The effect of ACTH and heat stress upon themetabolism of selected minerals in the bovine

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Authors Lox, Charles Douglas, 1936-

Publisher The University of Arizona.

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I

THE EFFECT OF ACTE AND. HEAT STRESS UPON THE METABOLISM OF SELECTED MINERALS IN THE.BOVINE

byCHARLES DOUGLAS LOX

A Thesis Submitted to the Committee onANIMAL PHISIOLOGI

In Partial Fulfillment of the Requirements For the Degree of.MASTER OF SCIENCE

.In the Graduate College. .THE UNIVERSITY OF-ARIZONA

1970

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library.

Brief quotations from.this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole, or part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship.In all other instances, however, permission must be obtained from the author.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below;

T, ;N, V/egner Assistant Professor

Y.Date

ACKNOWLEDGMENTS

The author wishes to thank Dr, G, H, Stott and Dr, Je D«Sehuh for their assistance and advice during the course of this project6

Thanks are also in line for Mr„ Brannick Biggs who aided, in the collection'of some of the samples, and to the personnel at the dairy research center who assisted at various phases of the research.

Most of all I wish to thank Dr, Thomas N, Wegner for his aid and encouragement, not only for making this project possible, but for insuring the author’s continuation in graduate school.

TABLE OF GOMOTS

PageLIST OF TABLEb *o » »» v <10 » e» VLIST OF ILLUSTRATIONS. * . * , . . . , . , . . . ....... viiABoTRAOT . . » e e $ . * e r . < ? c 0 * 0 e e <t » e e e e e- # »• . IL̂CINTRODUCTION * * . * * . , , . * , * , + , . . . . . . , . , 1REVIEW OF LITERATURE » * , * . . , , * . , . . . , . . . . . 3

General Concepts 3Physiological Aspects of Stress.. « • » » • • « . ae'* 3Jlrxier'alhS ISFactors Affecting Embryonic Survival . . . . * . * * .19ACCll.matXOn. * e . 20Mineral Analysis . « « • » • • « • • * » • • • * • • • 21

METHODS AND MATERIALS. . . , * , . . , 22Experimental Animals 22Procedure . c o o < i o c . c o 6 o « c o c o . @ o # * 22Analysis . . o . c . o c . o e o . @ @ » o 6 . » <► 0 o 23Statistical Analysis . » » » » « » <, « 0 « » » » » » 27

RESULl S ti . . o e o « > o 0 0 » 0 0 < t 0 0 0 0 0 0 » 6 0 « 0 o 0 2SPhysiological Reaction of Animals. + « , * , . . * * 28Minerals . * 0 0 0 0 . 0 * 0 0 * * 0 . 0 0 0 0 0 0 0 31Aboraaon o * » o o o * * * * 0 0 0 0 0 0 . 0 0 0 0 0 *̂7

DISCUSSION 000 00 o p o . o o o o o o e o 00 0 o 00 o o o 31APPm!MDIX . o « * * » o o o . » « c * * ̂° ̂ • * * . * * . * 72BIHLIOVtRAPHI 0 0 0 * 0 0 0 0 0 * 0 0 0 0 0 * 0 0 0 0 * 0 00 92

iv

LIST. OF TABLES

Table

2.3cAe

5.

6v

7c

8.

9.

■10,

11.

12. 13 c 1̂ -.15.16.

Physiological Condition of Experimental Animals at the Initiation of Experiments . . . . . . . . . . .Sample Dilution Factors for Atomic Absorption Analysrs . . . . . . . . . . . . . . . . . . . * . . .Percent Recovery of a Known Concentration. . . . . . .liineral Composition of Blood, Urine, Milk, and Feces of Heat Stressed Cow 673 . . . . . . . . . . . .Mineral Composition of Blood, Urine, Milk, and Feces of Heat Stressed Cow 729 . . . . * . . . . . . .Mineral Composition.of Blood, Urine, Milk, and Feces of Heat Stressed Cow 599Mineral Composition of Blood, Urine, Milk, and Feces of Heat Stressed Cow 64-0 .. . « , . . . . . . . .Mineral Composition of Blood, Urine, Milk, and Feces of ACTE Stressed Cow 608 . . . . . . . . . . . .Mineral Composition of Blood, Urine, Milk, and Feces.of ACTH Stressed Cow 609Least Square Means for Experimental Minerals in Blood and Ur m e . . . . . . . . . . . . . . . . . . . . . .Least Square Means for Experimental Minerals in Milk and Feces . . . . . . . . . . . . . . . . . . . .Least Significant Difference of Treatment for Blood. .Least Significant Difference of Treatment for Urine. .Least Significant Difference of Treatment for Milk . .Least Significant Difference of Treatment for Feces. «F Test Analysis for Equality of Variance . . . . . . .

v

Page

22

7373

74

76

78

80

81

83

85

868788899091

Table17.

18.

19.

20.

/

LIST OF TABLES - ContinuedVi

PageEffect of Heat Stress on Blood Calcium forCows 673 and 729 . 32Mean Effect of 250 Units ACTH on BloodSodium m 609. . . . . . . . . . . . . . . . . . . . . . 37Effect of Heat Stress on Mean Urine Phosphorus in 7 29. c . g . . . AOEffect of ACTH Stress on Fecal Phosphorusfor 608 and 609. . . . . . . . . . . . . . . . . . . . 46

LIST OF ILLUSTRATIONS

Figure Page1» Climate in the Environmental Chamber, , 292, Effects of 50 Units ACTI-I on Blood Calcium e o $ . » « « 333» Blood Calcium Means for 608 and 609 «- « „ » e «, . v « „ 33

Effect of Acute Heat Stress on Blood Phosphorus » . , . . 355= Effect of 50 Units ACTH on Blood Sodium 366, Effect of 50 Units ACTH on Blood Potassium, 387, Effect of 250 Units ACTH on Blood Potassium »<,<,». « 388, Effect of 50 Units ACTH on Urine Magnesium, „ * * * , * 399c Effect of 250 Units ACTH on Urine Calcium , , , « « « 39

10, Effect of Sub-Acute Heat on Urine Magnesium , ijl110 Effect of Acute Heat Stress on Urine Magnesium. • , .... U2 .12, Effect of 50 Units ACTH on Urine Phosphorus . . . . . . k3

13. Effect of Sub-Acute Heat on Urine Phosphorus. . . . . . UU,llt. Effect of 50 Units ACTH on Urine. Potassium,. . . . . . . . .1*515. Effect of Acute Heat Stress on Fecal Phosphorus . . . . lj.816. Effect of Sub-Acute Heat on Fecal Phosphorus. . . . . . U917. Blood and Urine Calcium Interactions. 5k

18. Milk and Fecal Calcium Interactions . . . . . . 1 . 5519. Blood and Urine Potassium Interactions. . . . . . . . . 5820. Milk and Fecal Potassium Interactions 5921. Blood and Urine Magnesium Interactions. . , .. .'. ,. . .. 6l22. Milk and Fecal Magnesium Interactions; . 62

.- v i i ‘

LIST OF ILLUSTRATIOBS - Continued Figure • , Page23= • Blood and Urine Phosphorus Interactions * « * , . * * 6424. Milk and Fecal Phosphorus Interactions. . . . . . . . 6525. Blood and Urine Sodium Interactions . . . . . . . . . 6826. Milk and Fecal Sodium Interactions. . . . . . . . . . 69

ABSTRACT

Calcium, magnesium, phosphorus, potassium, and sodium were measured in the blood, urine, milk, and feces of six dairy cows„ Three of the six cows were primed for one week prior to stressing with 50 units of ACTH (adrenocorticotropic hormone)„ One primed cow and one unprimed cow were placed together as a. group and were subjected to one of three stresses; sub-acute temperature stress for 2k hours (90-lOliF and 70-85% relative humidity), acute temperature stress for 2b hours (lOU-llSF and 78-85% relative humidity), or high levels of ACTH (250 units:, per day) for 72 hours „

Priming with ACTH caused significant differences in blood, urine, milk, and feces when compared to unprimed animals prior to stress*

When the animals were stressed, calcium patterns indicated a . general increase due to ACTH and a decrease due to heat. Temperature decreased the magnesium in all test substances except milk, ACTH in­creased milk and fecal levels of magnesium but decreased blood and urine levels. Phosphorus decreased in all the body fluids with both ACTH and temperature stress. Urine levels practically disappeared. Temp­erature stress decreased blood, urine, and milk sodium and increased fecal sodium. Blood sodium rose and fecal levels dropped due to ACTH, while urine/was. unaffected.. Milk sodium decreased in the ACTH stressed, primed cow, and increased in the unprimed cow.

■ •. . x Fetal abortion occurred following acute heat stress in both

primed and unprimed animals<, No changes were observed in the blood,milk, or fecal mineral content during the abortion periods Urine calcium:magnesium, and phosphorus increased, while sodium decreased, and potas-

, sium remained unchanged.From this study it is evident that elevated temperature and

ACTE alter the levels of various minerals in the bovine, which possiblycould be detrimental to the metabolism and physiology of the animale

INTRODUCTION'

Minera,! metabolism in higher vertebrates has not received the attention and scientific recognition that other biological products have in recent yearsc However<> with the introduction of some of the more modern analytical tools, such as the atomic absorption spectro­photometer, there has been a renewed interest in mineral research*

The role of some of the major mineral ions of the body as co­factors in the various biochemical pathways, as the main matrix com­pounds in the hard tissues of the body, and as part of the osmotic regulatory mechanism, is now well understood* There has also been a . great deal of work done on the mechanism of hormonal regulation of mineral metabolism, for example the action of parathormone on calcium and phosphorus levels, and aldosterone on sodium potassium ratios* However, the effect of high environmental temperatures on the normal homeostatic mechanisms of the body and the fate of minerals in the thermoregulatory process is still a wide-open field*

Arizona and other similar desert and tropic ecological habitats have extremely high summer temperatures* Much of the metabolic energy of the high producing dairy cow is directed toward cooling and thermo­regulation in the summer months and consequently a lot of the economic and scientific research effort of the Arizona dairy industry is directed towards understanding and minimizing the physiological stress of animals during this period. It is to this end and to an understanding of the

role of minerals in thermoregulation, namely, to follow the metabdlid fate of certain mineral ions in the lactating dairy cow subjected to high temperature and hormonal stresses that this project was under­taken.

REVIEW OF LITERATURE9

General Concepts One of the best recent porks which comprehensively covers the

minerals can be found in the treatise of Comer and Brormer (32, 33). This four volume work updates and modernizes the treatment of inorganic ions in biological systems*

The concept of a physiological response to stress goes back to the General Adaptation Syndrome, in which a sudden environmental change activates a homeostatic mechanism leading to a physiological response which results in the adaptation to the new condition. In this view, Robertson and others (123) list stressors in dairy cattle as: surgery,mastitis, milk fever, uterine prolapse, placental retention, with some variations possible*

J^mologisal AgRects of Stress Heat as a stress has been reported in the literature (28, 30,

76). Each paper deals with different facets of heat stress* The effect of heat as a stress and its action upon the animal, has been re­ported (14, 28, 30, 47, 76, 87) . Collins and Weiner (3.0) allocated an important position to heat, stating;

Environmental heat .presents, at all levels of biological organ­ization, a.stress that brings into' play in the homeothermic animal a, complex of nervous, endocrine,- neurohumoral, and motor functions combining to restore a constant body temperature and to adjust body fluid balance, energy metabolism, and behavior to the needs concomitant with survival in .the new environment.

3

4Findlay and Beakley (47) reported the rectal temperature reached

108F in cattle stressed at 105F. There was also an increase in the respiration and heart rate* Bianca (14) found that calves exposed to 400 had rectal temperatures of 42,$0, Respiration increased to 218 per minute with panting, and heart rate increase^ to 50 per minute, which he states is due to the increased demand' for oxygen in the respiratory muscles. After prolonged exposure to the heat the panting ceased and a deep "second phase" breathing set in, Collins and Weiner (30) concluded that a primary response to heat is characterised by increased body tem­perature, which in turn stimulates the brain causing evaporative cooling, panting, and vasodialation. They also felt (28) that the extent of change during heat stress depends upon the severity of the heat, ex-' posure time, and metabolic condition of the subjected animal. Hale and Mefferd (63) believed that the degree and time of exposure to heat was the deciding factor, acute heat caused the rise in body temperature, whereas chronic heat caused a homeostatic adjustment,

Anderson (3) found that during a pain stress in cattle, urine volume increased from 200-300%, The effect of stress upon the kidney was also reported by Ladell (83) in which heat exhaustion caused a darkening of the urine and a decrease in volume.of urine, Leithead (87) reported a failure of the renal tubules to respond to the anti-diuretic hormone of the posterior pituitary. Thus it seems a fine line exists between heat stress and heat exhaustion in regards to renal function. Oliguria is an early response to heat that is brought about by a change in renal blood flow, a decrease in glomerular filtration rate,•and ■

decreased interrenal resistance^ with the change in renal blood flow the prime cause of the Oliguria (28),

It is well known that" heat depresses growth in many species of animals, and that heat acclimated animals are smaller than animals' ' raised in cooler climates, This has been reported in Holstein, Brown Swiss, and Jersey calves by Johnson and Ragsdale (74) who found that continual exposure of cattle to 80? Is sufficient to generally retard growth. The fact that growth and metabolism are directly linked by .hormonal activation has led to interest in the effects of heat or stress o'n the endocrine glands, Timmer' (144) found that scalding stress in rats caused a decrease in growth hormone and an increase in thyroid stimulat­ing hormone (TSH), with the levels of TSH peaking 12 hours after the stress, .

