Influence of feeding and diet on growth hormone and diurnal patterns insulin in calves of plasma Mears, G. J. 1993.Influence of feeding and diet on diurnal patterns of plasma growth hormone and insulin in calves. Can. J. Anim. Sci. 73: 987-991. Plasma GH concentrations were lower after feeding than before (newborn calves, P < 0.05; steers, P < 0.01). Calves fed concentrate had higher plasma insulin concentrations than those fed hay (P < 0.01). Meal feeding milk or concentrate, but not hay, elevated plasma insulin (P < 0.05). Diet type and feeding time relative to sampling time must be considered when measuring GH and insulin. Key words: Diurnal patterns, growth hormone, GH, insulin, calves Mears, G. J.1993.Influence du r6gime alimentaire et de la distribution des aliments par repas sur I'6volution diurne des concentrations de GH et d'insuline chez les veaux. Can. J. Anim. Sci. 73:987-991. Les concentrations plasmatiques de GH 6taient plus basses aprbs le repas qu'avant (veaux nouveaux-n6s P < 0,05; bouvillons P < 0,01). Les veaux recevant des concentr6s affichaient des teneurs plasmatiques en insuline plus hautes que ceux qui consommaient du foin (P < 0,01). Le type d'aliment et l'heure de la distribution par rapport )r I'heure des pr6ldvements sanguins doivent Otre pris en compte dans les mesures des teneurs en GH et en insuline du plasma. Mots cl6s: Evolution diurne. hormone de croissance. GH. insuline. veaux The basal concentrations of circulatins hormones in ruminants fluctuate due to i number of intrinsic factors such as episodic hormone release and diurnal rhythm. Other factors such as ambient temoerature. diet and feed intake can also influenie basal hormone concentrations. Researchers measuring circulating hormones must consider the diurnal patterns of concentrations of these hormones and the effects of factors such as feedins and diet on these patterns. Growth hormone fgh; is released in an episodic manner in ruminants resulting in peak plasma GH concentrations every 1.3-2.0 h in Angus steers (Wheaton et al. 1986). Superimposed on this pattern is a diurnal rhythm (Ringberg 1978) that may (Vasilatos and Wangsness 1980; Wheaton et al. 1986) or may not (Chamley et al. 1974 Ringberg 1978) be influenced by meal feeding. Plasma insulin concentrations in ruminants also follow a diurnal pattern, which can be markedly influenced by meal feeding (Vasilatos and Wangsness 1980; Trenkle Can. J. Anim. Sci. 73: 987-991 (Dec. 1993) 1981). Ruminants respond to high levels of dietary grain or protein with greater concentrations of plasma insulin (Trenkle 1981), but it is not known if the type of diet influences the level of insulin response following meal feeding. The objectives of this study were to determine the effects of diet and meal feedine on diurnal GH and insulin patterns in Hols--tein calves. Five newborn Holstein bull calves (range : 2-10 d old) and 15 Holstein steer calves (range : 123-296 d old) were bled every 2 h over a 12-h period beginning at 07:00 h. Newborn calves were bucket-fed mllk at l0% of body weight (BW) at 08:00 and 15:00 h. Steer calves were fed solid feed at 08:00 and 15:00 h, with all feed consumed within I h of feeding on the day of this experiment. Within each group of three calves at birth, each calf was randomly assigned to one of three dietary groups. After 100 kg BW they received one of three diets: Diet 1, 85% grass hay and 15% barley-based concentrate; Diet 2, same as diet 1 but with enough rumen undegradable protein (formaldehyde-treated ggj
988 CANADIAN JOURNAL OF ANIMAL SCIENCE canola meal) added to raise the crude protein content by 10 g kg-'; and Diet 3, 85% barley-based concentrate and15% grass hay. Calves were restricted-fed to 95% of estimated daily ad libitum feed intakes (for details of feeding see Bailey (1989)). The amounts of the three diets offered were designed to provide equal qmounts of digestible energy per unit BWU'/). The amounts of digestible energy actually consumed were similar (P > 0.1) for the three diets (Bailey 1989). Calves were bedded on wood shavings in individual pens in a heated barn with water and trace-mineralized salts continuously available and handled in accordance with the guidelines of the Canadian Council on Animal Care. Blood samples were collected into heparinized vacutainer tubes by jugular venipuncture while gently restraining the calves in their pens. The steers were also used in another experiment in which they were frequently handled and sampled for blood. Consequently, they displayed little stress during sampling for the present study, allowing all samples to be collected during a 12- to 14-min interval at each sampling time. Blood samples were stored on ice between collection and centrifuging. Plasma was stored at -40'C until assayed for GH and insulin using standard double antibody homologous bovine radioimmunoassay (RIA) procedures (Mears et al. 1988). For the GH RIA, NIH-GH-BIS (0.81 IU mg-1) was used as the reference GH and for chloramine-t iodination, with rabbit-antibovine GH, lot 3-28-VII (supplied by the late Dr. I. Geschwind, University of California, Davis, CA) as the primary GH antibody. For the insulin RIA, bovine insulin (I-5500. 