, sixth-order polynomial functions were fit to each data set using least squares as previously described (4,17). Predicted values were used to estimate minima and maxima and their respective times. Significance was determined by the use of a general linear model (MODEL V A L U E = C O W MIN_MAX COW*MIN_MAX, where C O W is cow number and MIN_MAX is minima or maxima; 18). Alternatively, the circadian rhythm can be assumed to be sinusoidal and data can be fit to the equation a o + alcos(2~rt/24 ) + a2sin(2-rrt/24 ) where t is time. Estimation of the a i parameters allows the equation to be rewritten as a o + bocos(2rrt/24 + qb) where b 0 = (alz + a2) 1/2 and qb is the 4 quadrant t a n - l ( _ aa/al) (19). To examine higher frequency components of the data, a combination of methods was used (13,17,19). Spectral analysis was used to identify potential frequencies of interest. The raw data were then filtered to isolate specific frequency bands; averaging (low-pass) filters were used to remove higher frequencies, and subtraction of the lower frequency components from the raw data was used to remove lower frequencies (see Results).

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Techniques based on the analysis of the entire time series, such as spectral analysis, are based on the assumption that the time series is stationary. This assumption is seldom true in a strict sense for biologic signals, e.g., the time interval between peaks, episodes, or events varies and there are often abrupt changes in baselines. Alternatively, sequential windows of data can be fit to cosine functions, or individual peaks can be identified and time intervals between peaks can be analyzed. To identify peaks, all datum points that were greater than adjacent points were identified as potential peaks. Areas under potential peaks were determined by finding minima between potential peaks, fitting lines between minima, and calculating areas above fitted lines. Iterative calculations for areas were made until all areas were greater than 1 ng/ml per 15 min. To analyze peaks, histograms of time intervals between peaks were constructed and the average period between peaks was determined. To reduce the effect of errors in identifying peaks, the tails of histograms were excluded for calculating periods, i.e., time intervals less than or equal to 30 min and more than 120 min were excluded from analyses. These cutoffs were selected to exclude a total of 20% of identified peaks from calculations. Potential time of day effects were evaluated by determining the number of peaks and the average amplitude of peaks that occurred within sequential 3-hr intervals of the 24-hr clock. RESULTS

Circadian rhythm. Figures 1 to 3 show peripheral GH concentrations over 48 hr for the six cows along with sixth-order polynomial and cosine functions fit to data from individual cows. One high-amplitude peak of 55 ng/ml was seen at the end of the sampling period for Cow 11. This unique peak was excluded from all data analyses. All cows showed sinusoidal circadian rhythms. For all cows, polynomial functions were superior to cosine functions in terms of goodness of fit, however, both identified similar times for zeniths and nadirs. Averages of peripheral GH concentrations over 48 hr and of polynomial functions fit to data for the six cows show zeniths, which occurred around 630 hr, and nadirs, which occurred around 1820 hr, separated by approximately 12 hr (Figure 4; Table 1). The peak-to-trough amplitude of this circadian rhythm is about 1.2 ng. Midfrequency rhythms. Calculation of power spectra for individual cows using raw data indicated significant power at midfrequencies as well as at low and high frequencies. To better isolate the midfrequency components of the data, the polynomial functions fit to raw data were subtracted from the raw data, and results were smoothed by the use of a 13-point (low-pass) averaging filter (Figure 5). Cosine functions fit to smoothed data indicated substantial rhythms with periods around 5 to 6 hr for all six cows, 9 hr for five cows, and 15 hr for all six cows. Correlations of the sums of the two dominant midfrequency rhythms for individual cows with the filtered data ranged from 0.49 to 0.65 (Figure 5). Ultradian rhythms. Power spectra calculated for individual animals using raw data after the subtraction of polynomial functions showed evidence of rhythms with periods around 70 to 85 min; however, relative power was small compared with the lower frequency components of the spectra. Visual observation of the data suggested that the relative lack of power might be due to variability in the amplitude and the timing of the pulsatile releases of GH. To better characterize these potential ultradian rhythms, raw data were also analyzed by fitting cosine functions to 6-, 12-, 24-, and 48-hr data segments and by identifying individual peaks and calculating the average time interval between peaks. Cosine functions fit to data indicated rhythms with periods of 78 to 80 min. Results of fitting data to sequential data segments were consistent with the visual observation that

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Figure 1. PeripheralGH concentrationsover48 hr for the first two cowssampled with polynomial(dashedline) and cosine (dotted line) functionsfit to data. Peaks detected by the described algorithmare indicated by open circles. Cow 11 showed a unique peak of 55 ng/ml (*). Black bars indicate lights off. C, cow.