One non-hypophyseal endocrine gland which is involved directly with metabolism and heat is the thyroid, Thompson and others (143) de­scribed the effect of heat on thyroid function as characterized by a significant decrease in thyroxine secretion and protein-bound iodine, . Findlay (48) stated that the thyroid is involved in long term adaptation to heat and its depression is an initial reaction to heat, Itoh and Nishimura (73) likewise reported a thyroid shut down due to heat, and further,, because the thyroid hormone stimulates the adrenal, there was a decreased adrenal activity. Hale and Mefferd (63) refer to a decrease, in thyroid activity,.and a thyroid-adrenal interplay, .They stated, acute heat inhibited both, resulting in a decreased metabolic rate and a de­creased body heat production, Peterson (112) discussed the adrenal

. . - ■ . . ■ 6 hypertrophy which resulted from excess thyroxine# and showed hypo­thyroidism caused adrenal atrophy„

The mediation of a response to stress from any source involves the central nervous system, and therefore the involvment of ACTH from the anterior pituitary. Ganong and others (54-) reported that stress by cannulation in dogs seemed to show a decrease in AGTH secretion.This was also reported in humans subjected to a heat stress and exer­cise at 40.50 (l24)o Oh the other hand, surgical stress and fear or anxiety (130) has been reported to increase ACTH secretion (35, 154, 157)o Kotby and others (82) reported a sudden increase in ACTH after exposure to 340 in rats. Timmer (144) seemed to resolve some of the conflict when he observed that scald stress caused an initial rise in ACTH followed by a decrease one hour after the stress, which had not returned to normal after 24 hours.

The second, and the most important, major endocrine gland involved in stress and heat adaptation is the adrenal gland. References to this organ can be found dealing with all aspects of adrenal physio­logy and endocrinology. A very detailed account of the biology and chemistry of the adrenal can be found in Eisenstein (40), whose text gives an in depth review of the organ. .

It has been shown (117) that chronic stress caused a hyper­trophy of the adrenal and an increased output. Holzbaur and Newport (70) demonstrated that stress caused an increase in circulating cholesterol, pregnenolone, and progesterone, all of which are pre­cursors of the adrenal gluco and mineralocorticoids« Shima and Pincus (132) believed that the increases in corticosterone from cholesterol

is a result of pooled precursors and that AGTH or stress does not increase cholesterol levels. The fact that stress actuates or in­creases the levels of adrenal corticoids is well documented. Increases have been noted in 17-hydroxycorticoids, 17-ketocorticoids, and ll-deoxycorticoids (30, 97? 123? 145? 146? 151? 152).

One of the basic assumptions of a neurohumeral regulation' of the response to stress is that adrenal activation is brought about by . AGTH. Ganong? Biglieri and Mulrow (55) pointed out that a hypertrophied adrenal gland due to AGTH injections is more responsive to AGTH as far as glucocorticoid secretion is concerned. This mobilisation of the adrenal reserve would indeed be beneficial to the animal. AGTH does cause a rise in adrenal corticoids? especially cortisol and corticos­terone (16? 21? 77? 93? 155? 164). Mulrow and Ganong (106) reported that a dose of 2 units of AGTH stimulated cortisol and corticosterone release in dogs just as well as did 50 units. Ohlsen and Hokfelt (114) found that AGTH injections in man increased plasma cortisol? urinary 17-ketocorticoids and 17-ketogeniccorticoids,

Heat stress has its effect on the adrenal gland. Clark and others (27) reported a general adrenal hypertrophy due to heat. Collins and Weiner (30) stated that the bovine adrenal medulla is responsive to heat? by increasing the secretion of adrenaline. General.increases of corticoids in both man (29? 30? 61? 155) and in dairy cattle (76? 143) have been described. Moody (104) indicates an increase in adrenal pro­gesterone due to heat stress in cattle. Zarrow and others (164) found that rats stressed at 630 for 3 minutes shoved a large increase in both the.plasma and.adrenal;corticosterone levels? corticosterone being.the

major glucocorticoid found in the rat* It was also reported by Zarrowand others (163) that in the 2 day old rat plasma corticosterone morethan doubles three minutes after the 630 stress. Kotby and others (82) attributed this rise to the activation of the adrenal by pituitary AGTH. They found that heat caused an increase in urinary 17~hydroxyaorticoste- roids and 17-ketosteroids in man during the first three days of exposure, then the hydroxycorticoids returned to normal whereas the ketosteroids became depressed. Thus it may be that some of the adrenal steroids are inhibited by heat while others are activated, and this may further de­pend upon the degree of heat. Barlow, Angersborg and Keys (8) found that, a severe hyperthermia in dogs increased the blood 17™hydroxycorticosteroids significantly. Ghowers and others (25), working with dogs at 42-450, found a marked increase in the plasma cortisol after 45 minutes of acute heat, while gradual heat caused only very minimal changes. Yet Itoh and. Nishimura (73) stated there is no difference in urine steroid output in the rat at 20C and 320. This was true for both normal and hypophyseatom­ized animals. Exogenous AOTH was found to decrease the adrenal outputat 320, suggesting to Itoh that heat inhibits the adrenal itself.Robinson and MacFarlane (124) found similar results with decreasing urinary 17-*ketosteroids, indicating a decrease in adrenal activity and AOTH secretion. ' Bergman and Johnson (12) found, a depression in the plasma cortisol of cattle after 2 weeks at 84$Y Heilman (66) found a decrease in the 17~hydroxycorticosteroids in men heat stressed at 100F,

. If a continual secretion of corticoids were to occur after stress, possible damage to the organism might occur. Kitay,. Holub and Jailer (80) found that circulating levels of AOTH can inhibit the release of

further AGTH from the pituitary, so there must be a titer which regu­lates the normal release of AGTH, possibly stimulating the synthesis of AGTH. . A suppression of AGTH release occurs in man when circulating levels of AGTH are high (69, 3.4.0) „ Other workers (5, 13, 4-6, 78, 90) found that corticoid therapy or injection inhibits further adrenal activity by blocking AGTH'via a negative feedbacke

It has recently been reported (55) that one adrenal hormone is in part controlled by a system extraneous to the pituitary. This con­trol comes from the kidney and is in the form of the porteins renin and angiotensin. Several authors (64, 106, 134, 135) believe that, the major control of secretion of aldosterone is via the renin-angiotensin system, and have shown increased secretion of aldosterone after infusion of-renin extract in animals with the pituitary removed.

Aldosterone is required to sustain life. Its role in maintaining a proper salt balance between the retention and excretion of sodium and potassium is discussed in detail by Ganong, Biglieri, and Mulrow (55)» They state that changes in sodium and potassium in the blood can activate the adrenal cortex to secrete aldosterone, with a rise or fall in either mineral being sufficient to activate its release from the cortex.

The activation of aldosterone by the renin-angiotensin system was briefly discussed earlier. Numerous authors (88, 92, 133) have found that a, proper sodium to potassium ratio was maintained in hypophyse atom­ized man and rats, Hayslett' (65) has found that hypoaldosteronism occurs during renal failure. Osanky, van der Wal, and de Wied (3?) and Spark" et aL, (135) indicated that aldosterone, release was primarily controlled, by renin> with other modes of activation depending upon the levels of

renin at the time.It is known that ACTH stimulates aldosterone secretion in man

(36, 53, 105, 147, 157), in the sheep (16), in the rat (133), in the dog (106) and in cattle (30, 77), Ganong, Biglieri, and Mulrow (55) stated that no change occurs in the cells of the zona glomerulosa (which secrete, aldosterone) when injected with ACTH, Williams (157) found that ACTH promoted aldosterone, secretion by stimulating its synthesis in the adrenal cortex, but that continued injection caused a decrease in aldosterone production. In man, Muller, Riodel, and Manning (105) reported that the effect of ACTH on aldosterone, secretion is often masked by the other mechanisms which operate at the more mini­mal stimuli, and which are independent of ACTH, Tucci and others (147) have found that 80 units of ACTH given intravenously in man caused the greatest secretion of aldosterone after two days, Blair-West and others(16) working with sheep have found that ACTH injections at the levels found in Addisons disease caused an increase in the level of aldosterone. Mulrow and Ganong (106) found that it required from 100 to 1000 units of ACTH to stimulate the secretion of aldosterone in the dog. Kaplan and Bartter (77) report that adrenal slices from the bovine, when stimulated with ACTH on a cholesterol substrate, secrete aldosterone, but if the media was progesterone (instead of cholesterol), no secretion occurred.

This seems to agree with the statement of Williams (157) that ACTH may cause the synthesis of aldosterone from pooled adrenal pre­cursors.

It was found by Lamson and others (84) that neurotic patients subjected to insulin shock showed an increase in urinary aldosterone.

/ - ' . 11 The rise in aldosterone secretion and excretion due to heat is also well documented (31, 51, 8?)„ Robinson and MacFarlane (124) found that men exercising at 40,50 showed an increase in the urinary potassium to sodium ratio. This retention of sodium was indicative of Increased aldosterone secretion, Hellmark (66) found the levels of aldosterone increased in the urine of man when exposed to 100F, ̂ -

. There have been several reports of an aldosterone stimulating hormone (17, 38, 116) „ Palmore and Mhlrow (116) make a definite point that the pituitary is needed for an aldosterone response to sodium de­pletion in dogs, and the pituitary factor is not ACTH, Hypophysectomised animals, on a low sodium diet, were.injected with AGTH and secreted no aldosterone, although, there was an increase in general adrenalcortical secretion. When homogenized pituitary gland was injected, there was an increase in the secretion of aldosterone (116)» Blair-West and others(17) came to the same conclusion with sheep. They found high levels of aldosterone in animals which were salt depleted when the pituitary or kidneys were removed. No change in sodium or potassium occurred, eliminating ionic influences. This seemed to indicate a possible site of secretion other than the pituitary, Balfour et al* (?) found an inhibition of aldosterone from.the cerebral area. Dogs with the pituitary and kidneys removed had very low levels of aldosterone; when the forebrain was removed, the levels of aldosterone increased to a point equal to sodium depleted animals. This indicated that the brain might help prevent release of extraneous aldosterone.

It has been reported (89) that several other adrenal steroids have mineralocorticoid activity. Gaunt and others (56) felt that

12iS-hydroxydeoxycorticosterone caused sodium retention. It also had anti-diuretic properties and exhibited no effect on potassium excre­tion, Williams (157) stated that 11-deoxycorticosterone caused an acute sodium retention and potassium excretion, while corticosterone and most of the glucocorticoids had some aldosterone activity, George and others (5?) investigated the idea of: a sodium excreting hormone and found that progesterone caused a slight loss of sodium. They believe high levels of aldosterone during pregnancy may be a counter to this sodium excreting effect of progesterone,

• Minerals -The inorganic mineral composition of bovine blood has been

determined by many workers (24, 49, 85, 86, 100, 109, 126, 136), The mineral composition of cattle urine, reported by Spector (136), listed the values in Meg/ 24 hours (which is the normal process of reporting urine values). Corner and Bronner (33) stated that urine is the major path of excretion for metabolic magnesium. The determination of milk minerals has received as much attention as that of blood in cattle (79, 108, 113, 126, 136), Very little has been done with the total mineral content of fecal material, though several references to partial analysis are available (33, 39). Wrong and others (159) gave the mineral composition of human stool.

The hormonal influences on blood calcium have been observed in some animals. Comer and Bronner (33) stated that ACTH decreased the serum calcium in cattle, Ifatelson, Pincus, and Rannazzisi (ill, 112) reported that ACTH lowered rabbit serum calcium levels up to fifty

• / ■ 13percent, causing tetany and convulsions in one and a half to tx-ro and a half hour'se Comer and Bronner (33) stated that generally the corti- coids, including cortisone, decrease blood calcium,, Natelson, Pineus, and Rannazzisi (ill, 112) on the other hand stated that cortisone had no effect on blood calcium. Sellers and Hoepke (123) found that diethylstilbestrol lowers the serum calcium slightly. Reports (110, 111) indicated that parathormone increased blood, calcium and may be antagon­istic in action to AGTH, Eriesen ($2) found that beta melanocyte stimu­lating hormone (B-MSH) lowered the serum calcium levels in rabbits, the decrease amounted to 3 mg/100 ml in two hours, Natelson, Pincus, and Rannazzisi (111, 112) stated that thyroid stimulating hormone, growth hormone, luteotropic hormone, oxytocin, adrenaline, noradrenaline, insulin, glucagon, hydro cortisone, and corticosterone had no effect on the serum calcium levels in rabbits. They also stated that stress caused a 10 percent decrease in the calcium levels, Findlay and Beakley (47) found that extreme temperatures caused a high calcium/phosphorus ratio in Holstein cows, . Blincoe and Brody (18) and Brody et al, (20) reported no change in blood calcium due to heat.

Injections of diethylstilbestrol in cattle had no effect on serum magnesium levels according to Sellers and Hoepke (128), Thyroxine was found to decrease the blood magnesium (162)„ Scott and Dobson (127) observed that infusion of aldosterone into sheep caused a decrease in plasma magnesium, which agrees with the findings of Weil and State (156) that adrenalectomy increased serum magnesium levels, ■ The findings of Akgun, Rudman, and Mertheim (l), in regards to the effect of B-MSH in ' rabbits are noteworthy, since this hormone raised'the levels of plasma

14magnesium about fifty percent. This rise was attributed to the fact that the hormone is a. lipolytic agent and degrades some of the body lipid, freeing bound magnesium. It has been reported (18, 20) that high temperature has no effect on the blood magnesium in cows. How­ever, Yousef and Johnson (162) stated that 32G decreased the blood 'magnesium in dairy cattle, I

Matsuda (96) stated that AGTH increased blood phosphorus while deoxycorticosterone acetate and cortisone caused a decline. These find­ings seem.to be in antithesis, however, as the action of ACTH would be. through the adrenal cortex, thus any rise from the ACTH would be due to the action of the corticoids. Scott and Dobson (12?) found that aldosterone decreased the plasma phosphorus - in sheep. It was found in rabbits that insulin, adrenaline, glucagon, and parathormone all lower the phosphorus levels in blood (110), Yousef and Johnson (162) in addition to Goberdhan (58) concluded from their work with cattle that heat increased blood phosphorus,

Streeten and Solomon (138) observed that AGTH had no effect on the potassium in human erythrocytes. They also found that cortisone and hydrocortisone, had no effect on blood potassium. Mills and Thomas (103) found that both cortisone and cortisol had no effect on blood potassium in humans, Roberts and Pitts (122). did find that cortisone , decreased the levels of potassium in the blood of adrenalsatomized.dogs. Knight and others (81) observed a rise in serum potassium if large doses of hydrocortisone were given to men. They believed that the exchange was so great between the intra and extra cellular fluid, that the kidney could not excrete the potassium fast enough, resulting in the build up

in the blood. Sellers and Hoepke (128) observed no changes in the serum potassium levels of cattle when they injected diethylstilbestrol0 Insulinj, adrenalinep and glucagon lowered blood potassium, while nora­drenaline caused it to increase (110)„ Bauer and Rathschlag-Schaefer (10) using rabbits and Scott and Dobson (127) using sheep, found aldo­sterone decreased the blood potassium, Blinco and Brody (18) stated that heat has no effect on blood potassium of cattle, Ganong and others (54) showed that cannulation stress had no effect on the blood potassium in dogs. Kamal, Johnson, and Ragsdale (75) reported that blood po­tassium levels were lower at 270 than at 100 in cattle. They also reported (76) that a decrease occurred at 80F, Opposed to this, Barlow, Angersborg, and Keys (8) found that hyperthermia raised plasma potassium levels in the dog. Ladell (83) stated that heat exhaustion in man caused a decrease in blood electrolytes,

Knight and others (8l) found no effect on serum sodium when they injected dehydro cortisone in man, Diethylstilbestrol likewise had no. . effect in cattle (128), but thyroxine increased the sodium level (162), Aldosterone increased plasma sodium in sheep according to Scott and Dobson (127), as is expected with this hormone. Stress caused no change in the blood sodium of dogs (54)$ nor did heat in cattle (18, 162), but heat exhaustion did lower the levels of blood sodium in man (83).

