Environmental Heat Exposure on Cattle Plasma Catecholamine and Glucocorticoids 1

Transcription

1 Environmental Heat Exposure on Cattle Plasma Catecholamine and Glucocorticoids 1 M. B. ALVAREZ = and H. D. JOHNSON Department of Dairy Husbandry, University of Missouri, Columbia Abstract Nonlactating Holstein cows were exposed for varying times to environmental heat to determine changes in plasma epinephrine, norepinephrine, and glucocorticoids (principally hydrocortisone). Short heat exposures of 40 to 43 C caused average increases of 45 and 4270 in epinephrine and norepinephrine at 1 hour. Maximal increases (at 4.5 hours) of 127 and 847o were observed for epinephrine and norepinephrine. Glucocorticoids increased 3870 at I hour and declined to the control at 4.5 hours. More moderate 3-day exposure to 35 C increased epinephrine 70% at 6 hours and norepinephrine 3570 at 2 hours, which persisted throughout the 72-hour exposure. Glucocorticoids increased 62g by the second hour of exposure, reached a peak of at 4 hours, then declined gradually to values not different from normal at 48 hours and remained at this level for duration of exposure. Long heat exposure of 24 days showed epinephrine and norepinephrine means of 91 and 70% greater at 35 than at 18 C. At 35 C ghieocorticoids were 1370 higher than at 18 C on the third day of exposure, then declined and were lower by day 24. These data show a high and sustained adrenosympathetic activity (indicated by increases in epinephrine and norepinephrine) during heat acclimation and a transient increase in plasma glucocorticoids, which then declined even though body temperatures remained elevated during long heat exposures. Received for publication November 1, a Contribution from the Missouri Agricultural Experiment Station. Journal Series 6229, approved by the director. 'Rockefeller Foundation Fellow. Present address: Institute Co]ombiano Agropecurio, Apt. Aereo 7984, Bogota, Colombia, South America. Introduction The temporal changes in plasma catecholamines of animals exposed to environmental heat stress are of fundamental importance to understanding temperature-induced changes in heat production and dissipation. Peripheral vasodilation, blood pressure, heart rate, respiratory activity (5, 16), and tureen motility (3) are examples of functional changes associated with thermoregulatory functions suggesting an increase in sympathetic activity of cattle upon exposure to environmental heat. Plasma epinephrine and norepinephrine during 4 hr of heat were reported by Robertsbaw and Whittow (14) in steers and by Yu-Cong and Sturkie (18) in chickens. But there are no comprehensive studies on cattle exposed to environmental heat for varying times. Objectives of our study were to determine effects of short and long exposures to environmental heat on plasma epinephrine, norepinephrine, and glucocorticoids (principally hydrocortisone) and to evaluate relative changes during heat acclimation. 189 Materials and Methods We studied nonlactating Holstein cows housed in the Missouri Climatic Laboratory. Experimental animals were fed twice daily a high energy ration of 45~; grain, 50~ ground hay, and 57o molasses. Animals were allowed to drink and eat ad libitum; feed and water intakes were recorded daily. Data are the results of three experiments. The first involved acute 4.5-hr exposure of six cattle to environmental temperature of 40 C, followed 16 hr later by 4.5-hr exposure to 43 C. This experiment was repeated 2 weeks later. Original plans were to repeat the 40 C exposure, but because of alteration in temperature controls we used 43 C. In the second experiment five cattle were exposed to 35 C for 3 days. For the third experiment three cattle were exposed to 35 C for 24 days. Two cattle were maintained at 18 C. All experiments were under the same laboratory management conditions. During

