(Wallace & Bassett, 1970; McAtee & Trenkle, 1971) were unable to find significant

Transcription

1 J. Physiol. (98), 37, pp With 6 text-figures Printed in Great Britain EPISODIC GROWTH HORMONE SECRETION IN SHEEP IN RELATION TO TIME OF FEEDING, SPONTANEOUS MEALS AND SHORT TERM FASTING BY P. M. DRIVER* AND J. M. FORBESt From the Department of Animal Physiology and Nutrition, University of Leeds, Leeds LS2 9JT (Received 5 May 98) SUMMARY. Blood samples were taken every 2 min (for at least 28 hr) from five castrate male and two anoestrus female ad libitum fed sheep. Analysis for plasma growth hormone (GH) showed that two of the males, on two occasions, had regular, although individually specific, patterns of GH secretion (peaks 35-5 hr). The other animals all had irregular patterns of GH release. 2. Throughout the experiments, meal sizes and frequency were recorded and it was found that out of eighty spontaneous meals of at least 5 g, 57 (7 %) occurred in the hour after GH peaks, which accounted for 5 % of the total time. Furthermore, on twenty out of twenty-four occasions GH levels were found to be falling before the 'expected' feeding time when fresh food was offered and the animals normally consumed a large meal. The removal of the food from three of the males for hr during an experiment prompted an increase both in the size and frequency of the GH peaks. After re-feeding, GH levels immediately fell and remained low for -2 hr. 3. We believe that these results show an association between GH secretion and meal feeding in sheep, and that GH secretion quickly responds to fasting. As GH levels fell before, or in the early stages of meals, this indicates a neural reflex in the inhibition of GH before a meal. INTRODUCTION Elevated levels of circulating growth hormone (GH) during periods of nutritional deficit have been reported in several species, e.g. pigs (Atinmo, Baldijao, Houpt, Pond & Barnes, 978); rabbits (McIntyre & Odell, 974); human (Merimee & Fineberg, 974); sheep (Driver, Brown, Scanes & Forbes, 976). However, several authors (Wallace & Bassett, 97; McAtee & Trenkle, 97) were unable to find significant differences in mean blood GH levels in ruminants following prolonged fasting, although Bassett (975) did report an increase in the frequency of oscillation of GH secretion in fasted sheep and a tendency for GH levels to be reduced at feeding, a result which has also been found in cattle by Bl6m, Hasle & Hove (976). * Present address: Department of Science, Lancashire College of Agriculture, Myerscough Hall, Bilsborrow, Preston PR3 ORY. t To whom reprint requests should be sent.

2 44 P. M. DRIVER AND J. M. FORBES The episodic release of GH in sheep over a 24 hr period has been shown by Davis, Ohlson, Klindt & Anfinson (977), amongst others, and has been related to feeding by Bassett (974a) although Chamley, Fell, Alford & Goding (974) were unable to confirm this. The significant fall, from a peak, of plasma GH found in castrate male lambs at feeding (in both restricted and ad libitum fed animals) (Driver et al. 976) prompted an investigation into the relationship between plasma GH levels and spontaneous meals (as opposed to induced feeding when fresh food is offered) and the effect of short term fasting in sheep. Some of the results presented in this paper have been published in abstract form by Driver & Forbes (978) and Driver, Adams & Forbes (979). METHODS Animals The study included three experiments using a total of seven sheep. The animals were housed adjacently in individual, raised wire mesh pens in a small room within an animal house. They were handled frequently and made accustomed to the blood sampling procedures before the experimental runs. On the day before the experimental run the animals were prepared with short jugular cannulae. Blood samples (3-4 ml.) were taken at 2 min intervals. Meal recording All the animals received a concentrate feed (5%O dried grass, 5o barley; High D pellets, Lincolnshire Farm Feeds, Grimsby) ad libitum and had a steady intake in the days preceding the experiment. Meal duration and size were recorded by the continuous recording of the weight of the feed bucket suspended from a strain gauge assembly similar to that described by Suzuki, Fujita & Shinde (969). Meal size could be measured to the nearest I g and meal duration to approximately 2 min, however, feeding bouts that began within min of the end of a previous bout were counted as part of the same meal. A meal initiated within min of the offering of fresh food was counted as being 'induced'. Measurement of GH Blood samples were collected into pre-heparinized tubes and kept in ice for no longer than 4 hr. The samples were centrifuged at 4 'C, the plasma removed and stored frozen at - 5 T until assay. GH was measured by a specific radioimmunoassay (Driver, Forbes & Scanes, 979) using NIH-GH-8 as standard. A plasma sample assayed times in one assay had a mean concentration of 6-9 ng/ml., with a standard deviation of Consecutive samples differing in GH concentration by more than 28 ng/ml. (4 S.D.) can, therefore, be accepted as showing a real change in GH level. RESULTS The first experiment involved two month old castrated male sheep (Suffolk 3 x (East Friesland S x Blackface Y) Y), A and B. Run lasted from 82 until 8 the next day (34 hr); animal A was recannulated at 725 on the first day and results up to midnight were not used. Run 2 took place 8 days later between 82 and 2 the next day (28 hr). Each animal had food and water available throughout the whole experiment. Refusals were removed and fresh food offered at 97 for run, and 93 and 92 for run 2. GH secretion and spontaneous meals are shown in Figs. and 2. One run was carried out on two 4 month old seasonally anoestrus ewes (C and D; Suffolk J@ x (East Friesland S x Blackface Y) Y). Blood sampling was carried out