Clark and Kenny (26) found that calcitonin had no effect on urinary calcium in dogs. Hale and others (62) reported that heat in­creased the calcium to phosphorus ratio in the urine. Hale and Mefford (63) stated that this increased calcium to phosphorus.ratio was not re­lated to whether the animal was fasted or not. They further pointed

16out that the corticosteroids block the natural effect of. heat on calcium excretion. Cortisone has been shown to increase urine calcium (32). .

Comer and Bronner (32) reported that prolonged adrenal stimula­tion decreased the excretion of magnesium, which agrees with Hale and Mefferd (63) who also found that normal rats showed 6 decrease in urinary magnesium when injected with ACTH. However, heat acclimated rats showed no change in magnesium excretion when injected with ACTH (63)« Brody and colleagues (20) stated that urinary magnesium varies with the total output of urine. This agrees with an earlier reference (33) that the urine is the major excretory path of magnesium. Again Clark and Kenny (26) found that calcitonin had no effect on magnesium excretion, but Hill and others (68) found a decrease due to cortisol. Mader and Iseri (94) and Scott and Dobson (12?) found that aldosterone increased the amount of magnesium in the urine.

Clark and Kenny (26) reported that parathormone increased, urine phosphorus, whereas calcitonin had no effect. The excretion of phos­phorus was not affected by fasting (63).

Aldosterone increases urinary potassium excretion (127), which is a partial function of this hormone. Reiman and Schwartz (120) found that deoxycorticosterone acetate had the same effect as aldosterone on- potassium, Eversole and Romero (43) reported that corticosterone and dehydrocorticosterone had no effect on electrolyte excretion, but Comer and Bronner (32) stated both hormones increase-potassium excretion, and that growth hormone- caused a decrease in the potassium excretion. It has been reported in man (103) and rats (43) that-both cortisone and cortisol increased the excretion of potassium. Stress by cannulation

did not affect the urinary potassium (54-) and Pare et ale (11?) found that rats under chronic environmental stress showed no flux in the urinary potassium,, Kamal, Johnson and Ragsdale (76) and Leithead (8?) reported an increase in urinary potassium due to heat in cattle and men* Robinson and MacFarlane (124) found the urinary potassium to sodium ratio increased during heat indicating aldosterone activity,Gordon and others (59) working with men observed an increase in potas™ . slum excretion at 88F and 4-0 percent relative humidity*'

Ingle, Li, and Evans (72) found that ACTH elicited no change in sodium excretion in rats, while Hail and others (68) observed a urinary sodium decrease due to ACTH, Growth hormone caused a retention of sodium (32)., while calcitonin had no effect (26), Comer and Bronner (32) stated that corticosterone and dehydrocorticosterone both caused sodium, retention and plasma excretion. This action may be due to conversion of these two steroids to aldosterone, Eversole and Romero (43) stated that these two hormones had no effect oh urinary sodium in the rat, but corticosterone is the major glucocorticoid in the rat and it may be utilized as such when injected. Knight et al. (81")' found that hydro­cortisone (cortisol) had no effect on sodium excretion in man, where others found that deoxycorticosterone acetate (122) and deoxycorticoster­one (120) decreased the excretion of sodium. Cortisone (32, 43) and cortisol (43) were both reported to increase sodium excretion in rats, and yet other workers found that cortisone (103, 122) and cortisol (68, I03) favored sodium retention in man and dogs, Leithead (87) stated that sodium may practically disappear' from the urine due to heat in man. Sodium retention due to. heat has been reported in many works,

18some involving cattle (30, 76)„ This effect is probably due to the increased secretion of aldosterone by the adrenal cortex*

Brush (21) has found that 200 units of ACTH caused a sharp de­crease in the milk yield of cows* Hahn and Turner (60) established that if both corticosterone and aldosterone were mixed and injected in the rat, an increase in milk yield occurred, suggesting a synergistic action with the lactogenic hormone„

Scott and Dobson (127) found that aldosterone infusion caused no change in the fecal mineral content of sheep* Wrong and Metcalfe- Gibson (160) analyzing human, feces found that aldosterone influenced the electrolyte distribution in feces more than it did in the urine* Collins and Weiner (30) stated that fecal sodium retention occurred during secondary aldosteronism and this may be an important sodium con­serving mechanism during heat exposure*

Comer and Bronner (32) stated that an increase in plasma mag­nesium of 4-6 percent followed adrenalectomy, Weil and State (156) also noted an increase in magnesium following adrenalectomy in dogs. Ester- green and Van Denmark (41) found a depressed serum sodium and elevated serum potassium in the bull calf after adrenalectomy, Estergreen (42) found the sodium to potassium ratio decreased, observing at the same • time that serum calcium remained stable. Thompson (142) found that adrenalectomy had no effect on plasma calcium and magnesium, Baumann and Kurland (11) found that adrenalectomy decreased blood sodium and caused a rise in potassium, magnesium, and calcium levels in both cats and rabbits, Conway and Hingerty (34) found increased plasma levels of potassium, magnesium, and phosphorus and decreased levels of sodium and

19calcium after adrenalectomy in rats0 Muscle levels of potassium and. magnesium Increased while sodium decreased (34-)«

Factors Affecting Embryonic Survival The effect of hormonal increases and heat stress on pregnancy

should be considered in any comprehensive treatment of stress.Milkovic and Milkovic (102) using the ascorbic acid depletion test, con­cluded that the placenta of rats is not permeable to ACTE, The increased . secretions of AGTH from the mother would thus have no effect on the fetus* Verlardo (153) working with rats found that 2 units of AGTH per day caused abortion in normal rats but not in those which were adrenalectomized.This same result was.obtained by Robson and Sharaf (125) who found that 5 mg/day for 2 days in both rats and rabbits caused abortion*. Abortion also occurred in hypophysectomized and ovareetomized rats and rabbits (with progesterone given to maintain the pregnancy) given 5 mg of cortisone- per day for two days. This led them to conclude that AGTH stimulated the adrenal cortex, which in turn led to the abortion.Horwarth and Hawk (?l) found that hydrocortisone also reduced the embryonic survival in sheep. Faulkner (44-) stated that sows can abort entire lit­ters due to heat or stress and that trauma was not a great cause of abortion. He further stated the death of the fetus and its associated extra embryonic membranes caused a loss of progesterone which maintains the pregnancy, and that this decrease In progesterone is the cause of the expulsion of the fetus. Shelton and Huston (131) reported that heat stress in the ewe caused fetal dwarfing and a reduction in the length of the'gestation period, Alliston, Egli, and Ulberg (2) noted abnormal ova

20

and cessation of development daring the cleavage phase of embryogenesis ' due to heat stress0 Cameron (22) noted that elevated body temperature caused abortion, Fernandez--Cano (4-5) found that increases of body temperature to 103F destroyed the embryos in normal rats but not in adrenalectomized rats.. He felt the adrenal cortical hormones may be responsible for the abortion, which was also the contention of Verlardo . (153) working with ACTH as well as Robson and Sharaf (125) using cortisone

AcclimationAny organism undergoing.physiological stress must either escape,

die, or acclimate. Escape is quite often impossible and death the final release when all other mechanisms fail. This leaves acclimation, the response mechanism of change, which allows the organism to perpetuate its existence. Many references on the process of acclimation are avail­able, with Collins and.Weiner (30) giving a good account of the endo­crinological factors involved. Work with cattle is also detailed (23, 48, 74, 76) with Kamal, Johnson and Ragsdale (76) giving an excellent review oh the entire process. Findlay (48) stated that the thyroid is involved in long term adaptation to heat, and that the depression of the thyroid due to heat is only the initial reaction. Kamal, Johnson and Ragsdale (75) found that higher adrenal and thyroid activity exists in heat accli­mated dairy heifers than in controls. Streeten and others (139) statedkthat aldosterone was paramount in acclimation, especially in electrolyte regulation. Henrotte (67). studying man and climatic adaptation found that potassium tolerance is higher in the hotter.months of the year, and that Europeans who are recent arrivals in India have a higher rate of

Z L

adrenal activity than Europeans who have resided in India for a period of time* He also found that plasma potassium levels were higher for African Negroes than Europeans or Indians, Europeans or Indians from the more tropical habitats had higher, levels of potassium than their counterparts from more cooler climes,

■■ ■ ■ ! ' . . . - ■ ' " ’ . 'Mineral AnalysisThe use of atomic absorption as an analytical tool has become

widely accepted as an accurate method of mineral analysis. The analysis ' of minerals in the blood is appearing more frequently in the literature (1/ 98, 126, 137, 142). Urine analysis is reported using the atomic absorption method (26, 137), and also milk analysis (108, 137)» The Perkin-Elmer handbook (118) gives methods to determine mineral content of the above biological fluids except milk, but does include feces.

METHODS AMD MATERIALS

Six Holstein cows were selected at random from the University of Arizona dairy research center. Each animal was examined for health, average milk production and state of pregnancy. This data is recorded for each animal in Table 1,

Table 1, Physiological Condition of Experimental Animals at the Initiation of Experiments.

Age ' Months MilkAnimal Y-M Pregnant In Lbs.

599 4™ 5 4+ 26608 4-2 : 4+ 25609 4-1 4+ 24640 3-4 3,5 18673 8—8 5+ 25729 4-2 5+ 31

ProcedureThe six cows were divided into three groups with two animals tc

each group. Blood, urine, milk and feces samples were taken from each animal. Blood was collected by venapuncture from the jugular vein using a two inch 13 gauge needle and collected in 100 ml plastic tubes. The blood was allowed to clot and. then was centrifuged for 30 minutes at

. V \ t. V. 22 - ' .

233500 RPMf s on an International centrifugemodal. SBV, The serum was drawn off and frosen at 00 in plastic vialse Urine was collected in plastic Whirl-Paks (Nasco), acidified to 3% with hydrochloric acid, and frozen at 00, Milk was collected in the Whirl-Paks from the same quarter of the udder in each cow for the.duration of the-experiment.Fecal material was collected with an-insemination glove and frozen .inthe Whirl-Paks as was the milk. The samples were taken between 12:00noon, and 1:30 PM to obviate any circadian, periodicity. Urine., milk,and fecal samples were always collected before the blood to avoid a rapid shift in urine mineral content which might occur because of anxiety during jugular'puncture.

Samples were taken daily for a period of seven days to serve, as a pre-stress base line. One animal of each group was primed with 50 units of Lyophilized Adrenocorticotropic Hormone (ACIH, GALBIOGHEM, , given intramuscularly), daily for seven days while the second animal of each group was untreated. During this'period.blood, urine,milk and fecal samples, were collected as before. At the completion of the. phase . the stress stimulus was. applied. . " • .

The animals were stressed either by severe heat or by large doses. .

of.ACTH, In the latter case, both the primed and unprimed animals (608 and 609) were injected with 250 units of AGTH intramuscularly.Blood, urine, milk and feces were collected every 6 hours, for the next 24 hours. At the. conclusion of this period, the.250 unit AGTH injection was repeated. Twenty four hours later blood, urine, milk,, and feces - were again collected, followed.by Injection of another 250 units of AGTH, Once again at the completion of 24 hours the.blood, urine.,, milk and

feces were collected. This was the last set of stress samples, total­ing 72 hours of massive dosages of ACTH for adrenal stimulation. Collection of the samples was continued, daily for the next seven days for observations on post stress recovery.

Two sets of animals were stressed by severe heat. One animal from each group (599 for group one and 673 for group two) was primed with 50 units of ACTH daily for a week. Blood, urine, milk and fecal samples were taken daily from both the primed and unprimed animals, dur­ing this priming period. Both the primed and unprimed animal were then placed in a controlled temperature environmental chamber which had been pre-heated to the desired experimental temperature and humidity. Blood, urine, milk and feces were taken every six hours as before. At the end of the 24 hour stress period both animals were removed from the chamber and returned to their pens. Samples were then collected over the next seven days.