2 - 190 ALVAREZ AND JOHNSON these short heat exposures, 40-ml blood samples were collected at 0, 1, 2.5, 3.5, and 4.5 hr of exposure. Blood was drawn through a polyethylene catheter inserted aseptically into a jugular vein the day before heat exposure. Blood samples were centrifuged and plasma eatecholamines extracted immediately and frozen. In experiment two, cattle were exposed to 35 C for 3 days following a 2-week adjustment at 18 C and 505 relative humidity. Blood sampling and chemical procedures were similar to the 4.5-hr exposure studies. We used similar procedures for the last experiment to obtain information on the adrenosympathetic system during long heat acclimation by exposing animals to 35 C and 50% relative humidity for 24 days. After a 3-week adjustment at 18 C, two eows were maintained as controls at 18 C and three were heat exposed (illness eliminated one cow). Epinephrine and norepinephrine were estimated fluorometrically. Twenty milliliters of plasma were added to 1 g of aluminum oxide as described by Grout (8), and fluorescence was developed as described by Euler and Lishajko (7). With each sample, three blanks (reagent blank, faded blank, and nonoxidized blank) and an intenaal standard were analyzed. Each sample was analyzed in duplicate with an Aminco-Bowman spectrophotofluorometer. We used two sets of activation/fluorescence wavelengths-400/500 m/z and 420/550 m/~-and the formula of Price and Price (13) for differential calculation of epinephrine and norepinephrine. Recovery of added epinephrine and norepinephrine ranged from 82.0 to 100.1%. The ghicocorticoid extraction technique was a modification of techniques by Bergrnan (4), Mattingly (11), and Stewart et al. (15). Five milliliters of plasma were extracted with dichloromethane at a 4:1 ratio of solvent to plasma. A 20-min extraction was followed by a.1 N NaOH wash. The sample was dried and partitioned ha 75% methanol-ether. An activating wavelength of 470 m/z and a fluorescent wavelength of 530 m V measured fluorescence. During heat exposure we measured rectal temperature with a clinieal thermometer immediately after withdrawal of corresponding blood samples. Statistieal analyses were by standard statistical programs (variance and regression analysis). Treatment effects were tested by Duncan's multiple range test. JOURNAL OF DAIRY SCIENCE VOL, 56, NO. 2 Results In the first short experiment the influence of environmental temperature on plasma epinephrine, norepinephrine, glucocortieoids, and rectal temperature expressed as percentage change to exposure time is in Fig. 1. Statistical significance of means is in Table 1. At 40 C (Table 1) plasma epinephrine and norepinephrine increased in the first hour of heat exposure at a rectal temperature of 39.3 C and were highest at 4.5 hr of exposure at a rectal temperature of 40.6 C. We observed the same pattern at 43 C treatment. After 1 hr o exposure and at subsequent periods the percent of epinepb_riaae response to heat was greater than that of norepinephrine. Analysis of variance indicated the 40 and 43 C values were not different so figures were averaged in Fig. 1. Epinephrine averages at both 40 and 43 C were 1.1, 1.6, 1.9, 2.1, and 2.5 /~g/liter. Differences between 0 hr (con- z 140 " I00,.) z w Tr /J / 1 / I I i I ~ "....'" :""..../ -\ "\ \ \ I I I I 0 I EXPOSURE TIME, hours... / Tr - E NE... G... FIG. I. Average changes in plasma epinephrine (E), norepinephrine (NE), glueoeorticoids (G) and rectal tempexature (Tr) during exposure of 40 to 43 (3. (The Tr percent ehange scale was used to illustrate pereentage changes in rectal temperature which were of lower magnitude than the hormones. The --20 to 140 scale was used for the hormones.)