3 GH AND FEEDING IN SHEEP 45 6 A I2 ; k n " I M, Wh n A ~~~~~~~~~~~~~~~~~ - I I B t Fig. A, B, experiment, animal A. Episodic GH secretion and meal feeding over 34 (A) and anml 28 (B) reeie hr. The arrows fehfoat3echmrigfg. indicate the offering of fresh food.- Because I shwste of missing samples, H n ma results obtained before midnight from sheep A have not been used. Time represents midnight. the~~ 8 r Time (h between 72 and the next day (28 hr). The refusals were removed at 925 and the animals received fresh food at 93 each morning. Fig. 3 shows the GH and meal patterns. Two runs were carried out on three 5-6 months old castrate male sheep E and F (Suffolk 6' x Blackface Y) and G (Suffolk 6' x Greyface Y). Animal F was recannulated at 23 'on run and blood samples between 8 and 22 were taken by venepuncture from this animal. Run 2 was carried out 9 days after run. Blood sampling was done from 72 to (28 hr) in each run. Refusals were removed at 925 and the animals given fresh food at 93 on each of the 2 mornings of each run. On run 2 the food was removed from the animals at 73 and the same food put back

4 46 P. M. DRIVER AND J. M. FORBES 5 - A X 8 I - CU~~~~~~~~~~ e B I t 2 Time (h Fig. 2. A, B, experiment, animal B. Legend as for Fig.. at 33 ( hr fast). Results for animals E, F and G are given in Figs. 4, 5 and 6, respectively. Episodic GH secretion The profiles ofgh secretion for animals A and B are given in Figs. A, B and 2 A, B. A regular pattern ofgh secretion usually in the form of a double peak occurring every hr was evident. A difference in the secretary patterns between the two sheep was apparent, although the patterns from the same animal during the two experimental runs were almost identical. Sheep A tended to have a low basal level (4-6 ng/ml.), but large peaks (45-7 ng/ml.); sheep B had a slightly higher basal level (7-5 ng/ml.) but lower, broader peaks (4-5 ng/ml.). No regular pattern of GH secretion was found in ewes C or D (Fig. 3 A, B) and the general GH levels are much lower for C (peak 8-4 ng/ml.) than A or B. Ewe D had very high peak levels (5-2 ng/ml.) with a great deal of oscillation. Again, no regular pattern of GH release was found in any of the three sheep E, F or G (Figs. 4A, 5A, 6A) and peak levels (6-2 ng/ml.) were lower than sheep A

5 GH AND FEEDING IN SHEEP E 2 'a - ICA 5 I E cm 2: 5 8 ' Time (h) 4 8 Fig. 3. A, B, experiment 2, animals C (A) and D (B). Episodic GH secretion and meal feeding over 28 hr. Note the different GH scale for animal D (B). The arrows indicate the offering of fresh food. and B. On run 2 (Figs. 4B, 5B, 6B) GH levels began to increase in both size and frequency -2 hr after the removal of the food at 73, and continued to increase until the food was returned, at which time GH levels fell sharply. Relationship between GH and the offering offresh food Animals E, F, G were fed at exactly 93 each day, for 2 weeks before run, while sheep A, B, C, D had been fed between 95 and 945 before experiments and 2. Each run encompassed two normal times when the refusals were removed and fresh food offered to the animal, giving a total of twenty-four such occasions throughout the three experiments. On twenty of these occasions (see Figs. -6) GH levels were declining from a peak between h to 2 min before the usual feeding time. Of the four other occasions two are from animal C (Fig. 3A) in which GH levels were low some time before feeding and remained low, while the other two are from animal B (Fig. 2A, B) and are inconclusive. Relationship between OH and spontaneous meals The patterns of GH concentrations and spontaneous meals, shown in Figs. -6, were examined and meals divided into those which started during 2 min periods when GH was falling by more than -3 ng/ml. and those which started when GH was a) N.(E ) 4 ph Y 37