pulsatile releases of GH were erratic; there was no indication of any consistent drift in phase over time. The identification of individual peaks (Figures 1 to 3) was dependent on the area criterion used to identify peaks; however, similar results were obtained for areas in the range of 0.75 to 1.25 ng/ml per 15 min. In addition, when the tails of the histograms of pulse intervals were discarded in calculations of average pulse intervals, similar results were obtained for areas in the range of 0.3 to 3 ng/ml per 15 min. Estimated average pulse intervals ranged from 71 to 81 min. Distributions of peak numbers and amplitudes were generally uniform throughout the day. In conclusion, all three methods used to estimate the period of the ultradian rhythm yielded essentially identical results for individual animals. Predicted periods of around 80 min were consistent for all six animals (Table 2). DISCUSSION Examining rhythms in biologic variables can yield important insights concerning mechanisms regulating secretion and concerning interactions with environmental and other biologic variables. Unfortunately, optimal conditions for examining rhythms are difficult to predict and implementing such conditions experimentally can be challenging. One particular problem involves sampling rate. If samples are not taken frequently enough to characterize the highest frequency rhythm, the results obtained can be completely erroneous. Even when sampling constraints are met, rhythms in physiologic variables are still often difficult to detect because the rhythms are not true oscillations with fixed frequen-

GH R H Y T H M S

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Figure 2. PeripheralGH concentrations over 48 hr for the second two cows sampledwith polynomial(dashed line) and cosine (dotted line) functions fit to data. Peaks detected by the described algorithm are indicated by open circles. Black bars indicate lights off. C, cow.

cies and amplitudes. Under these conditions, if a rhythm is to be detected, baseline fluctuations must be minimized and system inputs must be stable. In this experiment, in an attempt to meet these constraints, lactating cows were completely acclimated to their environment and housed under rigidly controlled conditions, and an automated blood sampling system was used to reduce the effect of human presence and to comply with stringent sampling constraints (12). Examination of GH secretion in lactating cows is of particular interest because of the positive effect of exogenous GH on milk production. Recently, the Food and Drug Administration approved the use of recombinantly derived GH by dairy farmers for increased production. Knowledge of the physiologic mechanisms underlying GH secretion may allow treatment regimens for increased production to be optimized. Circadian r h y t h m . In this study, peripheral concentrations of GH exhibited a very distinct and consistent sinusoidal circadian rhythm with the zenith around 630 hr and the nadir around 1820 hr. The peak-to-trough amplitude of this circadian rhythm, about 1.2 ng, represents 25% of the daily mean concentration. The peak in concentration around 630 hr coincides with the expected onset of activity. The existence of a sinusoidal circadian rhythm for GH has not been reported for any species and is in contrast with prior findings for lactating cows. Vasilotas and Wangness (8) and Mollett and Malven (9) found no evidence of a circadian rhythm for GH in lactating cows. Other studies, using 1-hr sampling, also found no evidence of a circadian rhythm (20-22). Evans et al. (23), also using 1-hr sampling, identified a circadian secretory pattern consisting of one or two peaks per day, depending on the photoperiod. The range and mean of GH values reported in this study are consistent with values found in prior studies (8,9,20-23).

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Figure 3. Peripheral GH concentrations over 48 hr for the third two cows sampled with polynomial (dashed line) and cosine (dotted line) functions fit to data. Peaks detected by the described algorithm are indicated by open circles. Black bars indicate lights off. C, cow.

To our knowledge, a sinusoidal circadian rhythm for GH has not been demonstrated before this study. Detection of this rhythm was probably a direct result of experimental conditions. The animals were completely acclimated to a tightly controlled environment, and an automated blood sampling system was used to reduce the effect of human presence and to comply with stringent sampling constraints (12). In related experiments, we dem-

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GH RHYTHMS TABLE

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1. LEAST

SQUARE

MEANS

OF

GH

CONCENTRATIONS CIRCADIAN

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RHYTHM.

TIMES

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a Concentrations and times predicted by individual sixth-order polynomial functions (n = 6) were used to calculate means. b.c Within columns, means not sharing common superscripts differ (P < 0.001).

o n s t r a t e d t h a t t h e s e c o n d i t i o n s a l l o w e d the d e t e c t i o n o f c i r c a d i a n r h y t h m s a n d u l t r a d i a n r h y t h m s w i t h p e r i o d s o f 120, 80, a n d 9 0 m i n for c o r t i s o l (4), p r o l a c t i n (13), a n d t h y r o i d hormones (14), respectively. Midfrequeney rhythms. The physiologic significance of the large proportion of varia t i o n at m i d f r e q u e n c i e s is n o t clear. P r e c i s e a n a l y s i s o f t h e m i d f r e q u e n c y c o m p o n e n t o f t h e d a t a is d i f f i c u l t b e c a u s e t h e t i m e series are o f i n s u f f i c i e n t d u r a t i o n to a l l o w a n a c c u r a t e

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1200 1800 2400 0600 1200 1800 2400 0600 1200 Time of Day (h) Figure 5. GH data for individual cows after the subtraction of polynomial functions from raw data and smoothing to remove high-frequency components of the data. Cosine functions were fit to this filtered data. Shown (dotted line) are the sums of the cosine functions with periods (minutes) having the maximum ('q) and next to maximum (r2) amplitudes. C, cow.