3 HEAT AND PLASMA CATACHOLAMINE 191 TABLE 1. Effects of short heat exposure (40 and 43 C) on average rectal temperature and plasma coneentrations of catecholamines and glucocortieoids. Criteria Environmental Exposure Rectal temperature time temperature Epinephrine Norepinephrine Glucocortieoids (C) (hr) (C) (gg/liter) (gg/liter) (gg/100 ml) * 1.0 ~ ca a" 2.6 a 3.5" be 1.8 Cd 2.8 ~ 3.1 "b ~b 2.1 ~bc 3.1 bed 2.5 b~d ~ 2.5 "b 3.3 "b 2.2 ~d ~ 1.9" 2.4 bed a 1.8 Ca 2.9 " 3.1 ~b ~ 2.0 b~ 3.2 be 2.9 "b~ ~ 2.0 be 3.4 "b 2.4 Cd ~ 2.6 ~ 3.7" 2-2 a * Means with same superscript letters in same column are not significantly different (P <.05). Duncan's multiple range test was used to test for significance of means. trol) and averages of experimental values at each exposure time were statistically significant (P <.05). Respective average values for norepinephrine were 1.9, 2.7, 3.0, 3.2, and 3.5 /~g/liter. Differences between 0 hr (control) and averages of experimental values at each exposure time were also statistically significant (1) <.05). A positive correlation (P <.01) was found between rectal temperature and epinephrine (r =.76) and norepinephrine (r -=.74). A positive correlation (P <.01) was also found between epinephrine and norepinephrine (r =.81). Animals displayed elevation in plasma glucocorticoids in the first hour of exposure at both 40 and 43 C (Table 1, Fig. 1). MaMmal values were observed at 1 hr of exposure and declined gradually to near normal 3.5 hr after exposure to environmental heat. Fig. I illustrates that plasma glucocorticoids increased (Table 1) in relation to rectal temperature during the first hour of exposure, after which plasma glucocortieoids declined and rectal temperature continued rising. A negative, though not significant (r =.35), correlation was found between rectal temperature and plasma concentration of glucocorticoids. TABLE 2. Means of plasma glueocorticoids, epinephrine, and norepinephrine during 72 hr of exposure to moderate environmental temperature of 35 C. Criteria Environmental Exposure Rectal temperature time temperature Epinephrine Norepinephrine Glucocorticoids (C) (hr) ( C ) (~g/liter) (/zg/liter ) ( ~g/100 ml ) a* 38.6 ca 1-0 b 1.3 "b 2. 0~ 2.7 "be 2"4a 3 "9be "b 1.5 ab 2.8 "be 5.4" " b 1.7" 2.9 "b 5.(P "b 1.7" 3,0 ~ 4 "7~b "b 1.7" 2.9 "b 4 "5"b "b 1.8" 2.9 "b 3.4 cb ~b 1.8" 2-4 "be 3-4cb ab 1.8" 2.7 "b~ 3.4 ca "b 1.7" 2.7 ~bc 3.3 ~d "b 1.8 ~ 2.7 "be 3. 4~d * Means with same Duncan's multiple range superscript letters in same cohman are not significantly" different (P <.05). test was used to test for significance of means. JOURNAL OF DAIRY SCIENCE VOL, 56, NO, 2

4 1~2 ALVAREZ AND JOHNSON In the second experiment we reduced the environmental temperature from 40 to 35 C for animal survival. A 3-day exposure to more moderate heat of 35 C showed that elevated plasma catecholamine persists for 3 days whereas plasma glucocorticoids declined following a transient elevation (Table 2). An elevation (P <.05) in plasma norepinephrine was observed during heat exposure from 2 to 72 hr compared to the 0 hr (control), and plasma epinephrine showed an increase (P <.05) at 6 hr (Table 2). Plasma eateeholamines remained elevated during the 3-day exposure. Results of 3-day moderate heat exposure experiment were similar to those of the first experiment and confirmed the observation that heat exposure brought about a sustained increase in adrenosympathetic activity (epinephrine and norepinephrine). A correlation (P <.01) was found between plasma concentration of epinephrine and norepinephrine (r =.84). As in the first experiment, glucocorticoids began to rise during the first hour of exposure, reached a peak of 5.4 /~g/100 ml (120% increase) after 4 hr, then declined gradually to values not different from normal at 48 hr and stayed at this level in spite of continued heat stimulus and elevated