26.8IU mg-r. Sigma St. Louis, MO) was used as the reference insulin and for chloramine-t iodination, with guinea pigantibovine insulin (65-101, Miles Laboratories, Elkhart, IN) as the primary insulin antibody. A11 samples were assayed in one RIA for each hormone. Intra-assay coefficients of variation were 6.2% for the insulin and7.6% for the GH RIA. Mean plasma hormone concentrations for the treatrnent groups were compared using the SAS General Linear Model Procedure for analysis of variance with time of collection and treatment group (Newborns, Diet 1, Diet2. Diet 3) as the main effects. When there was a significantf (P < 0.05) for time of collection the Student t test was used to determine which collection time mean hormone concentrations were different from one another. Plasma GH concentrations for steer calves fed Diets 7, 2 and 3 were similar (P > 0. 1) and are pooled for presentation in Fig. 1. They were much lower than those for newborn calves (P < 0.001). An effect of calf age on plasma GH concentrations has previously been shown (Roy et al. 1983). Therefore, the higher plasma GH concentrations for newborn calves were probably a result of their younger age, rather than their milk diet. The diurnal pattern of plasma GH concentration for both newborn calves and steers was influenced by meal feeding (Fig. 1). Plasma GH concentrations were lower (P < 0.01) in steers for all diets after both the morning and afternoon feedings, as was found by Vasilatos and Wangsness (1980) for Holstein cows, Wheaton et al. (1986) for Angus steers, and Trenkle (1989) for sheep. Newborn bull calves had lower (P < 0.05) plasma GH concentrations after the morning milk feeding with a trend (P < 0.1) towards lower concentrations at the time of the afternoon milk feeding. The 15:00 h blood samples were taken only a few minutes after the afternoon feeding. As a result the next samples were taken nearly 2 h after feeding. If the depression in GH concentration in newborn calves was of short duration, as found by Bassett (1914) for lambs only a few days old, it may have been missed with our sampling schedule. On the other hand, Ringberg (1978) and Driver and Forbes (1981) found a depression in GH concentration even before the afternoon feedings for bull calves and sheep. The small depression in plasma GH concentration in newborn calves by the time of afternoon feeding indicates that this may also have occurred in the present study. A drop in plasma GH prior to feeding could be accounted for by the involvement of neural
MEARS DIURNAL PATTERNS OF GH AND INSULIN IN CALVES 989-28 24 q. P- - -.q \,r-td --",.-- 20 i E16 (') g,t2 -(t8 3.0 2.5 2.0 E o) L te.s A 1.0 c 0.5 ++ ll Diets 1, 2, 3 Newborns T Diets 1, 2 Diet 3 Newborns f+ tl 7:00 09:00 1 1 :00 13:00 15:00 17:00 19:00 Time of day (h) Fig. 1. Plasma GH (upper panel) and insulin (lower panel) concentrations over a 12-h period for five newborn Holstein bull calves fed milk and 15 Holstein steer calves fed Diets 1.2. and 3. Arrows indicate times of feeding. Symbols indicate the probability of the values being different from the value obtained prior to the last feeding ( o, P > 0.05; o, P < 0.05; r, P < 0.01). pathways (Driver and Forbes 1981; Trenkle 1e89). Plasma insulin patterns in newborn calves demonstrated a meal feeding effect (Fig. 1) with elevated (P < 0.05) plasma insulin concentrations following both daily milk feedings. A similar increase in plasma insulin was reported for young lambs following milk ingestion (Bassett 1974). Plasma insulin concentrations in steers fed Diets 1 and 2 save no indication of a diurnal pattern and were similar (P > 0.1). Therefore they were pooled for presentation in Fig. 1. Overall mean plasma insulin concentrations for Diets 1 and 2 were similar (P > 0.1) to the overall mean insulin concentration for newborn calves. By contrast, plasma insulin concentrations for steers fed high concentrate Diet 3 were significantly (P < 0.01) higher than those for Diets 1, ard2. and demonstrated I
990 CANADIAN JOURNAL OF ANIMAL SCIENCE a meal feeding effect with elevated plasma insulin (P < 0.05) following both daily feedings. This is similar to the reported effect on plasma insulin concentrations of meal feeding high concentrate diets to mature cattle and sheep (Vasilatos and Wangsness 1980; Trenkle 1981). Elevated plasma insulin following milk ingestion in young ruminants occurs as a result of absorbtion of carbohydrates as the end-products of digestion (Bassett 1974). With the development of a functional forestomach in older ruminants, elevated plasma insulin following ingestion of high concentrate diets occurs as a result of circulating volatile fatty acids, the endproducts of digestion (Trenkle l98l). Lack of an insulin response to meal feeding hay diets in this study could be explained by the slower ruminal digestion of roughage, which would modulate flow of digestive end products into the circulation and fail to stimulate production of insulin. The episodic release ofhormones, coupled with