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PLASMA ADRENOCORTICOTROPIC AND
GLUCOCORTICOID HORMONE LEVELS
DURING AGING IN CATTLE

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
Gail Daniel RiegIe
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ABSTRACT

PLASMA ADRENOCORTICOTROPIC AND GLUCOCORTICOID
HORMONE LEVELS DURING AGING IN CATTLE

by Gail Daniel Riegle

Pituitary—adrenal function was estimated in dairy and
beef cattle ranging from three days to fourteen years in
age, by biological assay of plasma adrenocorticotropic ac-
tivity and chemical assay of glucocorticoid hormones. Le—
vels of adrenocorticotropic activity were consistently low
(less than 2.0 mU ACTH activity/100 ml plasma) and in most
instances barely detectable in bulls from one to thirty
months of age. Adrenocorticotropic activity was increased
in the older cattle, reaching a peak value of 15.4 mU ac-
tivity/100 ml plasma. Substantial amounts of plasma adre-
nocorticotropic activity were assayed in all animals above
three years of age. Levels of plasma cortisol (6.0 to 9.0
ug/lOO ml plasma) and corticosterone (6.6 to 13.9 ug/lOO
ml plasma) exhibited no consistent alterations related to
increased adrenocorticotropic activity. These data are in-
terpreted to reflect adrenocortical insufficiency or insen-

sitiveness to ACTH during aging, with concomitant increased

Gail Daniel Riegle

adrenocorticotropic secretion in order to maintain blood
levels of glucocorticoids within the normal range. This
interpretation was supported by increased percentages of
blood eosinophils, decreased percentages of plasma albu-
min, and increased percentages of plasma gamma globulins

in the older bulls.

PLASMA ADRENOCORTICOTROPIC AND GLUCOCORTICOID

HORMONE LEVELS DURING AGING IN CATTLE

BY

GAIL DANIEL RIEGLE

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1963

 

@390 7 7
4/34/44

ACIQIOWLEDGMENTS

I would like to acknowledge and express my gratitude

My advisor, Dr. J. E. Nellor for guidance and the pro-
vision of facilities to carry out this study;

Dr. E. B. Romanoff for assistance in developing and
adapting the laboratory procedures used in this work;

The Michigan Artificial Breeders Co-op and the Michi—
gan State University Dairy Farm for allowing me to use
their animals in this study;

Miss Susan Mahoney for assistance in hematologic and
protein measurements;

The National Institutes of Health for supplying the
Research Fellowship which supported me financially;

This project was supported in part by grants from Smith,
fline and French Company and the Benson and Henry Fords;

My wife, Barbara, whose sacrifices and encouragement
were necessary to complete this study;

Without the contributions of the individuals and groups
mentioned, as well as many others, this study would not

have been possible.

ii

INTRODUCTI

MATERIALS

RESULTS .

TABLES . .

FIGURES .

DISCUSSION

SUMMARY .

LITERATURE

TABLE OF CONTENTS

ON . . . . . . . . . . . .

AND METHODS . . . . . . .

Plasma adrenocorticotropic

Plasma glucocorticoids

Hematology

Plasma Proteins

o o o a e a o o o o e o o

CITED . . . . . . . . . .

activity

Page

18

3O

3O

35

36

39

41

50

59

74

77

LI ST OF TABLES

Table Page

1. Plasma Adrenocorticotropic Activity in

Bulls . . . . . . . . . . . . . . . . . . . 41
2. Plasma Adrenocorticotropic Activity and

Glucocorticoid Hormone Levels in Cows . . . 43
3. Plasma Glucocorticoids in Bulls . . . . . . 44
4. Hematology in Bulls and Cows . . . . . . . . 46
5. Total Plasma Proteins in Bulls and Cows . . 48
6. Plasma Protein Components by

Electrophoresis . . . . . . . . . . . . . . 49

iv

 

Figure

1.

LIST OF FIGURES

Response of Incubated Rat Adrenal

Tissue to ACTH . . . . . . . . . .