6 48 P. M. DRIVER AND J. M. FORBES 2 -S B I A C, N - I ) - 6 E) cm IS 3 2 ' N._A a 2 Time (h) Fig. 4. A, B, experiment 3, animal E. Episodic GH secretion and meal feeding over 28 hr (A) and the effect of hr fasting (B). The bar indicates the fasting period. The arrows on the time scale indicate the offering of fresh food (93). rising by more than -3 ng/ml. between successive samples. The GH data were examined in relation to (a) spontaneous meals of at least 25 g and (b) spontaneous meals of at least 5 g (Table ). (a) Excluding induced meals and all meals following periods of enforced fasting there were 32 spontaneous meals of at least 25 g during a total of 269 hr of observation. In this time there were min periods when GH was rising and 44 when GH was falling. The expected number of meals occurring during periods of falling GH, if the two are unrelated is 32x 44 = The observed number of meals during periods of falling GH was 4. The deviation ofobserved from expected frequency is very highly significant by the X2 test (P < -). When the same method of analysis was applied to indvidual sheep, all showed a greater number of meals starting during declining GH levels than would be expected

7 GH AND FEEDING IN SHEEP 49 A ^ 2 E C" -S 2 - o E 3 3'-, 2 ' N -i a) 2 Time (h) Fig. 5. A, B, experiment 3, animal F. Legend as for Fig. 4. The large downward pointing arrow indicates the time the animal had consumed all its food. if meals started at random in relation to changes in GH level, but these deviations were statistically significant only in sheep B (P < '5), C (P < '5) and E (P < ). (b) As many of the changes in GH levels during 2 min periods were small and did not appear to be part of a major spike, and the 25 g meals included some 'nibbling', the data were re-analysed according to whether meals of at least 5 g started during hr after the peak value of a spike. Spikes are defined as values above ng/ml. in all the experimental runs with the exception of the following. Sheep B, run 2-5 ng/ml.; sheep D - 25 ng/ml.; sheep C and sheep F, run -5 ng/ml.: 33 spikes occurred during the 269 hr of free access to food. Of the eighty meals of 5 g or more, fifty-seven occurred during the hour after GH peaks, and twenty-three outside this post peak period. The number of meals occurring during the hour after a GH peak that would be expected by chance is 4, thus the observed number of meals was significantly greater (P < -) than the expected number. The results showed that meals occurred more frequently than can be ascribed to chance, when GH levels were falling than when GH levels were rising, and more 4-2

8 42 P. M. DRIVER AND J. M. FORBES E C s 2 A 338 u 2 m - w._ N IN - 4 U) 3 B' 2 I - 3 _la -2 '( - 2 Time (h) Fig. 6. A, B, experiment 3, animal G. Legend as for Fig. 4. TABLE. (a) Number of spontaneous meals of at least 25 g starting during 2 min periods when GH levels were rising or falling. (b) Number of spontaneous meals of at least 5 g starting during hr periods after GH peaks. Totals indicate observed incidence of meals and the frequency expected if meals were completely independent of GH secretion. *, includes only meals occurring before enforced fasting (a) Number of meals over 25 g Animal/ GH GH Figure run rising falling A Al 3 4 2A BI 2 lb A B B2 2 3A Cl 95 3B Dl 4A El 3 5 5A Fl 3 8 6A Fl 6 7 4B E2* B F2* 6B G2* 2 5 Totals Observed Expected 28 4:}p<o- Base line (b) Number of meals over 5 g - hr after peak Other P < -