Water and good alfalfa hay were available ad libitum during the stress period. Temperature, humidity and any pertinent observations as to behavior under the stress were recorded. Blood and urine samples were also taken very shortly after the initial priming or entry into the heat chamber to determine if any rapid shifts in mineral concentrations occurred. y

Of the four heat stressed animals, one group (599. and 640). was subjected to a lesser degree of heat (90-10AB, 70-85% RH) to simulate a normal summer environmental condition which could be encountered in the Tucson area. The other group (673 and 729) was subjected to an

25acute heat stress (104-118P, 78-85% EH) which would simulate the most severe climatic conditions,

. 42§ixsisMineral analysis of the blood serum, urine, milk and feces for

calcium, magnesium, potassium, and sodium was carried out using a Perkin-Elmer model 290B Atomic-Absorption Spectrophotometer. Inorganic phosphorus was determined in all samples using a Bausch and Bomb Spectronic 20 spectrophotometer by the colormetric method of Piske and Subbarow ,(50), blood and urine phosphorus is detailed in Hawks (115).All phosphorus determinations were allowed to develop for exactly five. ■ minutes' before measuring, and read at 675

Blood serum and urine were diluted directly with triple dis­tilled water to a measurable concentration for the Atomic-Absorption process. One gram of fecal material was wet ashed in a micro Kjedahl digestion flask using 5 mis of 3;1 concentrated nitric to concentrated sulfuric acid. This mixture was heated at a setting of 4 on a multiple burner electric heater (Laboratory Construction Go. type A) for two hours. After cooling, 5 mis of 70% perchloric acid was added and heated at a setting of 7 for thirty minutes. This solution was then transferred quantitatively with'washings•to a 100 ml volumetric flask and brought to volume. This solution was used for both the Atomic- Absorption analysis and for inorganic phosphorus.• The milk used for calcium, magnesium, potassium, and sodium was diluted directly with triple distilled water to the correct factor. Two mis of milk were wet ashed, using the same procedure as for the feces. This ashed mixture

2 6

vas brought to 100 mis volumetrically, and used for the determination of inorganic phosphorus in the milk sample«, The direct dilution values for calcium and magnesium were checked against those values obtained from the same sample using the Galeein method of milk calcium and magnesium determination as per Mtailianas (113)} and found to correspond quite well (82,5 mg/100 ml direct and 80 mg/100 ml for the Calcein method)„

All the standards and stock solutions used in the Atomic- Absorption process, as well as the research samples were prepared accord­ing to the directions found in the Perkin-Blmer Analytical Methods (118), Lanthanum oxide (Mathesons Coleman and Bell) used as a chelating agent, was.added to all calcium and magnesium samples to be measured. Absorp­tion wavelengths, fuel mixtures, and slit settings were also found in the Perkin-Elmer book as was other miscellaneous details for the use of the instrument. All the chemicals used were of analytical grade and were new when stock solutions were made. All analyses of samples ty Atomic-Absorption were done as a series. For example, all the blood samples from one animal were done sequentially at one time.

Table 2 in the Appendix lists the dilution factors used for all minerals, in all samples analyzed.

Recovery determinations were run on all samples for calcium, - -magnesium,- potassium and sodium. These values can be found in the %Appendix, Table 3.

Least squares analysis of variance was used to evaluate thedifferences in minerals. Analysis was made between the treatments, the periods, and treatment by period interactions. The acceptability of this test was determined by use of the F test for equality of variance. Inter and intra animal differences of treatment and periods were determined by use of Fishers Least Significant Difference test, where:

Y = The Meant = T Value from Students T DistributionpS = Mean Square of the ErrorN = Number of Samples for the Mean

RESULTS

Physiological Reaction of Animals •Blanca (15) -stated that severe heat stress is that point -where

the functional reserves of thermoregulatory mechanisms are being ex­hausted and body temperature rises continually. The upper limit of severe heat is a fine point indistinguishable from excessive heat, which is the point where death occurs*

During the course of this research, two categories of heat stresswere defined* In group I, a sub-acute heat stress was applied to animals599 and 640* In group II, an acute heat stress was applied to animals 673 and 729« The degree of stress as expressed by temperature and humidity are found in Figure 1.

Spector (136) stated that the heat tolerance of cattle for a 24 hour period is 410 and 51 percent relative humidity. This temperature allowed 100 percent survival.of all test animals. The sub-acute stressed animals on this experiment were exposed to this threshold for two 3 hour periods, with a 6 to 8 hour space between. Both animals appeared to accept these temperatures quite well. Both sweated slightly and exhibited some panting. Rectal temperatures were not taken. Sali­vation was slight and water taken freely end often. The urine of both animals became very dark during the last six hours of heat stress.

The second group of heat stressed animals, 673 and 729, were maintained at temperatures higher than the 410 threshold of Spector for the entire 24 hour stress period (Figure 1),

. . 28

Acute Heat120

80 EH-

1000700 1300010019001300

Sub-Acute Heat110

100

0700 1300010019001300

90

80ECLH-c+S

70

60

TIMEFigure 1. Climate in the Environmental Chamber

Speotor also stated that the critical rectal temperature for. the cow is Rectal temperatures of both acute heat stressedanimals, 673 and 729, were elevated above this critical point after 18 hours in the heat chamber c

Both 673 and 729 reacted quite vigorously to the heat to which they were subjected. Moderate sweating: occurred after six hours in both animals. After 12 hours, cow 673 had a rectal temperature of 107,5F, was sweating heavily, panting very rapidly, and exhibited no interest in water, Cow 729 had a rectal temperature of 106F, exhibited the. same heavy sweating and panting, however, consumed water copiously. After 18 hours, 673 was still sweating and panting, salivation was heavy, but the rectal temperature had dropped to 105F, An absence of fecal matter was noted, with only loose watery material in the lower colon of 673, Cow 729 had a severe diarrhea, and the rectal temperature was still 106?, at this time, At the completion of the 24 hour stress, a. deeper secondary breathing had set in as was noticed by Bianca (14), Although no counts were made, it was quite noticeable that the respiration rate as gauged by flank movement had dropped from the rapid respiration rate noted at the 18 hour observation period. Both animals appeared to be in very poor physical condition at this time. There was a complete absence of feces, with only a dark and stringy mucous material present in the lower colon. Both animals had rectal temperatures of 110F plus. Both animals were ataxic. Muscular coordination was abnormal, At this time (after 24 hours of heat stress), cow 673 went down in the heat chamber. The experiment was terminated at this point. The animals were cooled with water and circulating air. After 2 hours both animals were able

31to move slowly to their pens. Cow 729 was down for the next 2 days and required prodding to force her up. The urine was thick and slight in amount, while the feces was hard and brittle. Cow 673 never went down after the hunt chamber, and showed relatively normal excretory and behavior patterns, although body movements were slower than normal. It is of interest that 673 was the ACTH primed animal. Whether this more rapid recovery was due to the pre-injections of ACTH is not known and should be the subject of further investigation. Both animals aborted 5 month fetuses.

Both ACTH stressed animals, 60S and 609, showed little response to the daily injection of 250 units of ACTH, except for a moderately heavy drinking from the 12th through the 18th hour. Both of the animals appeared to be exceptionally active throughout the first 2A hours of the experiment. This was noted even when the 0100 hour samples were taken. This hyperactivity may be due to increased glucocorticoid levels as a result of the adrenal stimulation by the injected ACTH.

MineralsThe results of the F test from an analysis of variance of the

data, showed that major changes occurred in calcium, potassium, mag­nesium, phosphorus, and sodium of blood, urine, milk, and feces in both heat stressed and ACTH stressed animals. The only exception was no sig­nificant change in the urinary sodium content. These data are recorded by treatment, period, and interactions in Table 16. Changes in specific minerals based on the least significant difference are recorded in Tables 12, 13, 14 and 15.

Priming with 50 units of ACTH for one week caused significant changes in mineral patterns in the bovine although subsequent changes . in mineral composition due to heat stress or ACTH stress were variable within the same period of stress. Blood calcium, potassium, magnesium, phosphorus, and sodium (Table 12); urinary calcium and potassium (Table 13), milk calcium, potassium, magnesium, phosphorus, and sodium (Table 14); and fecal calcium (Table 15) showed significant changes' due to the ACTH priming,

ACTH generally caused a rise in blood calcium (Table 12),Priming with 50 units of ACTH daily caused a rise in serum calcium in cow 599. This increase was noticeable one hour after injection and was still rising after 2.5 hours (Figure 2). In both primed 608 and un­primed 609, 250 units of ACTH caused a significant rise in blood calcium between periods II and III (Figure 3),

Acute heat stress caused significant decreases in the blood calcium of primed 673 and unprimed 729.

Table 17. Effect of Heat Stress on Blood Calcium for Cows 673 and 729.

Animal PeriodI II III 17

10.95 8.91 , 7.55 8.46 ± . 4310.56 9.57 8.80 9,31

Period I is the baseline, period II is the priming.period, period III the stress, and period IV. is the' recovery. ■ A H values are the mean* in mg/100ml.

673729

Mg/10

0ml

Mg/100ml

33

14

13

12

11

10

Time in HoursFigure 2. Effects of 50 Units ACTH on Blood Calcium.

12

10

I II III IV

A --------A609

© ---- Q608(Primed)

PeriodFigure 3, Blood Calcium Means for 608 and 609.

34The priming dose of ACTH (50 units) injected intramuscularly.

caused no change in serum, magnesium in the cow. However, the stress dose of 250 units daily for 3 days, caused a slight rise in primed 608. and a slight decrease in unprimed 609, Each change was significant (Table 12), Possibly the increase in the primed animal was due to the adrenal hypertrophy from the ACTH priming, where the unprimed animal could not follow this trend.

The sub-acute heat stress caused no significant change in serum, magnesium. Acute heat stress caused a decrease in the concentration of serum magnesium. Although the net changes are only in the realm of ,3 to ,4 mg/100 ml, this constitutes a 20 percent drop (Tables 4* 5, 12),

ACTH priming caused a significant decline in 'serum phosphorus. The stress dose of 250 units of ACTH caused significant decreases in serum phosphorus (Tables 8, 9, 12) for unprimed 609, but had no effect on the primed animal.

Acute heat caused significant decreases in blood phosphorus.The effect was slightly greater in the primed than in the unprimed cow as is seen in Figure 4«

ACTH caused a very sharp rise in blood sodium, almost doubling 30 minutes after injection of 50 units in cow 599 (Figure 5). Two hundred fifty units of ACTH likewise increased the blood sodium of un­primed 609, but had no effect on the primed animal.

Ig/lOOml

729

O *0673

(Primed)

12Time in Hours

Figure Effect of Acute Heat Stress on Blood Phosphorus.

Mg/lOOml

36

100

900

800

700

600

400

300

200

100,

0 1 35 1.5 2 2.5Time in Hours

Figure 5. Effect of 50 Units ACTH on Blood Sodium

37Table 18. Mean Effect of 250 Units AOTH on Blood Sodium in 609,

PeriodI II III W

850,0 390,0 600,0 379*16

Due to the extreme variation within individual periods, the above mean valuegxare not statistically significant. However, it is felt that they do represent an important physiological trend. For example, the sodium level in the blood of 609 rose 650 mgs/lOOmls in 6 hours of the stress (Table 9),

AOTH priming caused a rapid decline in blood potassium in animal 599 (Figure 6), A significant decrease in blood potassium was found to occur following 250 units of AOTH (Table 12), Individual changes are evidenced in Figure 7* ■ •

Acute heat stress caused a significant decrease in the serum potassium of primed 673 (Table 12).

Urinary calcium showed some significant increases due to AOTH. AOTH priming in cow 608 caused a slight increase in the mean values(Table 13), Further stimulation with the stress dosage of 250 unitscaused a 550 percent increase in 6 hours (Figure 9).

AOTH seems to have a varied effect on urinary magnesium. Fifty units injected into cow 599 caused a rise from 4*4 mgs to 16,8 mg/lOOmi (Figure 8). Cow 608, however, significantly decreased urine magnesiumduring priming, and increased slightly with the. stress dose of 250 units of AOTH (Table 13), '

Mg/lOO

ml Kg/lOOml

38

22

Time in HoursFigure 6. Effect of 50 Units ACTH on Blood Potassium.

0— 0 609y\ 608 (Primed)

20

Time in HoursFigure 7* Effect of 250 Units ACTH on Blood Potassium,

Mg/lOO

mI Mg/lOOml

39

12

10

0 5 1 2Tim© in Hours

Figure 8. Effect of 50 Units ACTH on Urine Magnesium.

6

608 (Primed)609

5

3

2

1

0

360 12 60Time in Hours

Figure 9. Effect of 250 Units ACTH on Urine Calcium.

40Sub-acute heat, caused an increase in magnesium excretion, with

the unprimed animal (640) excreting greater amounts of magnesium (Figure 10)„ During acute heat stress magnesium in the urine practically disappears in both the primed and unprimed animal (Figure 11). Urinary magnesium reached a low level the last 12 hours of heat exposure

i(Table 13). . .I

ACTH priming Increased urinary phosphorus in cow 599 (Figure 12), but had no effect on 673. Stress levels of ACTH had no effect on either primed or unprimed animals (Tables 4, 8, 9, 13).

Heat caused significant retention of phosphorus in all animals on this experiment (Tables 4? 5, 6, 7, 13). In all cases the phosphorus practically disappeared from the urine (Figure 13). Animal 729 showed no detectable phosphorus in the urine the last 18 hours of acute heat stress.

Table 19. Effect of Heat Stress on Mean Urine Phosphorus in 729.

I IIPeriod

III I?38.73 33.30 1,24 122.54

ACTH did not significantly effect urine calcium over the stress period, but a sharp rapid rise was noted after 2 hours when 50 units of • ACTH was injected as a priming dose (Figure 14).

ACTH priming caused a significant rise in the milk potassium and magnesium (cow 608), but showed no further response when stressed

Mg/100ml

AX

0 — 0 599 (Primed)

A —A 64°

20,

10

6 180 12 24Time in Hours

Figure 10, Effect of Sub-Acute Heat on Urine Magnesium.

Mg/lOOnl42

20

673 (Primed)16

729

12

8

4

0

60 12 18Time in Hours

Figure 11, Effect of Acute Heat Stress on Urine Magnesium,

Mg/lOOml

43

30 _

20 _

15 _

100 5 1 1.5 2

Time in Hours

Figure 12. Effect of 50 Units AGTH on Urine Phosphorus.