Plasma ACTH with Aging . . . . . .

Plasma Corticoids with Aging . . .

Red Blood Cells with Aging . . . .

Total Blood Leucocytes with Aging

Blood Eosinophils with Aging . . .

Blood Lymphocytes and Neutrophils with

Aging . . . . . . . . . . . . . .

Total Plasma Proteins with Aging .

Plasma Albumin and Gamma Globulins with

Aging . . . . . . . . . . . . . .

Page

50

51

52

53

54

55

56

57

58

INTRODUCTION

The field of adrenal endocrinology was conceived when
Addison described the clinical picture of adrenocortical
insufficiency in 1855. The following year Brown—SeQuard
carried out the first adrenal experiments when he studied
the effects of adrenalectomy.

The major impetus for research in pituitary-adrenal
endocrinology followed the isolation and identification
of the active constituents of the bovine adrenal cortex
(Swingle and Pfiffner, 1930; Grollman and Firor, 1933, Ken—
dall et a1., 1937; and Wintersteiner and Pfiffner, 1936).

Macchi and Hechter (1954) demonstrated control of ad-
renocortical glucocorticoid hormone production depended
almost entirely on hypophyseal adrenocorticotropic hor-
mone. Kupperman e1 a1., (1955) and many other workers
have shown the inhibiting effect of adrenal glucocorticoids
on hypophyseal-pituitary output of adrenocorticotropic hor-
mone. Recently Dorfman (1957), Flink (1961) and many
others have written excellent reviews covering many aspects
of pituitary-adrenal physiology.

Gross and microscopic considerations of pituitary and
adrenal glands associated with aging have been investigated
by many workers. Wolfe (1943) reported a relative decrease

in anterior pituitary eosinophils and an increased number

of pituitary chromophobes in aged female rats. There was
also less mitotic activity in the pituitary gland cells
from rats of advancing age. Wolfe interpreted these ob-
servations to be indicative of decreased functional activ-
ity of the anterior lobe of the pituitaries of aged rats.
The histological changes within the anterior pituitary
of the fowl with increasing age were described by Payne
(1949). He reported marked retrogression of all anterior
pituitary cell types with age. Pycnotic nuclei and mito-
chondrial changes were observed in from 50 to 75 % of the
basophils from older animals. There were also fewer acido-
phils possessing secretory granules in the pituitaries
from the older birds. By 2 to 3 years of age cavities
appeared in the posterior portions of the anterior lobe
and with advancing age the cavities were present in all
regions of the anterior pituitary. Spagnoli et a1. (1955)
observed increased vacuolation of the anterior pituitary
associated with increasing age in the Golden Hamster. Male
hamsters exhibited a decrease in acidophils and a corresp
sponding increase in basophils with increasing age. Al—
though the fore—mentioned workers reported histological
evidence of anterior pituitary abnormalities associated

with aging, Fortier (1958) reported no significant

alteration in the pituitary content of adrenocorticotropic
hormone during aging in the rat.

Rats beyond 700 days of age had increased absolute ad—
renal gland weights (Yeake, 1944), however, their relative
cortex volumes were decreased with increasing age. Payne
(1949) observed a reduction in size of the adrenal glands
of the fowl by 5 to 8 years of age. Dribben and Wolfe
(1947) reported increased width of the adrenal capsule and
a gradual transformation of reticular fibers to collagen
fibers within the adrenal cortex from female rats of ad-
vancing age. The change to collagen fibers was especially
noticeable in the zona reticularis. In mice beyond 1 year
of age, Blumenthal (1955) observed a progressive reduction
in adrenal gland mitotic activity. Blumenthal also found
increased proliferation of adrenal connective tissue with

senescence. Ninety percent of rats above 15 months of age

had spotted and granular adrenal gland surfaces (Ingle, 1956).