infrequent blood sampling, may have influenced these results since the samples may have been taken at any point from the peak to the trough of a release. Although unlikely, such fluctuations may have contributed to the lack of diet-generated differences observed for plasma GH. However, an influence on the hormonal response to meal feeding was most likely minimal, since meal feeding synchronizes random episodic releases of GH in groups of steers (Wheaton et al. 1986). As the time between feeding and sampling was uniform for all calves in the present study, the effect ofepisodic release would be minimized. In our experience. plasma insulin concentrations exhibit little episodic release pattern in steers. Hence, these results would be influenced very little by the release pattern of insulin. Plasma concentration of a hormone is the net result of secretion into the circulatory system minus clearance from the blood. Decreased secretion of GH (Trenkle 1989) and increased secretion of insulin (Bassett 1974; Trenkle 1981) occur following eating. Therefore, the decrease in plasma GH and the increase in plasma insulin following meal feeding reported here are more likely due to changes in the rate of secretion of these hormones. In conclusion, these results clearly indicate that type of diet and time of feeding must be considered, along with the usual factor of diurnal patterns of hormone release, when planning experiments involving the measurement of GH and insulin. Depending on the diet fed, meal feeding may play a major role in altering the diurnal patterns of GH and insulin concentration. Plasma GH concentrations are lower within 2 h of feeding either hay or concentrate diets to steers. They are also lower within 2 h of feeding milk to newborn calves, at least for the morning feeding. Plasma GH may even begin to decrease before the afternoon feeding in young calves. Plasma insulin concentrations are higher in calves fed a high concentrate as compared to a high hay diet. Meal feeding of a milk or concentrate diet elevates calf plasma insulin concentration, whereas meal feeding of a hay diet does not. Therefore, samples for GH and insulin determinations should be obtained at the same time each day, relative to feeding times. Also, calves should all be on the same diet for measurement of plasma insulin, unless diet is a variable in the experiment. Failure to consider the effect that diet and the time of feeding relative to time of sampling have on the diurnal pattern of GH and insulin release may explain some reports of apparent discrepancies in hormone levels. Technical assistance provided by F. A. Brown, calf care provided by our animal herdsmen, the gift of antisera to bovine GH by the late Dr. I Geschwind, University of California, Davis, CA, and the gift of NIH-GH-BI8 through the National Hormone and Pituitary Program of NIDDK, University of Maryland School of Medicine, Baltimore, MD are deeply appreciated. Bailey, C. B. 1989. Rate and efficiency of gain, from weaning to slaughter, of steers given hay, hay supplemented with ruminal undegradable protein, or concentrate. Can. J. Anim. Sci. 69t 691-705. Bassett, J. M. 1974. Early changes in plasma insulin and growth hormone levels after feeding in lambs and adult sheep. Aust. J. Biol. Sci. 27: r5'7-t66.
MEARS DIURNAL PATTERNS OF GH - AND INSULIN IN CALVES 991 Chamley, W.A., Fell, L. R., Alford, F. P. and Goding, J. R. 1974. Twenty-four hour secretory profiles of ovine prolactin and growth hormone. J. Endocrinol. 61: 165-166. Driver, P. M. and Forbes, J. M. 1981. Episodic growth hormone secretion in sheep in relation to time of feeding, spontaneous meals and short term fasting. J. Physiol. 317: 413-424. Mears, G. J., Vesely, J. A. and Cheng, K.-J. L988. Plasma insulin and growth hormone in growing lambs fed monensin. Can. J. Anim. Sci. 68: 165-171. Ringberg, T. 1978. Diurnal variation of growth hormone in bull calves. Acta Agric. Scand. 28: 409-410. Roy, J. H. B., Hart, I. C., Gillies, C. M., Stobo, I. J. F., Ganderton, P. and Perfitt, M. W. 1983. A comparison of preruminant bull calves of the Hereford x Friesian, Aberdeen Angus x Friesian and Friesian breeds. Plasma metab-olic hormones in relation to age, and the relationship of metabolic hormone concentration with dry-matter intake and heart rate. Anim. Prod. 36:237-251. Trenkle, A. 198f. Endocrine regulation ofenergy metabolism in ruminants. Fed. Proc. N: 2536-2541. Trenkle, A. 1989. Influence of feeding on growth hormone secretion and response to growth hormone-releasing factor in sheep. J. Nutr. 119: 6r-65. Vasilatos, R. and Wangsness, P. J. 1980. Changes in concentrations ofinsulin, growth hormone and metabolites in plasma with spontaneous feeding in lactating dairy cows. J. Nutr. 110: 14'79-1487. Wheaton, J, E., Al-Raheem, S. N., Massri, Y. G. and Marcek, J. M. 1986. Twenty-fourhour growth hormone profiles in Angus steers. J. Anim. Sci. 62: 1267-12'12. G. J.Mears Reseorch Stqtion, Agriculture Canada, P.O. Box 3000, Main, Lethbridge, Alberta, Canada TI J 481. Contibution no. 3879284, received 4 January 1993, accepted 12 July 1993.