rectal temperatures. Values increased when rectal temperature was 38.3 to 39.0 C and declined as body temperature was further elevated. The reason for greater increase in glucoeorticoids in the second experiment than in Experiment 1 is not readily apparent. Presumably, the rise in plasma glucocortieoids was due to activation of the adrenocorticotropin (ACTH)-releasing mechanism in the hypothalamus by therrnoreceptors in the skin (6). Plasma glucocorticoids returning to normal, in spite of continuing heat stimulus, indicates that ACTH secretion was not continuously stimulated by heat, or the stimulatory effect of heat on the ACTH-releashag mechanism was counteracted by continual glucocorticoid feedback. Glucocorticoid feedback may have been induced by unbound plasma glucocorticoid concentration because the binding capacity of transcortin decreased markedly as temperature increased from 4 to 37 C (10). A greater ratio of free to bound glucocorticoids may counteract the stimulant effect of heat on the ACTH-releasing mechanism. Table 3 presents data from the third experiment which studied eatecholamine and glucocorticoid activity in relation to long heat exposure. Duncan's test indicated at 35 C mean glucocorticoids became lower after 12 days of exposure. A comparison (analysis of variance) of 18 and 35 C values during the 24 days showed the 35 C values to be lower (P <.01). Means of plasma epinephrine for control animals were 1.1 /~g/liter of plasma and for experimental animals, 2.1 /~g/liter. This increase was statistically significant. The mean TABLE 3. Means of plasma epinephrine, norepinephrine, and glucoeorticoids during long heat acclimation at 35 C. Exposure Time (days) Environmental Temperature 18 C 35 C Control animals Experimental animals Tr E NE G Tr E NE G ~* 1.7 a 3.0 a " 3.0 ~ 3.4" ~ 1.7 ~ 3.0 ~ " 3.4 ~ 3.1 a ~ 1.(P 1.7 a 1.8" 2.9" 3.0 ~ " 2.3" 3.4 ~ 3.4" 2.5 ~ 2.4 b ~ 1.7" 3.0" " 3.8" 1.9 b " 1.7 a " 3.7 a 1.7 b " 1.6 a 3.0" " 3.0" 1.9 ~ ~ 1.8" 2.8" a 2.8 ~ 2.1 u Tr = Rectal temperature (C) G Glucoeorticoids (tzg/100 ml) E = Epinephrine (t~g/liter) NE = Norepinephrine (t~g/liter) ~* Means with same superscript letter in same column are not significantly different (P <.05) reflecting trends at each temperature. JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 2

5 HEAT AND PLASMA CATACHOLAMINE 193 plasma norepinephrine was 1.7 and 2.9 /zg/liter of plasma for control and experimental animals; this increase is statistically significant. General support for this i~nding is in the work of Hale et al. (9) who reported relatively high catecholamine excretion in men acclimatized to heat. Mechanisms responsible for increase in plasma catecholamines during prolonged heat exposure are not known. It might be caused by decrease in binding and inactivation of norepinephrine, an increase in release of norepinephrine from storage vesicles of sympathetic nerve endings or adrenal glands, or metabolic changes leading to an increase in free plasma eatecholamines without concomitant increase in sympathetic activity. It is possible that heat aeelimination was accompanied by a decrease in sensitivity to physiological actions of cateeholamines and that high plasma catecholamines allowed more amines to react with their reeeptor. Deerease in sensitivity was suggested by observing that calorigenie action of epinephrine in cattle was depressed by heat exposure (1, 2). In summary, our data suggest an elevation in deep body temperature, and perhaps surfaee temperature, as effective stimuli for sustained increase in plasma catecholamines since they responded similarly to rectal temperature upon heat exposures. Physiological significance of depressed plasma hydrocortisone concentration during heat acclimation is not clear. Glucocorticoids exert a stimulatory effect on heat production in cattle, and depressed adrenoeortical function