44

O ' Q 599 (Primed)

A A 640

20 _

12Time in Hours

Figure 13. Effect of Sub-Acute Heat on Urine Phosphorus,

Mg/lOOml

45

2000

1800

1600

1200

1000

800

600

4000 1 1.55 2

Tim in Hours

Figure 14. Effect of 50 Units ACTH on Urine Potassium.

with 250 unitsc Significant increases were noted in these minerals when stress levels of ACTH were injected into unprimed 609 (Tables 10) 14), The calcium levels of milk rose significantly only when 608 was primed; further stress with 250 units caused no changee Priming caused significant drops in the milk sodium of 673 and 608. The stress level of ACTH ■ further decreased, the mean values for 60S (Tables 10,.. 14)« Milk phosphorus .significantly decreased in all instances when ACTH was injected at either the prime or' stress levels (Tables 10, 14)«

Acute.heat stress caused significant rises in milk minerals.ACTH primed 673 and unprimed 729 had calcium increases of 32 mgs in 24 hours. Potassium rose only in unprimed 729? whereas magnesium rose ■ 10 to 20 percent in both animals. Phosphorus increased in both animals, while sodium rose in unprimed 673 (42 mgs) and dropped (63 mgs) in un­primed 729 (Tables 10, 14).

Fecal potassium levels increased in 60S when ACTH primed and stressed. The increase totaled about 30 mg/gram of feces. ' Fecal phos­phorus dropped significantly after 250 units of ACTH. The mean values were down approximately 50 percent in 60S and 609.

Table 20. Effect of ACTH Stress on Fecal Phosphorus for 60S and 609. -

PeriodI II III IF

608 5.63 3.64 2.94 5.56± °99609 7.91 4.56 3.95 3.23

/■ ■ 47. ACTH priming caused a significant rise in fecal sodium for 673,

whereas the sodium decreased in primed 608 and unprimed 609, when stressed with 250 units of ACTH (Table 15)« Two hundred fifty units ACTH caused a significant drop in fecal calcium of unprimed 609, with the mean decrease 182*85 mg/gram, of feces* A decrease also occurred in the stress period of primed 608, after a rise during the priming period*

Acute heat decreased the fecal phosphorus in unprimed 729 sig­nificantly, where sub-acute heat caused a rise in unprimed 640 (Figures 15, 16). Primed 599 decreased fecal phosphorus, with no effect on the mean values for primed 673 statistically, but individual samples, did indicate physiological reaction (Table 4). It required 12 hours for a noticeable reaction in primed 673, where unprimed 729 started to de­crease immediately, indicating that priming might aid in the retention of phosphorus in the feces of the bovine (Figures 15, 16). Acute heat caused fecal sodium to rise in both the primed and unprimed animals. Fecal calcium dropped significantly in both acute heat stressed animals and rose in the primed sub-acute stressed animal. This same occurrencewas noted for fecal magnesium, 673 and 729 dropping and 599 rising(Tables 6, 11, 15).

AbortionThe acute heat stressed animals, 673 and 729, both aborted 5

month fetuses, cow 673 from 48 to 72 hours and cow 729 from 72 to 96 hours after the cessation of the stress.

There were significant shifts in some of the mineral ions dur­ing the period of abortion. In both cows, the blood values for calcium.

Mg/Gran

43673 (Primed)

& -----& 7^9

6 13120Time in Hours

Figure 15. Effect of Acute Heat Stress on Fecal Phosphorus.

599 (Primed)4

3

2

1

0

0 6 12 18 24Time in Hours

Figure 16. Effect of Sub-Acute Heat on Fecal Phosphorus.

magnesium^ phosphorus^ potassium, and sodium were normal and showed ■ no appreciable change just prior to, or after the abortion.

Urinary calcium levels rose to peak levels just before abortion and returned to normal immediately afterward (Tables 4# 5). No major

• effects on urinary potassium due to abortion were observed-. Urinary sodium dropped noticeably after the abortion to abnormally low levels (Tables 4* 5), Urine magnesium in the primed animal remained normal, where in the unprimed cow, 729, it rose 300 percent and greater. The greatest changes were observed in the urine phosphorus levels of un­primed 729. The mean value for the abortion period in the primed animal rose over 100 mgs, and in the unprimed animal the mean rose over 200 mgs, with the highest individual value being 357,9 mg/100 ml just prior to the expulsion in 729 (Tables 4, 5, 10).

Milk minerals were relatively unaffected by abortion except that sodium rose slightly in cow 673 (Table 4),

The only fecal mineral to change drastically during abortion was potassium in unprimed 729, which increased 150 percent (Table 5),

DISCUSSION

Since a complete mineral balance study was beyond the scope and intent of this thesis, a total metabolic study was not conducted. Inter­pretations of the data must then of necessity rely on the patterns and general changes in the mineral composition of the various vascular compartments and excretory material. There are admitted drawbacks to this approach, mainly that no conclusions can be made concerning the ' . overall retention and metabolism of a particular mineral. However, the general patterns when compared with seven day baselines do allow a crit­ical discussion of the mobilization and excretion of minerals under short term stress treatments such as 24 hour acute heat or high levels of ACTH simulated stress. These mobilization patterns could be very important in assessing the homeostatic efforts of the stressed animal to sustain relatively normal thermoregulation or to maintain ionic equili­brium in the face of stressing factors. ,

It. was assumed that sub-acute and acute heat stresses were suf­ficient to initiate a release of ACTH from the pituitary. Cooper and Nelson (35) stated that ACTH levels in the plasma rose sharply after surgical stress in humans, peaked one half hour after initiation of stress and had not reached baseline after 24 hours. Asfeldt and Elb (6) believe that a major override or separate releasing factor is available during stress, and that this override will permit the release of ACTH regardless of the normal inhibitory processes working at that time.•

" ■ ' 51 ■' ■

■ ' . 52The selection of 250 units of ACTH as the amount for the stress -

dosage was based on unpublished past work at the University of Arizona, although Brush (21) used 200 units when he was working with cattle. It may be possible that the use of 50 units of ACTH for. the priming was excessive and caused exhaustion of the adrenal gland, reserve. This .is in part substantiated by the work of Macchi and Hector (93)$ who found that bovine adrenal glands in vivo responded to ,001 unit of ACTH/liter of blood, ,1 unit of ACTH/liter of blood evoked a maximum response, and 10 units caused no change over that of ,1 unit in the urinary corti­costeroid output. It should be pointed out that these doses (93) were perfused directly into the blood stream and that ACTH given intra­muscularly as it was in,this present research could require larger amounts for equivalent results.

The experimental evidence indicates that priming the lactating dairy cow with 50 units of ACTH daily for 7 days causes statistically significant differences in several mineral patterns in blood serum, urine, milk, and feces when compared with the unprimed group. This evidence further Indicates that these inorganic mineral ions may in part be regulated either directly or indirectly by the adrenal corticosteroids. Although the commercial ACTH could have traces of other anterior pituitary hormones, such as PSH, TSH, STH, Unpublished data at the University of Arizona tends to indicate this, for follicular growth on the ovaries of cows has been observed when ACTH was injected, indicat­ing the presence of PSH,

Calcium patterns indicated very noticeable and significant changes due to the treatments, used in this study. The total flux in

the /body materials which were tested aid in following the overall picture of the fate of this mineral as well as the other mineral ions followed«

Priming - caused significant increases in blood and urine calcium over baseline levels but had no effect on the milk and fecal levels. This same trend continued when the stress dosages of 250 units ACTH'were injected* Sharp rises occurred in the blood,, urine and fecal calcium following the ACTH stress, with the primed animal in all instances having higher levels than the unprimed animals. This might be interpreted that the adrenal was made more responsive to stress stimuli after low dosage ACTH stimulation. Increases in calcium levels due to ACTH injection were also recorded in rabbits by Matusda (96). However, workers found a decrease in blood calcium in rabbits after ACTH injection (l, 52, 112), The rising levels of calcium in blood and urine (Figure 1?) would suggest that the ion is probably being mobilized from some other source and is not simply an exchange between the com­partments, This mobilization of calcium from other body sources was observed when the hormone from the pars intermedia., MSH, was injected in rabbits. It was found that the calcium arose from lipid deposits which had been degraded (l). It is possible that the ACTH used in this experiment was not sufficiently purified and contained some MSH, or that ACTH itself may Cause this liberation of lipid bound calcium. Ganong (53) stated that excessive glucocorticoids (which would result from large ACTH injections) cause increased calcium excretion due to the breakdown of bone matrix. If this occurred in this experiment, then it

Mg/lOOrnl

Mg/l

OOml

54Blood

17.16.15.14.13.12.11.10.9.8e7*

673 729 608 609 673IX III III III III IV 729 608 609

IV IV IV

21.81.61.41.2,1.8.6.4.2

0Urine

Figure 17. Blood and Urine Calcium Interactions.1 — Baseline Keanj II — Priming Mean^ III = Stress Periodj IV = Recovery Period; * — Statistically Significant« 673—729 wore Acute Heat Stressed: 603-609 were ACTH Stressed; 673-603 were Primed.

Mg/Gra

n Mg/lOOol

55

Milk150, U O 130 120,

110 100, 90, 80, 70, 60 ,

50

II673III

J___

729III

608III

609 673III IV

729IV

608IV

609IV

450

400

350

300

250

200

150Fecal

Figure 18, Milk and Fecal Calcium Interactions,

56would be expected that the phosphorus levels would rise also, since it would be liberated from the bone at about the same time as the calcium, but this was not the case, for phosphorus levels decreased (Figure I?)®

In opposition to the ACTH results, acute heat caused significant decreases in blood, urine and fecal calcium levels, while increasing the milk levels (Figure 18)„ The decrease in blood.calcium in heat stressed Cattle Was also observed by Riek and Lee (121)e In all stress instances, ACTH and heat, the Hood calcium levels during the recovery period remained high, suggesting that once a shift or mobilization of calcium had been initiated, the re-establishment of body equilibrium requires an extended time span. Since the effects of ACTH and acute heat on serum calcium are seemingly opposite (Figure 1?),'it could imply, under these particular conditions, in these experimental cows, that acute heat acted as an inhibitory or masking agent to pituitary ACTH release or that some other factor of acute heat was affecting blood calcium.

Potassium patterns Indicated that stress treatments had an effect on this ion. However, the overall trend was not striking.Priming had a varied effect on blood potassium (Table 10) increasing levels in 608 and decreasing levels in 673. A very rapid effect of ACTH on blood potassium.was demonstrated when the level dropped lv mgs in one hour in the blood and rose 1550 mgs in 1,5 hours in the urine. The means for these substances during priming also showed this reciprocal, rapid, shift, with blood levels dropping and urine levels rising. Al­though the urine means were, not significant statistically, they seem to represent, a physiological change, which suggests that exogenous ACTH

/ 57 .( - - . - - .

causes an initial release of aldosterone, which establishes an equil­ibrium after several hours „ This wa,s further illustrated with the ACTH stress, the unprimed animal showed a significant decrease in blood potassium while the primed animal was unaffected* Urine levels of potassium remained relatively constant during ACTH stress*

Acute heat decreased blood, urine and fecal levels of potassium, with milk levels rising in the primed animal* In the unprimed animal, the only change which was significant was the very dramatic increase in milk potassium* Since this was the only change in the unprimed animal, it is possible that it represents a mineral concentration as the milk fluid volume decreased* It is known that severe heat profoundly affects the kidney, and this was taken into consideration during the course of the experiment. The decrease in the urine potassium of 673 in both the stress and recovery periods could be a result of renal failure, as has been found to occur in rats (65). Since 673 was the primed animal, the possibility exists that these changes in blood and urine potassium were facilitated or accentuated by priming. It was also the primed animal which reflected significant changes to heat stress. This would tend to indicate that adrenal response to heat is enhanced in the primed animal.

Magnesium levels generally reflected changes with the treatments imposed upon the animals. Although milk values were statistically sig­nificant, the total changes were so slight (Figure 22), that it is doubtful that a physiological reaction as a result of the stress occurred. Since all the changes were increases in the same order of magnitude, it would tend to lend credence to the suggestion that a decrease in milk pro­duction was the cause of the increase in mineral concentration per unit volume,

Mg/lOO

ml Mg/lOOml

58

25 _

20

Blood

15673 729 603 609

II III III III III673 729 608 609IV IV IV IV

1000 _

800

600

400 n mUrine

Figure 19. Blood and Urine Potassium Interactions.I = Baseline Mean; II = Priming Mean; III = Stress Period; IV = Recovery Period; * = Statistically Significant. 673-729 v/ere Acute Heat Stressed; 603-609 were ACTH Stressed; 673-608 were Primed.

Mg/Gra

m Mg/lOOml

59

Milk100 —

75-

50.

25

673 729 608 609 673 729 608 609II III III III III IV IV IV IV

200 _

150

100 _

50 _

0Fecal

Figure 20, Milk and Fecal Potassium Interactions.

»

6 0

' : . . ACTE stress increased the blood levels of magnesium in primed 608 and decreased urine levelsc Unprimed 609 showed decline in both the blood and urine levels of magnesium* The fecal magnesium levels in both primed and unprimed animals remained relatively unaffected* The apparent retention of magnesium in the serum of cow 608 was also re­ported in rabbits (l)Since heat caused a decrease in serum magnesium, the possibility is again offered that heat.inhibited ACTH release,The decrease in urine magnesium in both ACTH stressed animals was also reported in man and rats (62, 68). Since a magnesium upsurge occurred following ACTH priming (12,8 mgs in 2*5 hours), and then dropped to a low level for the rest of the priming period, and showed a slight rise during stress with 250 units of ACTH, it is possible that one of the adrenal steroids may have a partial role in the retention of magnesium. The continued adrenal stimulation with ACTH at stress levels, could have depleted the adrenal steroid, resulting in the slight rise in serim magnesium noticed during stress*

Acute heat stress decreased blood, urine, and fecal magnesium levels in both animals. The greatest decrease was in the primed cow, which may mean an augmented adrenal secretion due to the priming. Ex­cretory mean values of magnesium rose during, the recovery period. How­ever, this included the abortion phase, which undoubtedly influenced the data. This is discussed later on. Decreases in blood magnesium in cattle due to heat have been observed by others (58, 162), It has also been shown that urinary magnesium varies with the urine output (20).Since, the output was not measured in this experiment, this possibility cannot be substantiated. The very significant decrease in magnesium

Mg/lOO

ml Mg/lOOml

61Blood

3

2.5 J(

2

1.5 J

1

35

30

25

20

15

10

5 J

673 729 608 609 673 729 608 609II III III III III IV IV IV IV

UrineFigure 21. Blood and Urine Magnesium Interactions.