Jayne (1957) reported adrenal cortical degenerative changes
in 10 of 20 rats 1 year of age and older. In the older
rats there was a gradual decrease in size of the zona ret-
icularis with a corresponding increase in the size of the
fat laden zone. The adrenal cortex of the aging Golden

Hamster was shown by Meyers and Charipper (1956) to contain

a progressive increase in collagenous connective tissue, a
thickening of the capsule and degenerative changes involving
cellular atrophy, necrosis and hemorrhage. Jayne (1953)
observed histological alterations occurring in the adrenal
gland of the aging rat similar to degenerative changes
occurring after hypophysectomy.

Vezar (1960) studied differences with aging in the com-
pensatory hypertrophy of the remaining adrenal gland when
one was removed and reported significantly less ability to
compensate by adrenal gland enlargement in the older rats.
Adrenal glands from groups of 40 and 360 day old donor rats
were transplanted into 40 day old rats (Geiringer, 1956)
to determine if the glands from the older animals would
supply the functional needs of the recipient animals as
well as the transplants from the 40 day old rats. In these
studies there were no differences in growth, reproductive
capabilities, lactation or response to a water load test
for cortical activity between the rats with adrenal trans-
plants from the adult and 40 day old rats.

The possibility of utilizing changes in blood leucocyte
concentrations as a measure of pituitary-adrenal function
was recognized at an early date. Blood-cellular changes

after adrenalectomy in the cat were investigated by Zwemer

and Lyons (1928) and Corey and Britton (1932). After adre—
nalectomy, Corey and Britton (1932) observed an increase in
blood lymphocytes from 32 to 64% and a decrease in blood
neutrophils from 63 to 30% of the total white blood cell
counts. Replacement therapy with adrenal cortical extracts
restored the differential counts to preoperative levels.
Adrenalectomy reduced the total leucocyte count in the cat
from 12,400 to 7,000/cmm (Corey and Britton, 1932). On the
other hand, Shecket et al. (1935) studied leucocyte responses
of the rat to adrenalectomy and found that before the rat
died from adrenal insufficiency the total leucocyte count
increased from 11,300 to 17,500/cmm of blood. In these
studies lymphocytes were shown to increase from 81.2 to
88.4% of the total blood leucocytes. The increase in blood
lymphocytes was correlated with a corresponding decrease

in blood neutrophils.

Total red blood cell counts were increased after adre-
nalectomy in the rat (Dalton and Masson, 1940 and Dougherty
and White, 1947) and in the cat (Corey and Britton, 1932).
A slight decrease in red blood cells of the rat in response
to ACTH or glucocorticoids was observed by Dougherty and

White (1944).

Solomon and Shock (1950) demonstrated significantly
smaller blood neutrophil increases after administration of
ACTH in aged men compared to young men. These aged patients
also exhibited smaller decreases in blood eosinophils and
increases in blood neutrophils after injected epinephrine
stress.

The influence of hormones on lymphoid tissue in rats and
mice was investigated by Dougherty and White (1944). They
found a significant decrease in blood lymphocyte counts
following single injections of ACTH or glucocorticoid.

Blood neutrophils were shown to increase after ACTH or glu—
cocorticoid injection in rats, but this increase was of
smaller magnitude than the corresponding lymphopenia. Rein-
hardt and Li (1945) demonstrated an ACTH and glucocorticoid
effect on lymphatic tissue by the rapid decrease in thoracic
lymph lymphocytes observed following injections of ACTH or
glucocorticoids. Hormones of the adrenal cortex were shown
to affect cellular suspensions of thymus or marrow cells
(Schrek, 1949). Extracts of adrenal cortex, corticosterone
and 17-hydroxycorticosterone all produced a cytotoxic effect
in the cell suspensions leading to cellular disentegration.
The cytotoxic effect was demonstrable at low concentrations

of the hormones, but increased amounts of the adrenal

hormones did not augment the response. The cytotoxic
response was specific for the glucocorticoids as no effect
was observed when the cellular suspensions were mixed with
ACTH, desoxycorticosterone or various male and female sex
hormones. Desoxycorticosterone has also been shown to have
no effect on peripheral lymphocyte counts (Dougherty and
White, 1944).