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TABLE 2. ESTIMATED PERIODS OF ULTRADIAN RHYTHMS FOR INDIVIDUAL COWS BY SPECTRAL ANALYSIS, PEAK IDENTIFICATION, AND FITTING COSINE FUNCTIONS USING LEAST SQUARES. Ultradian Period (min) Method

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estimation of signal parameters. Estimation could be improved if the variation could be related to some underlying physical process analogous to the relation of circadian periodicities with day length. In addition, it is possible that the detected rhythms with periods of about 6, 9, and 15 hr are actually artifacts resulting from interactions of true underlying periodicities. As examples, the rhythms with periods of 9 and 15 hr could be beat frequencies of 12- and 3-hr rhythms, and the rhythm with a period of 6 hr could be a harmonic of a 3-hr rhythm. Ultradian rhythm. In this study, peripheral GH in lactating cows exhibited ultradian rhythms with periods around 80 min. The rhythms consisted of pulsatile spikes in peripheral GH concentrations with marked variation in amplitude and time interval between secretory events. This variation makes it difficult to detect the periodic nature of secretory events and can easily result in an underestimation of the importance of the periodic nature of GH secretion. Vasilotas and Wangness (8) reported eight to nine GH spikes per day in lactating cows. In contrast, Mollett and Malven (9) found that concentrations of GH over 72 hr did not show any tendency toward rhythmicity. Bines et al. (24), using 1-hr sampling, reported short-lived peaks of concentration at irregular intervals in high-yielding cows during peak lactation. The irregularity is probably due, at least in part, to the inadequate sampling rate for characterizing the ultradian rhythm. In humans, controversy exists as to whether GH secretion is episodic or periodic (1,2,25). Winer et al. (2), using a sensitive assay and sophisticated time series analysis techniques, concluded that: 1) pulsatile GH secretion was oscillatory rather than episodic; 2) GH pulses occurred with a dominant, but not strictly periodic, approximately 2-hr rhythmicity and were somewhat enhanced during sleep periods; and 3) an oscillation in the range of 120 min was a fundamental aspect of the system controlling GH secretion in humans. For undisturbed rhesus monkeys, Quabbe et al. (11) found rhythms with cycle lengths of 3 to 6 hr and a circadian rhythm that was detected by the use of autocorrelation analysis. No link was found to the sleep/wake cycle or to the slow wave sleep/rapid eye movement (SWS/REM) sleep stage cycle. In rats, GH peaks occur periodically approximately every 3 hr with no relation to the sleep cycle (26). For cows and sheep, individual sleep patterns vary widely and little time is spent in SWS. REM sleep is erratic, is normally of short duration, and is often observed after periods of drowsiness rather than after periods of SWS (27). Because of the erratic nature of sleep in cows, no attempt was made to relate individual secretory pulses with sleep stages. CONCLUSIONS Lactating cows acclimated to a rigidly controlled environment showed a very distinct sinusoidal circadian rhythm that peaked around 630 hr, an ultradian rhythm with a period of around 80 min, and evidence of an ultradian rhythm with a period of around 6 hr. The consistency of the circadian rhythm and ultradian rhythm with a period around 80 min

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indicates that these rhythms are probably intrinsic to mechanisms regulating peripheral GH concentrations in the lactating dairy cow. ACKNOWLEDGMENTS/FOOTNOTES To whom correspondence should be addressed: U.S. Department of Agriculture Milk Secretion and Mastitis Lab, Building 173, Beltsville Maryland 20705; Telephone: (301) 504-8451; 504-8213 Secretary; 504-9498 FAX.