I = Baseline Mean; II = Priming Mean; III = Stress Period; IV = Recovery Period; * = Statistically Significant. 673-729 were Acute Heat Stressed; 608—609 were ACTH Stressed; 673—603 were Primed.

Mg/Gra

m Mg/lOOmI

62

15

10 J

Milk

5 J

0,673 729 608 609 673 729 608 609ii in in in in n iv iv iv

125

100 _

75.

50.

25.

0 _Fecal

Figure 22. Milk and Fecal Magnesium Interactions.

/ . 63levels in the blood, urine- and feces during acute heat stress suggests some possible role for this ion in thermoregulation, ' The low magnesium levels could indicate an inordinate extracellular fluid expansion or tissue retention. Where this retention occurs is open to conjecture, since a total metabolic study was not carried out. It is also possible that the. ion may have been lost or'actively secreted in the saliva or sweat, •

The patterns of fluctuations in phosphorus during stress reflect some of the greatest changes, and the most Interesting total pattern changes along with sodium, • However, an evaluation of the significance of these changes is difficult, for there are indications of a very . sensitive phosphorus regulatory mechanism, especially in the urine.

Priming significantly decreased the phosphorus levels of blood, urine, milk and feces. The phosphorus level was almost immeasurable in the urine of animal 608, When the primed animal was further stressed with 250 units of AOTH, no further changes were noted in blood phosphorus, indicating a possible all or nothing reaction or that the priming dose was excessive for the response. There was a decrease in the urine and milk phosphorus in the primed animal when the stress dosage of AOTH was administered, while the levels rose in the same fluids in the unprimed animal. Fecal levels of phosphorus dropped in both animals. The rise which'was noted in the urine of unprimed 609 was mostly a result of a large surge the second day of stress (Table 9), while decreases were noted over the first 24 hours of stress and after the spike. It is not known if this surge is a result of the treatment or other environmental or external factors. If. the spike were a result of the AOTH treatment.

6410_ Blood

o 5-

0

II673 729 608 609 673 729 608 60?Ill III III III 17 IV IV IV

oH

125 -i

100 _

75

50

25

UrineFigure 23. Blood and Urine Phosphorus Interactions.

I = Baseline Mean; II = Priming Mean; III = Stress Period; IV = Recovery Period; * = Statistically Significant. 673-729 v/ere Acute Heat Stressed; 603-609 were ACTH Stressed; 673-608 were Primed.

Mg/Gra

n 00

^65

150_ Milk

125.

100

75 J

50

673 729 603 6C9 673II III III III III IV

m

729 603 609IV IV IV

10_

5W

0Fecal

Figure 24* lillk and Fecal Phosphorus Interactions*

I 66it would be expected that the level of phosphorus would remain high on the third day of stress, (it did not do this)s because it might be ex­pected that the condition which led to the spike was an exhaustion of the adrenal steroid which was responsible for the previously noted retention of the ion. Decreases in urine phosphorus were noted in rats by Hale and Mefferd (62), while Comer (32) reported an increase due to ACTH injections. The decrease in blood phosphorus found to occur due to AGTH was also found to occur in rabbits (110)„ •

Acute heat decreased blood phosphorus 85 percent in the primed animal and 77 percent in the unprimed animal which agrees with the re­sults of Riek and Lee (121). However, this is in opposition to the findings of others who also worked with cattle (20, 58, 162)„ These latter results were obtained at lower temperatures than were used in this experiment, which may account for this discrepancy. The sub-acute heat stressed animals seem to substantiate this, for there was a slight rise in blood phosphorus during the stress period (Table 6). Acute heat stress and ACTH stress both caused a decrease in blood phosphorus8 This parallelism between the two types of stress was not shown for cal­cium and magnesium. Some insight into the mechanism of stress on phosphorus may be gained by examining .urinary phosphorus patterns. Heat must activate the kidney to retain practically all the phosphorus passing through it, for in all instances, the phosphorus all but disappeared from the urine. This agrees with other work (33), where it was found • that the kidney has the capability of absorbing 99 percent of the phos­phorus passing, through it. Fecal phosphorus decreased due to acute heat in both animals. This could imply a general, phosphorus retention due to

67stress. Milk phosphorus level rose in unprimed 729 (which is opposed to the result in 673) and is the.only variation in the pattern. This rise in milk phosphorus, noted in the one animal,-may again be due to a decrease in milk production. As with magnesium, stress caused an almost across-the-board decrease in the blood, urine and fecal levels of phosphorus. It is possible that this mineral was also excreted in the saliva or sweat, or it may have an intracellular function such as ATP formation during thermoregulation. An increase in the intracellular concentration of phosphorus would show up as a decline in blood, urine and fecal levels.

Extreme changes in sodium occurred due to .the treatment (Figures 25, 26) although the changes had no statistical significance in the blood and urine due to the great variation within periods. These changes may, however, have possible physiological significance during stress. Priming had no great effect on the blood and urine sodium levels over the baseline values. Milk sodium decreased and fecal sodium increased due to priming. Since there was a slight rise in the mean sodium levels in urine and feces due to priming, this pattern suggests that priming levels of AOTH are sufficient to" cause sodium excretion. Stress levels of ACTH caused rises in blood sodium of an equal magnitude in both the primed and unprimed animals. The urine levels remained, about the same,A large decrease in fecal sodium occurred in both ACTH stressed animals. The sodium level dropped in the milk of the primed cow and rose in the unprimed cow. Whether this latter result is due to priming is not known, for the priming level caused a drop in .previous trials. Blood levels of sodium rose, due to ACTH stress while the fecal level's decreased and

Mg/lOO

ml Mg/lOOml

68Blood

600575 _ r i

525 _

475 _ ---

425 _

375 -

325

275673 729 608 609 673 729 608 609

II III III III III IV IV IV IV

700

550

400

250 _

100 n oUrine

Figure 25. Blood and Urine Sodium Interactions,I = Baseline Mean; II = Priming Mean; III = Stress Period; IV = Recovery Period; * = Statistically Significant, 673-729 v/ere Acute Heat Stressed; 608-609 v/ere ACTH Stressed; 673-608 v/ere Primed,

Kg/Gran

Mg/lOOal

69

230

170

Milk

120 O

II673 729 608 609III III III III

673 729 608 609IV W 17 IV

290

250

190

150

90 dtacFecal

Figure 26. Milk and Fecal Sodium Interactions.

urine levels were relatively stablec Because of this, possibly in the cow, the excretion of sodium is at least in part via the fecal material. This was further attested to by the results of acute heat stress, when blood levels of sodium dropped sharply in both animals, again were fairly stable in the urine (although a drop was seen in the primed animal) while fecal levels rose sharply; These results are in opposi­tion to the sodium pattern noted with ACTH stress (Figures 25? 26), which again suggests that heat inhibits ACTS release from the pituitary. The levels of urinary sodium did not vary, however fecal sodium rose during acute heat. This would suggest that sodium balance is in part regulated through the feces, and that aldosterone activity may be moder­ated at least in part through the colon, •The importance of fecal mineral excretion was observed in humans (160) when aldosterone was found to affect the fecal electrolytes greater than it did the urine„■

Both heat and ACTH caused important changes in the mineral com­position of bovine milk. However, it must be remembered that unusual metabolic and physiological demands are placed upon high producing dairy cows, and further investigation is required to more" fully understand the role of stress upon milk composition, .

High titers of adrenal steroids may be involved in fetal death, ACTH has been reported to c’ause fetal abortion (125, 153), this could occur by activation of the adrenal cortex. Cortisone (125) and hydro­cortisone (71) have been reported to:cause abortion, indicating that the adrenal steroids are in some way involved in this physiological insult. Heat has also been implicated in abortion (22, 45)« .

In the two acute heat stressed animals of this experiment, fetal abortion occurred during the recovery period. Mo significant changes

, I - ' ■' ' - :

in blood minerals were noted during the abortion period* However, it has been reported (33, 128, 158) that blood calcium and phosphorus de­crease during the period of parturition in cattle. Perhaps this did not occur in this particular instance due to the fact that both of these ions decreased significantly during the heat stress period, and, further loss of these minerals from the blood would cause detrimental effects. 1411k and fecal samples showed no major changes in mineral com­position during abortion* Major changes were noted in the urine mineral content during abortion* Calcium, magnesium, and phosphorus levels rose sharply and significantly, potassium, remained unchanged and sodium de­creased notably 24 hours after the loss of the fetus (Tables 4, 5). Tremendous excretion of phosphorus'occurred, which in relation to the previously observed trends of phosphorus excretion indicates that the loss of phosphorus is related to the pathological condition that occurred. Since the excretion of minerals in the urine was significant during abortion, although no large changes in blood, milk and feces occurred, this would suggest that.the urinary mineral loss arose from the dead fetus and its associated tissues, or from the breakdown of the maternal placen­tal tissue. The former seems a more plausible explanation since the excretion peaks occurred just before fetal expulsion, and returned to normal after the loss of the fetus.

It is not known if the shifts in minerals during this period are important as an ionic compensatory mechanism, or are due to the increased secretion of hormones from the posterior pituitary, which would occur dur­ing the expulsion process. In any case, they represent an interestingand possibly significant occurrence during birth and could possibly, as in the case of phosphorus, be an index of impending abortion.

APPENDIX

72

73Table 2. Sample dilution factors for atomic absorption analysis0

Calcium Magnesium Potassium Sodium

Blood 20% 20% 50% 5000%Urine 5% 200% 5000% 5000%Milk 100% 100% 500% 500%Feces 1000% 1000% 250% 1000%

Table 3* Percent recovery of a known concentration.

Calcium Magnesium Potassium Sodium.

Urine 100,00% 101,03% 101,30% 114.78%Blood 101.02% 106.45% 97,28% . 98,43%Milk 98,70% 97.87% 94.11% 100.00%Feces 81.25% 98.57% 95.23% 100.00%

Table 4= Mineral composition of the blood, urine, milk and feces of heatstressed cow 673=

Blood UrineDate Hour, Ca . K . Mg P Na Ca ' K Mg ' P ' Na

- ms/lOQmls me/lOOmls4/7 1330 10=8 21=0 2.40 6.34 450 2.6 750 20.0 ,07 5004/8 1300 9=2 18.0 . 2.32 6.28 700 * 750 17.2 0.00 7504/9 1300 10.4 20.5 2,48 7.02 600 1=8 900 23=6 0.00 5504/10 1300 12.8 23.5 2.64 6=34 600 .6 800 17=6 0.00 4004/11 1300 10.4 21.0 2,60 5=79 375 =8 800 24.0 0.00 4004/13 1130 10.8 19.5 2,48 7.02 650 .8 1050 21=6 .60 3504/14 1300 12.0 18.5 2,46 6.34 850 2.0 400 9(2 0.00 3004/15 1300 11.2 19,0 2.60 6,47 350 = 5 600 16|4 0.00 300Pt**! Z /T £1 1310____ w u u - x r - . , n -- e-ww_ _ _ _ _ 14/16 1300 8=4 19.5 2.20 6.00 500 = 5 1100 26.0 .31 2004/17 1300 9,2 19,0 2,30 5=49 300 1=7 700 13.6 .62 2004/18 1300 9=6 21.0 2,44 5.59 350 1.0 1150 25=2 .15 • 1004/19 1300 8.8 20.5 2.28 5,32 300 1,6 1200 19.2 .71 1504/20 1300 8.8 23.5 2.46 5.11 350 ,5 1000 25=6 0.00 504/21 1300 8.4 22,0 2.68 4=99 350 .4 1250 23=2 0.00 1504/22 1230 9=2 22,0 2.40 7.35 400 »4 1100 Iv̂* 0 10,50 150Stressed 1300 ---- —--4/22 1900 8.0 20.0 2.32 3.62 350 .5 700 11.2 2.21 6004/23 0030 7.6 19.5 2.08 1,66 300 .7 650 1,6 1.89 1004/23 0700 7.4 17=2 1.80 1.08 200 .6 250 ■ ,8 .77 254/23 1300 7.2 15.5 1.68 1.36 250 .2 350 .8 .77 25Post Stress--period underlined is time of fetal abortion-4/25 1300 7.8 17.0 2.00 7.44 250 1.5 450 25=2 .119.48 504/26 1300 7.4 22_?„5 2.40 6.47 ' 250 ,9 600 23.0 107.08 3004/28 1200 8.0 19.5 1.68 6.41 250 .3 300 8.5 28.88 124/29 1300 8.4 16.5 1.76 6.11 400 .3 450 4=4 20.78 3004/30 1300 8.8 19=5 17=4 5.89 300 .3 400 3.2 5=77 1505/1 1300 9=2 19.0 18.8 7.26 300 .2 300 5.2 10.03 1005/2 1300 9.2 21.0 19.6 8.01 275 1=3 650 10.0 14,47 250

Table 4 --Continued

Milk FecalDate Hour ,Ca ,K Mg P ,Na .Ca K . Mg , P Na

' • mg/lOOmls mg/lOOmls_______;______4/7 1330 ■ 80 60 13,2 79,5 145 380 62,5 108 6,43 • 604/8 1300 78 85 12,4 104.8 240 220 70,0 76 7,78 804/9 1300 96 40 12,2 118,1 160 360 70,0 90 6.90 754/10 1300 86 80 11,6 63.4 245 24.0 . 62,5 62 6.81 1104/11 1300 76 55 12.0 109.3 140 220 70.0 86 6.24 754/13 1130 70 . 55 11.0 91.2 165 370 60,0 92 6.33 1004/14 1300 70 60 10.8 78.4 170 360 65,0 90 7,10 604/15 1300. 78 60 11,4 104,8 155 360 65,0 86 6.52 75Primed 4/15 13104/16 1300,4/17 13004/18 13004/19 13004/20 13004/21 13004/22 1230 .Stressed 1300 4/22 19004/23 00304/23 07004/23. 1300 _Post Stress-period underlined is time of fetal abortion— ----- -------------- --- — — -4/25 1300 94 45 13.4 82.7 295 460 42.5 106 10.88 1304/26 1300 126 40 18.4 95.9 225 360 77.5 71 ' 7.78 904/28 4/29 .4/30 5/1 5/2

80 80 11.8 78,4 185 ' 320 62,5 90 6,05 100 .84 65 13.4 48,6 165 280 57.5 84 5.37 13058 55 10,4 76,2 155 260 57.5 78 3.47 11070 60 11. 6 56.9 170 360 60.0 98 6,81 13080. 55 11,6 78.4 155 280 60,0 72 3.96 12072 70 . 12,2 82,7 150 300. 55.0 70 5.86 11068 65 11.8 52,7 145 320 65.0 80 8,98 130

300' 62.5 78 7.97 14078 65 11.2 65,4 220 . 340 42.5 96 8.37 200

60 22.5 16 2.54 250110 65 14.8 91.2 195 60 60.0 16 2.05 250

1200 84 100 13.6 104.8 195 . 320 95.0 74 9.92 1001300 80 65 13.2 91.2 185 220 72,5 92 7.88 1101300 84 60 13.0 91.2 145 380 57.5 150 6.43 901300 84 . 70 12.6 100.3 135 300 55.0 94 4.60 1301300 92 70 12,6 95.9 140 220 62,5 94 3.96 130

<zVt

Table 5, Mineral composition of the blood, urine, milk and feces of heatstressed cow 729»

•Date -Hour GaBlood

K ' Mg mg/lOQmls

P Na Gaurine.