Lymphocyte response after starvation, histamine and
anaphylaxis stress in adrenalectomized mice was investigated
by Dougherty and Kumagui (1951). Animals stressed 2 hours
after adrenalectomy had significantly higher lymphocyte
concentrations in the blood 2 hours after stressing and
exhibited pronounced lymphocytosis 8 and 12 hours following
stress. Lymphocyte concentration had returned to normal in
these mice 24 hours following stress. These data were
interpreted to indicate that stress not only augments the
pituitary-adrenal system producing lymphopenia, but also
stimulates lymphatic tissue growth in some manner.

Eosinopenia was associated with adrenal hormone produc-
tion by Hills et a1. (1948). Following administration of
ACTH, human patients exhibited a decrease in blood eosin-
ophils associated with the expected lymphopenia and increased

neutrophil counts. Visccher and Halberg (1955) found that

blood eosinophil counts exhibited a diurnal variation very
similar to plasma glucocorticoid concentrations. Best and
Samter (1951) using injections of élucocorticoids or ACTH
in man found the maximum eosinopenia occurred 4 hours after
administration of the hormone. Forsham et a1. (1948) in-
vestigated the hematological changes in man following ad-
ministration of ACTH. A maximum response was obtained
within 4 hours producing a 74% reduction in blood eosin-
ophils, a less spectacular fall in lymphocytes and a non-
consistent rise in neutrophils. When ACTH was administered
to Addisonians there was only a 4% reduction in eosinophils.
However, a subsequent fall in blood eosinophils in the Ad-
disonians was produced by the administration of 20 mg of
hydrocortisone. However, Best and Samter (1951) reported
too many physiological variables effect blood eosinophil
counts to consider eosinophil counts alone as a reliable
measure of pituitary—adrenal physiology.

There is considerable evidence indicating a positive
correlation between plasma adrenal glucocorticoid levels
and relative and absolute amounts of plasma proteins. Al-
though serum albumin was found to be depressed in Addisonians

(McCullagh and Lewis, 1945) the relative concentrations of

all the globulin fractions tended to be increased. Jager et
a1. (1951) studying human patients exhibiting hypoalbu—
minemia found that injections of cortisone restored serum
albumin levels to normal in 5 of 7 cases. The serum gamma-
globulin concentration was reduced in patients following
cortisone or ACTH therapy (Jager et a1” 1951, and Vaughan

et a1., 1951).

White (1956) reported decreased amountsot protein were
released from the liver and spleen of adrenalectomized rats
compared to intact rats. The protein release was restored
to normal by adrenal cortical extract injection in adre—
nalectomized rats and augmented by adrenal cortical extract
or ACTH injections into intact animals.

Serum protein patterns from patients with Cushing's
syndrome were investigated by Lewis and McCallagh (1947).
Patients with Cushing's syndrome and normal subjects treated
with cortisone exhibited decreased levels of gamma-globulin.
Thorn et a1. (1950) reported a 10.2 gm% to 4.8 gm% reduction
in serum globulin of a multiple myeloma patient following
ACTH injections.

When quantitative chemical assays for biological quan-
tities of steroid hormones were developed many different

laboratories actively utilized these new tools to study

10

pituitary-adrenal function with aging. Pincus (1956) re-
ported a very significant decrease in the output of urinary
l7-ketosteroids in men and women with increasing age. In

the span of from 20 to 80 years of age, urinary l7-ketosteroid
excretion rates decreased from 8.5 mg/24 hours to 2 mg/24
hours among men and from 7.5 mg/24 hours to 1.0 mg/24 hours
among women. However, the ll-oxy—l7-ketosteroids were
maintained relative to the ll—desoxy-l7—ketosteroids indi-
cating retention of adrenal function. Johnsen (1956) pointed
out that while there was a decrease in urinary 17—ketosteroid
output for both men and women, there was too much overlap
between the sexes to determine if a sample was from a male