REFERENCES 1. Tannenbaum GS. Neuroendocrine control of growth hormone secretion. Acta Paediatr Scand $372:5-16, 1991. 2. Winer LM, Shaw MA, Baumann G. Basal plasma growth hormone levels in man: New evidence for rhythmicity of growth hormone secretion. J Clin Endocrinol Metab 70:1678-1686, 1990. 3. Lefcourt AM. Circadian and ultradian rhythm in ruminants: Relevance to farming and science. In: Chronobiology: Its Role in Clinical Medicine, General Biology and Agriculture, Part B, Hayes DK, Pauly JE, and Reiter RJ (eds). Wiley-Liss, Inc., New York, pp. 735-753, 1990. 4. Lefcourt AM, Bitman J, Kahl S, Wood DL. Circadian and ultradian rhythms of peripheral cortisol concentrations in lactating dairy cows. J Dairy Sci 73:2607-2612, 1993. 5. Shumway RH. Applied Statistical Time Series Analysis. Prentice Hall, Englewood Cliffs, NJ, 1988. 6. Brandenberger G, Simon C, Follenius M. Ultradian endocrine rhythms a multioscillatory system. J Interdiscipl Cycle Res 18:307-315, 1987. 7. Lavie P, Kripke DF. Ultradian circa 1 1/2 hour rhythms: A multioscillatory system. Life Sci 29:2445-2450, 1981. 8. Vasilatos R, Wangsness PJ. Diurnal variations in plasma insulin and growth hormone associated with two stages of lactation in high producing dairy cows. Endocrinology 108:300-304, 1981. 9. Mollet TA, Malven PV. Chronological profiles of prolactin and growth hormone in lactating cows. J Dairy Sci 65:211-216, 1982. 10. Mason JW, Wool MS, Wherry FE, Pennington LL, Brady JV, Beer B. Plasma growth hormone response to avoidance sessions in the monkey. Psychosomat Med 30:760-773, 1968. 11. Quabbe HJ, Gregor M, Bumke-Vogt C, Eckhof A, Witt I. Twenty four hour pattern of growth hormone secretion in the rhesus monkey: Studies including alterations of the sleep/wake and sleep stage cycles. Endocrinology 109:513-522, 1981. 12. Lefcourt AM, Bitman J. Method of automatic continuous blood sampling with remote alarm for clogged catheters. J Dairy Sci 68:2108-2114, 1985. 13. Lefcourt AM, Akers RM, Wood DL, Bitman J. Circadian and ultradian rhythms of peripheral prolactin concentrations in lactating dairy cows. Am J Physiol 267:R1461-RI466, 1994. 14. Bitman J, Kahl S, Wood DL, Lefcourt AM. Circadian and ultradian rhythms of plasma thyroid hormone concentrations in lactating dairy cows. Am J Physiol 266:R1797-RI803, 1994. 15. Barnes MA, Kazmer GW, Akers RM, Pearson RE. Influence of selection for milk yield on endogenous hormones and metabolites in holstein heifers and cows. J Anim Sci 60:271-284, 1985. 16. Akers RM, McFadden TB, Beal WE, Guidry AJ, Farrell HM. Radioimmunoassay for measurement of bovine alpha-lactalbumin in serum milk and tissue culture media. J Dairy Res 53:419-429, 1986. 17. Bennett, RJ. Spatial Time Series. Pion Limited, London, 1979. 18. SAS Institute Imc. SAS/STAT Guide for Personal Computers, Version 6, SAS Institute Inc., Cary, NC, 1987. 19. Chatfield, C. The Analysis of Time Series. Chapman and Hall, New York, 1984. 20. Koprowski JA, Tucker HA, Convey EM. Prolactin and growth hormone circadian periodicity in lactating cows. Proc Soc Exp Biol Med 140:1012-1014, 1972. 21. Sutton JD, Hart IC, Morant SV, Schuller E, Simmonds AD. Feeding frequency for lactating cows: diurnal patterns of hormones and metabolites in peripheral blood in relation to milk fat concentration. Br J Nutr 60:265-274, 1988. 22. Zinn SA, Purchas RW, Chapin LT, Petitclerc D, Merkel RA, Bergen WG, Tucker HA. Effects of photoperiod on growth, carcass composition, prolactin, growth hormone and cortisol in prepubertal and postpubertal holstein heifers. J Anim Sci 63:1804-1815, 1986. 23. Evans NM, Hacker RR, Hoover J. Effect of chronobiological alteration of the circadian rhythm of prolactin and somatotropin release in the dairy cow. J Dairy Sci 74:1821-1829, 1991. 24. Bines JA, Hart IC, Morant SV. Endocrine control of energy metabolism in the cow: Diurnal variations in

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the concentrations of hormones and metabolites in the blood plasma of beef and dairy cows. Horm Metab Res 15:330-334, 1983. 25. Weltman JY, Veldhuis JD, Weltman A, Kerrigan JR, Evans WS, Rogol AD. Reliability of estimates of pulsatile characteristics of luteinizing hormone and growth hormone release in women. J Clin Endocrinol Metab 71:1646-1652, 1990. 26. Willoughby JO, Martin JB, Renand LP, Brazeau P. Pulsatile growth hormone release in the rat: failure to demonstrate a correlation with sleep phase. Endocrinology 98:991-996, 1976. 27. Ruckebusch Y. The relevance of drowsiness in the circadian cycle of farm animals. Anim Behav 20:637643, 1972.