9 GH AND FEEDING IN SHEEP 42 frequently during the hour after the peak values of GH than at other times. Because of the frequency of sampling (2 min) it is not possible to say, in many cases, whether GH was falling before the meal started or whether the decline started in the early part of the meal. DISCUSSION Episodic GH secretion These data confirm the findings of other workers (Chamley et al. 974; Bassett, 974a; Davis et al. 977) that sheep in common with other ruminants (cattle, Blom et al. 976; goats, Tindal, Knaggs, Hart & Blake, 978) secrete GH in an episodic fashion with several peaks over 24 hr. Sheep A and B showed regular, and individually specific, patterns of secretion consisting of a double peak every 3-5 hr, similar to those found in rats (Tannenbaum & Martin, 976) and baboons (Steiner, Stewart, Barber, Koerker, Goodner, Brown, Illner & Gale, 978). Davis, Ohlson, Klindt & Everson (979) similarly found that individual mature rams had patterns of GH secretion that were observed to be more repeatable within animals on different occasions, than between animals on the same occasion. The overall levels of GH were lower than those reported here and, as no details of feeding are presented, nutritional correlates of the GH data of Davis et al. (979) are not available. Other studies (see above) on ruminants failed to show any obvious temporal rhythm and this was the case in the results from sheep C-OG. While the episodic release of GH is evident, no regular pattern of secretion can be discerned. As the regular peaks of secretion found in animals A and B, run, were reproducible 2 weeks later (run 2) it is unlikely that this is a chance finding. Possible explanations are the age and sex differences between the animals; such differences in GH levels and secretion have been shown in sheep (Davis et al. 977) and in cattle (Trenkle, 97 a). Eden (978) has reported that the episodic release of GH declines in rats as they get older but that it persists in males longer than in females. Other cues, such as feeding (see below), may be responsible but at this time we cannot give any satisfactory explanation as to the reasons for regular secretary pattern of GH in some animals but not in others. Relationship between GH and the offering offresh food The findings that GH fell when lambs were fed diluted milk and also fell during the sham feeding of adult sheep led Bassett (974 b) to postulate that GH release involves a neural reflex pathway. In twenty out of twenty-four cases in these experiments GH levels were falling before the time at which fresh food was normally offered (93) and it appears reasonable that the sheep might 'expect' to be fed at this time, after which they nearly always take a large meal. These results lend support, therefore, to the concept of a type of conditioned response to expected feeding which inhibits GH release. Effect of short-term ( hr) fasting upon GH secretion Prolonged fasting does not appear to affect circulating GH levels inruminants (Wallace & Bassett, 97; McAtee & Trenkle, 97) although restricted fed ruminants have significantly higher levels of circulating GH than ad libitum fed animals and the

10 422 P. M. DRIVER AND J. M. FORBES levels fall at feeding (Bassett, 972, 974a, b; Hove & Blom, 973; Blom et al. 976; Driver et al. 976). The results from animals E-G clearly show that episodic GH secretion increases during a short period of food deprivation. It is worth nothing that animal F (run, Fig. 5A) had consumed all its food by midnight and then effectively produced its own fast; GH levels increased over this period but fell before the expected feeding time and appeared normal after feeding. Ewe D (Fig. 3B) hardly ate throughout the experiment (reason unknown), the GH levels showing a great deal of oscillation with very high peaks. Again the levels fell at feeding and remained low after the animal had consumed a large meal. Several workers (see Introduction; Hart, Bines, Morant & Ridley, 978; Trenkle, 978) have reported elevated GH levels in ruminants kept under feeding regimes which would cause relative energy deficit; the results presented here indicate that the previous findings may be the combined effects of a short-term increase in GH secretion induced by short-term energy deficit between feeding times. The experiments in which GH levels were measured at feeding in ruminants show a fall in circulating levels at feeding (Bassett, 972; Hove & Blom, 973). Two reports (Bassett, 974b; Trenkle, 97 b) suggest that short-term fasting has no effect upon ovine GH. However, both reports examine mean plasma values. The episodic GH secretion patterns found in our experiments indicate that each animal has a specific individual type of secretary pattern and the use of mean values may not give valid results unless GH secretion has been synchronized by feeding time or some other cue. Also the fasting of animals which may have been in relative energy deficit and may therefore already have an increased GH secretion, may not further increase GH levels. Why GH levels are not increased after long-term fasting is puzzling. Perhaps a long fast produces an unphysiological situation in which other hormones become more important. The half life of GH is reduced in fasting (Trenkle, 976), but a possible explanation is that GH is converted into biologically active molecules more quickly than under normal conditions. There is growing evidence (I. C. Hart, personal communication; P. M. Driver, unpublished results) that ruminant GH radioimmunoassays measure only immunoreactive pituitary GH and possibly not a biologically active form. In starvation, more GH may be converted into the active form thus giving no apparent change in circulating radioimmunoassayable GH levels. Relationship between GH secretion and spontaneous meals The discovery of a relationship between induced feeding and GH, and between induced GH secretion and suppression of food intake (Driver et al. 979) prompted us to investigate the association between episodic GH release and spontaneous meals (Table ). In the experiments reported here the majority of spontaneous meals were preceded by peaks of GH, but it is not possible to say with certainty in all cases whether GH levels started to fall before or after the initiation of the meal. In either case, the GH peak might be a response to mild energy deficit and a reflex inhibition might occur in anticipation of this deficit being rectified by feeding. The significance of increased GH secretion in response to lack of readily available sources of energy is not clear because although GH is lipolytic in vivo (Rao & Ramachandron, 977) this is a slow action and may only be permissive.