or female subject. Relative concentration of urinary andro-
sterone, the major metabolite of testosterone, was found to
decrease at a much faster rate with increasing age than

the other l7-ketosteroids (Johnsen, 1956, and Pearson and
McGavack, 1955) indicating a more rapid loss of testicular
androgen secretions than adrenal androgen. The decrease in
urinary androsterone output was found to be directly cor-
related with decreased concentrations of testosterone in

the human spermatic vein (Hollander and Hollander, 1958).
Johnsen (1956) reported greater differences in biological

androgenecity of urinary extracts between young and old men

11

than differences in total urinary l7—ketosteroids. Levels
of urinary l7-ketosteroid metabolites after ACTH stimulus
were 20% less in elderly patients compared to young subjects
(Romanoff et a1., 1957).

Diurnal variations in plasma 17-ketosteroids were studied
by Migeon et a1. (1957) and Duboff (1957). Plasma 17—keto-
steroids varied in a diurnal fashion with peak values ob-
tained at about 8 a.m. However, the diurnal pattern of
plasma 17-ketosteroids was not nearly as uniform as the
pattern for plasma 17—hydroxy-corticosteroids.

Borth et a1. (1957) demonstrated 30% more 17-ketosteroid
excretion in men than women of the same age. Urinary 17-
ketosteroid values determined for eunuchs (Hamburger, 1948;
Johnsen, 1956; Furman et a1., 1958; and Hamilton et a1.,
1959) were found to be consistently lower than similarly
aged intact men and usually closely paralleled urinary 17-
ketosteroid production from women of like age. In all cases
this decrease in urinary l7-ketosteroid output was as severe
for the eunuchs as for intact men, as the aging process
continued.

An intensive study of urinary steroids and aging from
617 subjects was carried out by Pincus et a1. (1954). While

urinary l7-ketosteroid output was found to decrease sharply

 

12

with advancing age, neutral reducing lipids, adrenal glu-
cocorticoids and their metabolites, exhibited only a
slight non-significant downward trend with increasing age.
Romanoff et a1. (1957) reported no change in relative oc-
currence of the characteristic urinary corticoids in young
and elderly subjects after ACTH administration. In another
study Romanoff et a1. (1958) found that if the lesser
amounts of corticoid metabolites present in the urine of
elderly subjects were compared to urinary creatinine excre-
tion from the various aged subjects, the quantitative dif-
ferences in urinary corticoid metabolites related to age
disappear.

The adrenal gland response to an intravenous infusion
of ACTH was studied by Samuels (1957), Nugent et a1. (1959)
and Tyler et a1. (1955). The older subjects achieved higher
levels of plasma l7-hydroxycorticoids in response to 25 IU
of exogenous ACTH than the young subjects. However, Samuels
(1957) demonstrated that humans beyond 50 years of age have
a smaller distribution volume for l7-hydroxycorticoids and
the hormones have a longer half life in peripheral circu—
lation. Because of these alterations, plasma 17-hydroxy—
corticoids require substantially more time to be lowered to

the initial level present before ACTH stimulus in the

13

elderly subject. West et a1. (1961) demonstrated a pro-
gressively slower removal rate of cortisol from the circu-
lation with advancing age. When these alterations in cor-
tisol utilization were considered, the older patients
showed less responsiveness to exogenous ACTH. Adrenal l7-
hydroxycorticoid production of men, 50 years of age or more,
was shown by Moncloa et a1. (1963) to be less for all levels
of exogenous ACTH tested. This decreased response to exo-
genous ACTH was interpreted to indicate that adrenal gland
response capacity and sensitivity to ACTH diminished with
age.

Eik—Nes et a1. (1955) tested the adrenal response to
exogenous ACTH in clinical Addisonians by measuring plasma
l7-hydroxycorticoids. None of the Addisonians exhibited a
significant increase in l7-hydroxycorticoids from exogenous
ACTH. Eik-Nes suggested that Addisonians with measureable
l7-hydroxycorticoid levels showning no significant response
to ACTH have adrenal gland remnants which are probably al-
ready maximally stimulated by endogenous ACTH.