K Mg mg/lOQmls

P Na

A/7 1330 10.0 16,5 2.14 7.96 400 1.0 1050 21,2 37.5 5504/3 1300 9,2 18,5 2,16 8.01 650 1.8 850 20.4 110,2 6004/9 1300 10.8 20,0 2,52 7.35 400 , 4 650 19.2 98.1 2504/10 1300 10.8 20.0 2,40 6.47 400 .7 600 18.0 62.1 4504/11 1300 10.0 15,5 2.20 ■4.92 300 =3 300 8.4 21,3 3504/13 II30 11.2 17.5 2,40 4.92 500 .3 625 18.8 55.7 5004/14 1300 12.0 17.5 2,44 7.44 250 .5 600 13.6 41,4 7504/15 1300 10.4 17.0 2,44 5.59 350. .6 650 16.0 35.5 5504/16 1300 9,8 17.5 2.48 5.41 400 .7 1200 20.8 46.3 4004/17 1300 10.4 18.5 2.48 6.00 350 .5 1150 24.4 43.6 3004/13 1300 10.0 19.5 2.36 6.60 350 .8 1800 37.6 31.8 1504/19 1230 9,2 19.0 2.48 6.34 300 .6 1200 30,0 50.9 2004/20 1300 8,8 20.5 2.64 6,95 300 ' .4 650 15.2 4.9 1504/21 1300 8.8 19.5 2.56 7.10 300 ,5 400 13.6 21.2 4004/22Stres

1230 10.0 ised 1300—r—---

20.0 2.48 8.11 400 *4 1150 19,2 98.3 2004/22 1900 9,4 22,5 2.44 3,67 300 . 4 1300 3.2 5,0 6004/23 0100 8.6 17,5 2,20 1.88 250 .4 700 2.4 0.0 5004/24 . 0700 ■ 8.8 19,5 2.12 1.99 400 .2 200 .8 0,0 3004/23 1300 8.4 ■ 17; 5 2.08 2,25 300 ,3 500 1,6 0.0 150Post stressed— ■period underlined is time of fetal abortion— — «>««» •*»«

4/25 1300 10.0 18.5 2.16: 7.02 200 3.8 600 60.0 148,9 2004/26 1300 9.2 19.5 2.20 5,45 300 4.0 600 60.0 357.8 5004/28 1230 .. 8.8 16.0 2.00 4.55, 300 ______.6__- -950..__38<p. 196,5 . 504/29 1300 9.2 18.0 2,24 4.69 250 .6 550 16.0 38.9 4004/30 1300 9.6 19.5 2.16 5.69 325 .6 650 26.4 63.5 5005/1 1300 8.8 19.5 2.16 6.22 300 ,6 500 19,2 25,1 3505/2 1300 9,6 18,5 2,08 7.02 450 1.6 550 15,2 27,2 350

Table 5 — Continued

Milk FecalDate Hour ' Ca K Mg P Ha . Ca K • Mg P Na

. Dig/lOOmls mg/lOOmls ______ _4/7 1330 60 55 9,4 56,9 195 240 105 50 6,1 904/8 1300 80 45 11,6 100,3 195 320 65 66 8,2 1104/9 1300 64 45 10.8 65,4 170 460 55 100 11.3 804/10 1300 66 50 11,2 71.9 165 320 48 76 10.7 1004/11 1300 78 60 10.6 107,1 200 200 38 60 6,8 1104/13 1130 62 40 10.8 15.8 170 240 70 74 4.9 704/14 1300 78 55 11.4 82,7 200 280 48 88 5.3 804/15 1300 66 60 10.8 107.1 215 330 48 90 4.6 1004/16 1300 62 , 90 10.2 100.2 195 240 45 78 4.3 2104/17 1300 62 85 10,2 102.6 170 320 52 72 3,2 1404/18 1300 62 105 10,0 95.9 215 200 52 70 3.0 1304/19 1230 56 80 9.4 91.2 180 180 70 60 5.0 1204/20 1300 58 70 9.4 84.9 185 300 80 84" 4.5 904/21 1300 54 60 8.8 93.6 185 280 68 70 4.0 1404/22 1230 62 70 10,2 59.1 170 240 58 74 6,5 130Stressed . -1500— -----— — ~— — ---— — — — --- — ---— -------- -— — — — — — — -4/22 1900 240 55 64 3.3 2304/23 0100 86 110 11.4 116.0 110 180 42 54 2,5 350.4/24 0700 140 ' 28 50 3.3 2304/23 1300 94 75 12.2 127.5 135 80 92 22 2,0 360Post stressed-;-period underlined is time of fetal:abortion-—4/25 1300 140 80 25.0 109.3 160 360 165 116 4.8 1004/26 1300 92 50 13.8 100.3 210 220 190 58 6.4 804/28 . 1230 .___ 78 60 11.2 91.2 160 280 152 90 9.8 704/29 1300 66 70 10.8 100.3 235 280 135 112 5.7 1004/30 1300 72 50 11.2 109.3 195 200 90 102 5.7 1405/1 1300 66 70 10.0 95.9 260 260 65 106 5.7 1105/2 1300 72 55 10.8 107,1 215 320 80 88 6.8 120 3

Table 60 Mineral composition, of the blood., urine, milk and feces of heatstressed cow $99» -

Date HourBlood

Ca K Mg Pmg/lOOmls

Na Ca KUrine

Mg Na

2/11 1200 10.4 21,5 2.44 6,4 500Primed2/112/112/112/112/112/18

1230130013301400143015001200

10.411,2

13.210.0

Stressed 2/18 2/18 2/19 2/19 2/19

2/20

1300-

22.517.5

19.021.5

2.602.36

2.602.32

5.86.1

5,95,6

950350

150450

*6 400 4*4 10.3 350

.4 750 9.2 30,7 350

.6 1950 12.0 41,1 600

.7 800 16.8 22.1 350

.5 600 8,8 29.0 450

1400 9.2 18.5 2.32 5.5 500 .5 650 13.2.... 35.7 4501800 9,2 17.5 2.36 6.1 450 .5 800 . 17.2 34.7 5000001 9,2 17.5 2.48 5.8 450 , 4 650 21.2 6.7 4000600 9.6 17,5 3.50 4.8 375 .5 800 27,2 3.6 5001200 9,0 20.5 2.68 6,4 375 ,6 650 20.0 5.0 400

1300 9.0 18,5 2.48 5.6 450 .4 650 18.0 28.5 400

Continued

Table 6 -s* Continued

Milk FecalDate Hour Ca - K Mg P Na Ca K Mg. P Na

mg/lOOmls_________________ ._______ mg/lOOmls__________2/11 1200 Primed 1230

.2/11 1300 2/11 1330 .

2/11 1400 '2/11 1430 2/11 15002/18 1200 80 60 11.0 72.0 260 180 72.5 78 4.47 140Stressed 1300— — — -----_______— .— _— ----- ;— ------ ---------------2/18 14002/18 1800 170 70,0 104 .28 1102/19 0001 .130 110 11.5 88.1 160 180 72.5 90 , 27 1002/19 0600 210 72.5 126 .14 802/19 12Q0 140 95 11.0 77.0 150 290 65.0 I64 0.00 70Post Stress-2/20 1300 103 ■ 60 11,0 69.5 265 210 125,0 118 . 29 70

Table 7= Mineral composition of the blood? urine, milk and feces of heatstressed cow 640.

Blood UrineDate Hour Ga K Mg p . Na Ga K Mg P Na

mg/lOOmls mg/lOOmls2/18 1200 9.6 17,5 1.92 6.1 550Stressed . 1300-r-2/18 1800 10,0 18,5 1.96 5,2 . 800 1,6 ■ 900 " 18.0 28.5 9502/19 0001 . 9.2 18,0 1.88 6,0 500 .6 950 28,4 16,1 1000 ■2/19 0600. 9.6 21.5 1,92 6,2 650 1.1 800 50.0 3.5 7002/19 1200 9.2 20,0 1.88 4.6 550 1,4 900 80.0 ,8 250Post stress ----2/20 1300 9.6 17.5 2.20 6.6 450 1.4 1250 40.0 96.8 100

Milk . FecalDate Hour Ga K . Mg P Na Ga K .Mg P Na

ms: A 00ml s mg/lOOmls2/18 1200 112 115 u, 0 73.0 65 200 65.0 82 5.43 180Stressed 1300— ►*»-»« i*~"i« *“<■— »2/18 1800 220 50,0 100 5.75 1802/19' 0001 115 120 14.0 66,0 115 190 60.0 120 5.91 1602/19 0600. 260 67,5 132 6.34 1602/19 1200 112 105 14.0 58.0 140 280 52.5 126 6.97 210Post pvitJoo "nwe,*"grrt,u2/20 1300 105 70 16.0 80.5 160 320 175,0 156 7.-50 140

Table 8* Mineral composition of the blood* urine* milk and feces of AGTHstressed cow 608,

Date HourBlood.

,0 a K Mgmg/lOQmls

NaUrine

Ga K Mg ;_______ mg/lOOmls

Na

3/103/113/143/163/173/18

133013001300130013001300.

11,611,6.10.010.812.811.2

Primed 3/18 1310 —3/19 1230 14,43/20 1300 12,43/ a 1300 13.23/22 1300 • 14.03/23 1200 14.03/24 1300 13,23/25 1230 12,4Stressed 13003/23/263/263/263/273/28Post3/293/303/314/14/24/3

190001000700124013001330

Stress — 1300 1230 1230 130013301300

13.616.4* 14,0 19.2 16,8 16.812.815.616.412.013.29

17.5 a , 5 18.0 19.0 20.015.5

19.0 19.0 18,0 15.0 19 16

2.442.482,402.242.082.08

5 5

17,020.019,015.017.018.0 15.518,0 17,0 17. 16, 15. 17,

,0,5,5,5

48 56 80 72 80

2.68 2.52

7.848.056,6,5.6,

80658032

8.016,006.885.795.495.236.22

350500300350300500

1050650400400350450700

.7

.9

.51.9,5,6

1,21,83.21.2 .7

1,4.9

500250850450750550500750700600550750400

18.04.8

32.412.020,020.0

18,10,42,13,18,68,

2,88 4.76 500 5.5 800 ' 10.0 00.3

400450350500400350

6,0 00.0 6009.6 00.0 12005.2 00.7 9009.2 00.2 6006,4 00,0 90011.6 00,0 8005.6 00.0 1200

9002,68 4.62 800 1,7 700 8.4 00.0 7002,68 4.48 500 1*7 650 13.2 00.0 5502.56 5.19 850 .6 450 10.4 00.6 3002,56 5.59 450 1.4 750 24,8 00.3 175-2,93 5.27 450 1.5 700 4,8 00.3 500•2.76 5.79 500 .7 700 4,4 00.3 9002.64 7.71 550 1.1 750 18.0 40.0 650 •

. 2.64 7.71 550 .5 800 7,2 20.7 ?2.28 9.30 350 2.4 750 8.0 22,4 6002.48 6.22 450 ' .7 400 12,8 8.4 6002.80 5.79 - 500 .8 950 8.8 3.2 400 £

Table 8 — Continued

Date Hour. Milk

Ca X Mg P________■ mg/lOOmls

NaFecal

Ca X •/ . Mg P mg/lOCtala

Na

3/10 1330 82,5 203/11 1300 87,5 303/14 1300 95,0 453/16 1300 90,0 403/17 1300 100,0 503/18 1300 82,5 40Primed 3/18 13103/19 12303/20 13003/21 13003/22 13003/23 12003/24 13003/25 1230Stressed 13003/253/263/263/263/273/28Post3/293/303/314/14/24/3

1900

8,0 59,3 1958,2 52.0 1859.8 71,0 1959.5 113,4 2109.8 90.1 1908.5 52,0 215

410 60.0 72 8,7 11058 7.6 12074 6.7 9088 2,6 10070 4,0 10080 4,2 90

370 27.5360 57.5340 45,0220 72,5390 57.5

88.0 55 9.6 74,7 19590.0 50 11.6 50.7 16578.0 ' 50 9.8 54=6 17082.0 65 10.6 58.5 16578.0 40 ' 10,4 31.0 17082.0 42 9.8 127.5 13576.0 40 10.4 11,6 128

440 61,2480 105,0480 57.5500 72.5380 65.0300 65.0240 52,5

80 4.0 68 4.6 74 4.680 4.4 72 3.882 2,7 58"' 1,4

300 70,0 80 2,2

120120100901108015090

0100 92.0 80 11.2 62.3 160 250 77.5 76 1.4 800700 270 75.0 92 2,3 901240 90.0 •40 10,6 46.5 135 260 70,0 74 2.2 1101300 84.0 55 12,8 74.7 110 • 380 105.0 56 3,8 1101330 , 74,0 40 10.8 78.5 115 420 95,0 62 5.8 80

Stress —1300 66.0 40 7.8 34.9 140 640 112,5 102 10.8 601230 72.0 30 9.2 94,8 130 500 97.5 68 7.7 60

. ■ 1230 78.0 25 9.2 62.3 135 380 65.0 74 4,4 901300 80.0 35 8.8 50,7 140 260 55.0 66 2.2 1001330 78.0 45 8.4 46,5 160 ■ 240 57.5 64 3,2 1401300 76.0 45 8.4 58,5 145 220 75.0 70 2,6 100

Table 9o Mineral composition of the blood, urine, milk and feces of ACTHstressed cow 609,

Date Hour• Blood

Ca ■ K Mg B . mg/lOOmls

NaUrine

Ca K ' -Mg' P mg/lOOmls

Na

3/10 1300 3/11 13003/1-4 13003/16 1300

1300 1300

3/19 12303/20 1300 3/21 1300 3/22 1300 3/23 1200 3/24 13003/25 1230Stressed 1300 3/25 19003/26 0100 3/26 07003/26 12403/27 13003/28 1330Post Stressed —

15.511.6 10.8 13,6 12,4 12,09,28,89.68,88,88.48.49.69.211,9.9,9,

19.021.018.5 19.0 19.020.519.5 20.0 18.019.519.5 17.019.521.020.016.517.5 19.5. 17,0

12 28 08 00 08 08

2.12 2.16 2.24 08 04 88 92

3/29 1300 10.0 18,03/30 1230 9,2 19.53/31 1230 9.6 18.04/1 1300 9,2 18.54/2 . 1330 9.6 18.04/3 1300 10.0 17.0

,16,92.96,80,76,04,12,20,32,32,32,36

6.376.015.235.14 5.50 6.80 6.60 6.47 7.71 5.4!5.155.23 4.694,4-55.414.695.065.79 4.835.79 6,34 6.175.795.79 5,23.