during heat acclimation might contribute to depression in heat production observed during prolonged heat exposure (17). Deep body or surface temperature does not provide continual stimulus for glucocorticoid activity. Since glucocorticoid elevation response was not maintained throughout 4.5 hr of environmental heating, continuous stimulation of nonspecifie stressor such as ACTH (12) was not evident. The initial skin thermal-receptor increase was probably the stimulus for ACTH release through synaptic connections with the corticotropin releasing factor (CRF) mechanism in the hypothalamus (6). These data provide a graphic temporal study of adrenosympathetie responses to environmental heat in cattle and clearly establish that epinephrine and norepinephrine persist at an elevated level in the plasma, whereas the glucocortieoids following significant vari- ances subsequently decline to values lower than controls even though body temperature remains elevated during all heat exposures. References (1) Alvarez, M. B Relation of environmental temperature to the activity of the adrenosympathetie system of cattle. Ph.D. Diss. University of Missouri, Columbia. (2) Alvarez, M. B., W. D. Robertson, L. Hahn, M. K. Yousef, and H. D. Johnson Physiological responses of bovine to catecholamine. J. Anim. Sei., 26:940. (Abstr.) (3) Attebery, J. T., and H. D. Johnson Effects of environmental temperature, controlled feeding and fasting on rumen motility. J. Anim. Sci., 29:734. (4) Bergman, R. K Effects o~ a prolonged high environmental temperature on the glueocorticoids in the bovine. Ph.D. Diss. University of Misso~lri, Columbia. (5) Bianca, W., and J. D. Findlay The effect of thermally-induced hyperpnoea on the acid-base status of the blood of calves. Res. Vet. Sci., 3:38. (6) Chewers, I., H. T. Hamme], J. Eisenman, R. M. Abrams, and S. M. McCann Comparison of effect of environmental and preoptie heating and pyrogen on plasma cortisol. Amer. J. Physiol., 210:606. (7) Euler, U. S. V., and 17. Lishajko Improved technique for the fluorometrie estimation of catecholamines. Acta. Physiol. Scan&, 51:348. (8) Grout, R Catecholamines in urine. In M. Reiner (Ed.) Standard Methods of Clinical Chemistry. Vol. III. Academic Press, New York. (9) Hale, H. B., E. W. Williams, and J. P. E~lis, Jr Catecholamine excretion d~ring heat deacchmatization. J. AppI. Physiol., 18:1206. (10) Lindner, H. R Comparative aspects of cortisol transport. Lack of firm binding to plasma proteins in domestic ruminants. 5' Endocrinel., 28:301. (11) Mattingly, D A simple fluorometrle method for the estimation of free ll-hydrexycorficoids in human plasma. ]. Clin. Path., 15:374. (12) Mitra, R., and H. D. Johnson Bovine plasma growth hormone levels as influenced by environmental heat. J. Dairy Sci., 53: 652. (Abstr.) (13) Price, H. L., and M. L. Price The chemical estimation of epinephrine and norepinephrine in human and canine plasma. 5. Clin. Lab. Med., 50:765. (14) Robertshaw, D., and G. C. Whittow The effect of hyperthermla and localized heating of the anterior hypothalamus on JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 2

6 194 ALVAREZ AND JOHNSON the sympatho~adronal system of the ox (Bos taurus). J. Physiol., 187:351. (15) Stewart, C. P., R. Albert-reeht, and L. M. Osman The simultaneous fluorometrie mierodetermination of eortisol and corticosterone in plasma. Clin. Chem. Acta., 6:696. (16) Whittow, G. C, The effect of hyperthermia on the systemic and pulmonary circulation of the ox (Bos taurus). Quart. J. Exp. Physiol., 50:300. (17) Yousef, M. K Hormonal effects on gaseous metabolism and thyroid ftmetlon of cattle at various temperatures. Ph.D. Diss. University of Missouri, Columbia. (18) Yu-Chong, Lin, and P. D. Sturkie Elfeet of environmental teanperature on the eateeholamines of chickens. Amer. J. Physiol., 214:237. JOUI~NAL OF DAIRY SClENCt~ VOL. 56. NO. 2