Patterns of diurnal rhythmecity of l7-hydroxycorticoids
have been established (Migeon et a1., 1956). Pincus et a1.
(1954) reported delayed l7-hydroxycorticoid diurnal rhyth-

micity in senile subjects. The effect of exogenous ACTH on

l4

diurnal l7-hydroxycorticoid patterns was studied by Nugent

et a1. (1959). Continuous intravenous infusion of ACTH at
rates from 0.8 to 1.4 IU/24 hours maintained plasma 17-
hydroxycorticoids above maximal levels obtained spontaneously
during a diurnal variation. There were no diurnal l7-hyro-
xycorticoid variations when ACTH was infused at rates ex-
ceeding 0.8 IU/24 hours. In males, it was calculated that
the maximal normal diurnal variation can be achieved by a
secretion of ACTH of less than 4.0 IU/day for a period of
less than 4 hours.

In cattle, studies of pituitary-adrenal function asso-
ciated with aging have not been numerous. Perfused bovine
adrenal glands were shown (Hechter, 1953; and Bush, 1953)
to secrete nearly equal quantities of cortisol and corti-
costerone. Both cortisol and corticosterone were shown to
increase markedly when ACTH was added to the perfusion media.

Adrenal gland abnormalities have been associated with
many metabolic and reproductive disturbances in cattle.
Cupps et a1. (1954 and 1956) associated reproductive mal-
functions in male and female dairy cattle with histological
evidence of adrenal gland degeneration. Animals with repro-
ductive troubles showed degranulations of cells in fasci-

cular and reticular zones. These zones also contained

15

smaller cells which had many more pycnotic nuclei than had ad—
renal glands from normal cattle. The adrenal glands of
ketotic cows are larger and exhibit degeneration of the
epithelial cells (Shaw et a1., 1948 and 1949). Saarinen

and Shaw (1950) reported increased total fat in the adrenal
glands of ketotic cows. Adrenal glands from these ketotic
cows also exhibited a gross structural flabbiness that could
not be explained on the basis of changes in water or fat
content. Although adrenal glands from cows in early ketosis
were enlarged, cows suffering from chronic ketosis had
atrophied adrenals (Hatziolos and Shaw, 1958).

Grant (1960) reported higher ACTH content in bovine
pituitaries than in pituitaries from other species. He
described the bovine pituitary'as"a stressed gland, full of
compact cells." Shaw et a1. (1948) found anterior pituitary
abnormalities in ketotic cows. General cellular degenera-
tion together with numerous vacuoles was evident in the
anterior lobes from ketotic cows.

Although blood hemoglobin and hematocrits from ketotic
cows were normal (Shaw et a1., 1948), ketotic cows had
increased concentrations of blood neutrophils and decreased
concentrations of blood lymphocytes and eosinophils which

could suggest an increased adrenal gland function in ketotic

16

cows. However, in another study Shaw et a1. (1954) reported
blood eosinophils were not decreased as much in a ketotic
cow following epinephrine injection as in a normal cow in-
jected with epinephrine. Shaw (1947) demonstrated a more
marked hyperglycemic effect from ACTH in normal cows com-
pared to ketotic cows.

0n the basis of urinary glucocorticoids, Puntriano(l952)
reported decreased adrenocortical activity in ketotic cows.
Holcombe (1957) measured urinary "reducing corticoids" in
cattle and sheep. While absolute amounts of urinary "re-
ducing corticoids" were depressed with aging, similar to
glucocorticoid excretion in older humans, the differences
were not significant with increasing age. Urinary "reducing
corticoids" in cattle also showed indications of a typical
diurnal pattern.

Schalm (1961) studied changes in leucocytes in cattle
with aging. The only significant alteration was in eosin-
ophils which increased from 0.8% to 12.0% in dairy cattle
from 4 months to 14 years of age.