11 GH AND FEEDING IN SHEEP 423 It is clear that as GH secretion is inversely linked with insulin release in ruminants (see Bassett, 975; Halse, Blom & Hove, 976; Forbes, Driver, Brown, Scanes & Hart, 979), all the findings presented here might possibly be explained as being a consequence of insulin secretion or inhibition in response to feeding and fasting. Much of the evidence from ruminants to date relates to an increase in plasma insulin at or after ingestion (Lofgren & Warner, 972; Bassett, 974a; Hove & Bl6m, 973; Blom et al. 976) while our results indicate that GH levels fall before or in the early part of a meal. In rats Strubbe, Steffens & de Ruiter (977) showed that insulin is low up to 5 min before a meal but increases upon the ingestion offood. Alloxan-diabetic sheep maintained on protamine-zinc insulin still have episodic GH secretary patterns which are related to spontaneous meals (P. M. Driver, unpublished observations). It therefore seems unlikely that the GH secretary profile and the relationship with the initiation of spontaneous meals is a direct consequence of insulin secretion, although GH, insulin and possibly glucagon may all be involved in the mechanisms which initiate feeding in sheep. In summary, therefore, we believe that the results presented here indicate () that episodic GH secretary rhythms in sheep are individual and that mean results can be of little value; (2) that GH secretion will respond quickly to short-term fasting and slight energy deficit; (3) that there is a neurological reflex to inhibit GH secretion in anticipation of 'expected' feeding; and (4) that inhibition of GH secretion may also occur around the time of initiation of spontaneous meals. We should like to thank Dr W. B. Brown, Dr J. Falconer and Mr G. B. Adams for their assistance in the collection of blood samples, and Mr J. Saltmarshe for assistance with the radioimmunoassays. REFERENCES ATINMO, T., BALDIJAO, C., HOuPT, K. A., POND, W. G. & BARNES, R. H. (978). Plasma levels of growth hormone and insulin in protein malnourished V8. normal growing pigs in response to arginine or glucose infusion. J. anim. Sci. 46, BASSETT, J. M. (972). Plasma glucagon concentrations in sheep: their regulation and relation to concentrations of insulin and growth hormone. Aust. J. biol. Sci. 24, BASSETT, J. M. (974a). Diurnal patterns of plasma insulin, growth hormone, corticosteroids and metabolite concentrations in fed and fasted sheep. Aust. J. biol. Sci. 27, BASSETT, J. M. (974b). Early changes in plasma insulin and growth hormone levels after feeding in lambs and adult sheep. Aust. J. biol. Sci. 27, BASSETT, J. M. (975). Dietary and gastro-intestinal control of hormones regulating carbohydrate metabolism in ruminants. In Digestion and Metabolism in the Ruminant, pp , ed. McDONALD, I. W. & WARNER, A. C. I. Armidale, N.S.W. Australia: University of New England Publishing Unit. BL6M, A. K., HALSE, K. & HOVE, K. (976). Growth hormone, insulin and sugar in the blood plasma of bulls. Interrelated diurnal variations. Acta endocr. Copenh. 82, CHAMLEY, W. A., FELL, L. R., ALFORD, F. P. & GODINO, J. R. (974). Twenty-four hour secretary profiles of ovine prolactin and growth hormone. J. Endocr. 6, DAVIS, S. L., OHLSON, D. L., KLINDT, J. & ANFINSON, M. S. (977). Episodic growth hormone secretary patterns in sheep: relationship to gonadal steroid hormones. Am. J. Physiol. 233, E DAVIS, S. L., OHLSON, D. L., KLINDT, J. & EVERSON, D.. (979). Estimates of repeatability in the temporal patterns of secretion of growth hormone, prolactin and thyrotropin in sheep. J. anim. Sci. 49,