450 .6 50 3.2 17,1 200350 .6 450 9.6 12.1 300300 .6 25 ' ,8 40,8 100250 .6 550 3,6 15.6 150300 .7 500 5.2 22,9 300350 .8 600 9.2 74.9 250400 1.2 500 2.8 20,0 600250 1.1 800 19,6 37.8 350400 .8 500 18,8 15.1 300350 .7 300 11,2 17.8 250400 .6 375 8,0 16.2 400350 .5 400 6.8 23.4 650550 .9 350 7.4 35.8 250100 1.1 400 9.2 22.4 50550 ,8 300 5.2 15.2 300700. .5 200 7.6 4.5 400600 1.3 1000 8.0 17.7 750400 1,6 550 12,8 124.0 100250 - .8. 500 6.0 38.8 250200 .8 650 12,4 43.6 300325 1,1 350 6.8 23.7 150500 1.6 . 600 19.2 30.6 350400 1.7 500 8.8 20.9 300350 1.3 900 13.6 20.0 400500 1.2 800 9.2 14.1 200

Table 9 — Continued

Date Hour Ca K,

3/10 1300 57 103/11 1300 72 53/14 1300 60 53/16 1300 70 103/17 1300 70 103/18 1300 65 103/19 1230 74 403/20 1300 64 353/21 1300 54 303/22 1300 42 203/23 1200 80 353/24 1300 - 86 403/25 1230 . 100 55Stressed 1300 —3/25 19003/26 0100 70 503/26 07003/26 1240 76 553/27 1300- 66 453/28 1330 74 30Post Stressed3/29 1300 58 303/30 1230 30 203/31 1230 54 404/1 1300 86 454/2 1330 58 254/3 1300 110 35

MilkMg P. Na Ca

7,5 40,6 335 3507,8 82,5 255 3808,5 67.0 280 2908,8 82,5 250 1909,2 82.5 235 2009.0 82,5 245 44011.6 97.8 245 54010.8 102.3 245 56010.2 102.3 250' 6409.6 84.9 250 58011.8 93.7 245 38013.0 147.0 230 24013.4 93.7 130 160

26013.8 84.9 260 200

30014.6 102.0 225 22013.6 102,0 245 . 34014.2 76.2 180 24010.8 25.0 220 2407.2 50.2 290 200

10.6 12.5 265 20012,6 89.5 190 3009.4 138,0 225 32011.0 89.5 200 220

.FecalK • -Mg P Na

mg/lOOmls_______55.0 68 12,5 90

122.5 62 . 4 9557.5 68 8,9 9082.5 68 3,2 80 .70,0 78 6,3 8077.5 86 4.1 5095.0 80 7.6 13077.5 83 5.3 12095.0 102 4.8 11075.0 100 3.2 12082.5 88 3.6 10057.5 68 4.4 10062,5 70 3.2 16077.5 94 4.7 9082.5 98 2.3 10085.0 126 - 4.2 11070.0 84 4.1 9090.0 70 4*. 4* 10085.0 80 4.0 9067.5 74 3.1 8080.0 68 3.6 11065.0 . 76 . 2.9 10080.0 88 4.7 10072.5 78 2.9 15077.5 104 2.2 120

Table 10, Least Square Means for Experimental Minerals in Blood and Urine,

Period AnimalGa

Blood K Mg

Me/lOOmlP Na Ca

Urine K Mg

MsrAOOmLP. Na

Period I 673 10,95 20.12 2.49 6.45 571.87 .98 757.14 18.51 ■ ,08 435.71(Base) . 729 10,56 17.81 2*34 6.55 431.25 ,56 765.90 16.00 38.73 414.00

608 11,33 18.58 2.28 6.81 383.33 .85 558.33 25.06 18.37 408.33609 12.08 19,60 2.10 5.71 850,00 .68 525.00 6.90 31,37 250.00Period II(Primed) 673 8.91 21,07 2.39 5,62 392.85 .87 1071.42 21,02 1.74 142.85(Primed)

729 9,57 19.21 2.49 6,64 342.85 .53 920.00 20.08 38.30 289.99608 13,37 17.71 2.65 6.23 571.42 1.48 607.14 7.65 .13 885.71609 8.96 19,30 2.10 6.71 390.00 .83 460.71 10.65 23.72 400,00Period III(Stress) 673 7.55 18.06 1.94 ' 1.93 275,00 ,49 487.49 3.60 1,40 187.49729 8.80 19.25 2.21 2,44 312,50 .32 674.99 5.60 1,24 387.49608 16.13 17,41 2.71 4.98, 591.66 2,06 675.00 11.80 .27 520.83609 9,66 18.58 1.94 5.03 600.00 1.00 491.66 8.13 37,09 308.33599 9,24. 18,30 2.66 5.73 430.00 .50 710.00 19.76 17.14 450,0064.O 9,36. 19.10 1.90 5.67 600.00 1.60 900.00 37.68 19.70 450.00Period IV(Recovery) 673 8.40 19.27 1.91 6.80 289.28 .68 450.00 11.35 43,77 166.14729 9.31 18,50 2.14 7,38 303.57 1.07 628,57 33.57 122.54 335.7160S 14.00 16.90 2.58 5.80 450,00 1.68 679,99 23,04 18.37 669.99609 9.60 18.16 2.27 5.85 379.16 1,28 633.33 11,66 • 25.48 283.33

co.Vi

Table 110 Least Square Means for Experimental Minerals in Milk and Feces,

Period Animal ' MilkCa ' ' K Mg P

Mg/lQQmlPeriod I(Base) 673 79*25 61.87 11.82 93.69

729 70.28 52.85 10.82 84.49608 89.58 37.50 8.95 72.63609 65.83 8.33 8.45 72.93

Period II(Primed) 673 73. H 64.28 11.82 67.72

729 59.42 . 80.00 9.72 89.65(Primed) 608 82.00 48.92 10.31 58,37

609 71.42 36.42 11.48 103.10Period III(Stress) 673 94.00 65.00 13.00 78.31

729 90.00 92.50 11.80 121.50608 8-5.00 53,75 11.35 65.00609 71.50 45.00 14,05 91.27599 135.00 102.50 11.25 82.556/0 113,50 / 112.50 . 14.00 62.00

Period IV(Recovery) 673 92.00 64.28 13,82 94.58

729 83.70 62.14 13.25 101.91608 74.80 35.00 8.68 57.84609 66.00 32,50 10.26 67.44

FecesNa Ca K Mg P Na

- . •____ ;_________ Mg/Gram_________

177,50 313.75 65,62 86.25 6.77 79,37191,42 298.75 59.37 75.49 7.23 92.50193,33 348.33 53.33 73.66 5.63 101.66266.66 308.33 77.50 71.66 7.91 80.83

160.71 302,85 59.64 81.77 5.78 118.57185.71 251.42 60,71 72.57 4.35 137,14161.14 402.85 68.34 73.42 3.64 110.00227.85 442.85 77.85 84.42 4.56 120.00

207.50 190.00 46.87 51.50 5.23 210,00122,50 160,60 54.37 47,49 2,79 292.00130.00 313.33 82.08 73.33 2,94 93.33227,50 260.00 81.66 92.00 3.95 96.00155.00 . 210.00 71.50 115.20 1.02 102.00127,50 234.00 57.50 117.20 5.95 178.00

188.57 322.85 66.07 97.71 7.35 111.42204.99 274,28 125.35 96.00 6.40 102.85140.99 404,00 77.50 75.20 5.65 90.00231.66 246.66 73.75 81.33 3.23 109.99

8 7

Table 12e Least Significant Difference of Treatment for Blpod0

Period Paired Inter- . Period Paired Inter- LSD of Mean action LSD III Mean action LSD Treatment

CALCIUM^ : : : ± -8« ^

609 ± •S« % 6 6 ±'-91 ±POTASSIUM

" I S i a ± ia2 ' ..■■± 1 -48 * •5S

609 19 I 3 0 . i 1 *12 Ilfs + le 2 1 i 062

mcmsiuM7I I m t •°12 l:9I ± •°16 ± •°06

w z a o ± -012 ± •0i2 ± •°07599 2.66640 1.90 ± o39

PHOSPHORUSIf if

w If ^ : if. „ SODIUM

599 430.0640 . . 600.0 + 112,34

8 8

Table 13, Least Significant Difference of Treatment for Urine«

Period Paired 'Inter- Period Paired Inter-Animal II Mean action LSD III Mean action LSD

-CALCIUM% ; « ± -so ■ ± .90

60S 1,48 4. 58 2,06 4. <73609 ,83 ~ 1,00 -

POTASSIUM599 710.0640 900,0

+ 1.05599 .50640 1.60

MAGMESIUM729 ± 10-8 ' 1 % i 12-0

609 ICL65 - 1 9 J " 18 ll3 1 9°8

PHOSPHORUS33: % 1 ^ 7 i : %

6 ^ 23:72 ±35-3 ±38.0

± 90,6

LSD of Treatment

± .37

± .37

+ 5.0

± 4.9

±19.5

± 19,0

39Table L4» Least Significant Difference of Treatment for Milk,

Period Paired Inter- Period Paired Inter- LSD ofAnimal II Mean action LSD III Mean action LSD Treatment

CAL0im-l673729

73.1459.42 + 12.4 94.090.0 ± 23.2 ± 6.7

608609

82.0071.42 + 12.4 85.0

71.5POTASSIUM

± 16,4 ± 6.7

673729

64.2880,00 ± 10.7 65.00

92.50 ± 20.0 ± 5.8

608609

48.9236.42 ± 10.7 53,75

45.00MAGMESIUM

±14.1 ± 5.8

673729

11.829,74 + 1.62 13.00

11,80 ± 3.0 ± .88

608609

10.3111.48 + 1.62 11.35

14.05PHOSPHORUS

± 2.1 ± .88

673729

67.72 . 89.65 + 19.3 78.31

121.76 ± 37.0 ± 10,7

608609

53,37103.10 ± 19.8 65,5091.27

_ SODIUM

±26.1 ± 10.7

673729

160.71185.71 + 28,8 207.5

122.5 ± 53.9 . ±15.5

608609 .

161.14227.85 ±28,8 130.0

227.5 ±38.1 ± 15.5

' . 90Table 1-5» Least Significant Difference of Treatment for Feces,

Animal II Mean

673 302.85729 251.42608 402.85609 442.85

673 59.64729 60.71608 68.34609 77.85599640

673 81.77729 72,57608 73.42609 84.42

673 5.78729 4.35608 3,64609 4.56599640

673 118.57729 137.14608 110.00609 120.00599640

Period Paired Inter- Period Paired Inter­action LSD III Mean action LSD

CALCIUM± 26,4 160 IbO ~ H0,5

±26.4- 26o[oo - 28e5POTASS IUI1

+ 17,8 54.° 37 ~ 23.6

± ^ . 8 ± 19.2

71.5057.50

PHOSPHORUS2.79

1.025.95

SODIUM

102.00178.00

± 6.6

MAGMBSIDM ± i4-7 ± 19-5

± U > 7 IzM ± 15-9

± 1.85 5.E + 2.45

± 1.85 : : 2.94 ± 2,00

+ 1,49

± 23-2 ±- 30-7

± 23-2 ± 25-°

i, 30.2

LSD of Treatment

+ 43.4

+ 45.1

± 9 ,2

+ 9,6

± 7.6

± 7.9

± .96

± .99

+ 12.0

+ 12.5

91Table 16, F Test Analysis for Equality of Variance,

Mineral SampleBLOOD

Value for;Period Treatment Interaction

CalciumPotassiumMagnesiumPhosphorusSodium

0.01N.S.0.010.01H.S.,

0.010.010.01N.S.0.05

0.01N.S.0.01N.S.N.S,

URINECalciumPotassiumMagnesiumPhosphorus.Sodium

N.S.N.S.0.010.01N.S.

0.050.010.050.01N.S.

0.05N.S.0.010.05N.S.

MILKCalciumPotassiumMagnesiumPhosphorusSodium

0.050.010,01N.S.0,01

0.010.010.010.010.01

0.050.050.010.050.05

FECESCalciumPotassiumMagnesiumPhosphorusSodium

0,010,010.010.010.01

0.010.01N.S.0.010,01

N.S.0,010.01N.S,0.01

N.S. — Not Significant

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