12 424 P. M. DRIVER AND J. M. FORBES DRIVER, P. M., ADAMS, G. B. & FORBES, J. M. (979). Response of plasma growth hormone to short term fasting and spontaneous meals in sheep. J. Endocr. 83, 5P. DRIVER, P. M., BROWN, W. B., SCANES, C. G. & FORBES, J. M. (976). Serum GH levels in growing lambs at two daylengths and two levels of feeding. J. Endocr. 69, 44P. DRIVER, P. M. & FORBES, J. M. (978). Plasma growth hormone and spontaneous meals in sheep. Proc. nutr. Soc. 37, 9A. DRIVER, P. M., FORBES, J. M. & SCANES, C. G. (979). Hormones, feeding and temperature in sheep following cerebroventricular injections of neurotransmitters and carbachol. J. Physiol. 29, EDEN, S. (978). The secretary pattern of growth hormone: an experimental study in the rat. Acta physiol. 8cand. 4, (suppl) -58. FORBES, J. M., DRIVER, P. M., BROWN, W. B., SCANES, C. G. & HART, I. C. (979). The effect of daylength on the growth of lambs. 2. Blood concentrations of growth hormone, prolactin, insulin and thyroxine, and the effect of feeding. Anim. Prod. 29, HALSE, K., BLOM, A. K. & HOVE, K. (976). Growth hormone related to insulin and sugar in nocturnal blood plasma of lactating cows. Acta endocr. Copenh. 73, HART, I. C., BINES, J. A., MORANT, S. V. & RIDLEY, J. L. (978). Endocrine- control of energy metabolism in the cow: comparison of the levels of hormones (prolactin, growth hormone, insulin and thyroxine) and metabolites in the plasma of high- and low-yielding cattle at various stages of lactation. J. Endocr. 77, HOVE, K. & BLOM, A. K. (973). Plasma insulin and growth hormone in dairy cows: diurnal variation and relation to food intake and plasma sugar and acetoacetate levels. Acta endocr. 73, LOFGREN, P. A. & WARNER, R. G. (972). Relationship of dietary caloric density and certain blood metabolites to voluntary feed intake in mature wethers. J. anim. Sci. 35, MCATEE, J. W. & TRENKLE, A. (97). Effect of feeding, fasting and infusion of energy substrates on plasma growth hormone levels in cattle. J. anim. Sci. 33, MCINTYRE, H. B. & ODELL, W. D. (974). Physiological control of growth hormone in the rabbit. Neuroendocrinology 6, 8-2. MERIMEE, T. J. & FINEBERG, S. E. (974). GH secretion in starvation: a reassessment. J. clin. Endocr. Metab. 39, RAO, A. J. & RAMACHANDRON, J. (977). Growth hormone and the regulation of lipolysis. In Hormonal Proteins and Peptides, vol. iv, pp. 43-6, ed. LI, C. H. New York: Academic. STEINER, R. A., STEWART, J. K., BARBER, J., KOERKER, D., GOODNER, C. J., BROWN, A., ILLNER, P. & GALE, C. C. (978). Somatostatin: a physiological role in the regulation of growth hormone secretion in the adolescent male baboon. Endocrinology 2, STRUBBE, J. H., STEFFENS, A. B. & DE RUITER, L. (977). Plasma insulin and the time pattern of feeding in the rat. Physiol. & Behav. 8, SUZUKI, S., FUJITA. H. & SHINDE, Y. (969). Changes in the rate of eating during a meal and the effect of the interval between meals on the rate at which cows eat roughages. Anim. Prod., TANNENBAUM, G. S. & MARTIN, J. B. (976). Evidence for an endogenous ultradian rhythm governing hormone secretion in the rat. Endocrinology 98, TINDAL, J. S., KNAGGS, G. S., HART, I. C. & BLAKE, L. A. (978). Release of growth hormone in lactating and non-lactating goats in relation to behaviour, stages of sleep, electroencephalograms, environmental stimuli, and levels of prolactin, insulin, glucose and free fatty acids in the circulation. J. Endocr. 76, TRENKLE, A. (97 a). Growth hormone secretion rates in cattle. J. anim. Sci. 32, 5-8. TRENKLE, A. (97 b). Effect of diet upon levels of plasma growth hormone in sheep. J. anim. Sci. 32, -4. TRENKLE, A. (976). Estimates of the kinetic parameters of growth hormone metabolism in fed and fasted calves and sheep. J. anim. Sci. 43, TRENKLE, A. (978). Relation of hormonal variations to nutritional studies and metabolism of ruminants. J. dairy Sci. 6, WALLACE, A. C. & BASSETT, J. M. (97). Plasma growth hormone concentrations in sheep measured by R.I.A. J. Endocr. 47, 2-36.