Nellor (1958a) reported the quantitative detection of
adrenocorticotropic activity in bovine plasma. Indications
of increased adrenocorticotropic activity with increasing
age in dairy bulls were observed by Nellor (1958b) and Sutton

(1960).

17

At this point it becomes clear that there is strong
evidence indicating alterations in pituitary-adrenal
functions with aging. Pituitaries and adrenal glands from
many species show degenerative changes with aging. Reduc—
tions in circulating blood leucocytes suggest adrenocortical
hypofunction with advancing age. There is a definite de-
crease in adrenal l7—ketosteroid secretion in older humans,
strong evidence indicating less glucocorticoid responsive-
ness to exogenous ACTH, and a tendency to lose diurnal
patterns of plasma glucocorticoids with aging.

Since the symptoms indicating adrenocortical abnormal-
ities are especially prevalent in cattle, the present study
concerns the effects of increased age on the level of plasma
adrenocorticotropic activity in cattle, along with simul-
taneous estimation of adrenal response to plasma adrenocor-

ticotropic activity by analysis of plasma glucocorticoids.

MATERIALS AND METHODS

Pituitary-adrenal physiology was evaluated in the fol-
lowing catthe: fifteen Holstein Friesian bulls ranging in
age from 3 to 14 years from the Michigan Artificial Breeders
Co-op; five Holstein Friesian bulls ranging in age from 1
to 16 months and two Holstein Friesian cows 5 and 7 years
of age from the Michigan State University Dairy Farm; one
3 year old Hereford cow, one 6 year old Angus bull, one 5
month old Hereford bull before and after castration and one
Hereford calf 3 days old from the herd of the Endocrine
Research Unit, Michigan State University. All the cattle
were used to being handled and great care was taken to
handle them in a routine manner before sampling was attempted
to minimize possibilities for a stressful condition af-
fecting their pituitary-adrenal systems.

Heparinized external jugular samples of approximately
500 mls were taken from 8:30 to 9:30 a.m. to obtain con-
sistency in time of sampling and reduce errors due to di-
urnal hormonal patterns. All samplings were spaced at
least 30 days apart. The blood samples were immediately
placed in an ice bath until they could be returned to the

laboratory for centrifugation which was completed as rapidly

l8

19

as possible. Centrifugation of the plasma from the formed
elements and all subsequent handling of the plasma prior
to assay was carried out at 3°C.

Plasma adrenocorticotropic activity was assayed from
plasma, plasma concentrated by lyophilization, and plasma
separated into protein fractions by Cohn's cold ethanol
No 6 method (Cohn et a1., 1946), modified by using step-
wise precipitation of proteins in the II + III fraction.
Most of the information presented in this paper concerns
assay of the 12-25% fraction of II + III and the IV-l pre-
cipitate. Plasma to be used for adrenal glucocorticoid
analysis was extracted immediately after centrifugation
or stored at -10°C until it could be assayed.

Routine laboratory analysis of blood samples including
red and white cell counts, white cell differential counts,
total plasma proteins, plasma protein component concentra-
tions after electrophoresis and hematocrits was conducted
on as many samples as possible.

Plasma adrenocorticotropic activity was assayed by

the ig_vitro technique of Saffran and Schally (1955). For

 

each assay, eight Long-Evans rats of either sex weighing

between 180 and 250 grams were sacrificed by decapitation.

20

The adrenal glands were rapidly removed and placed on a
filter paper moistened with Krebs—Ringer bicarbonate glu-
cose medium in a Petri dish which was kept cold by an ice
bath. The adrenal gland pairs from each rat were kept
separate on the filter paper. The adrenal glands were dis-
sected free of fat and extraneous connective tissue leaving
the adrenal capsule intact for easier handling. The adre-
nal glands were then carefully quartered with a small scis—
sors on a filter paper saturated with cold Krebs-Ringer
bicarbonate glucose medium. The eight quarters from each
pair of glands were distributed onto eight sectors of a
filter paper moistened with Krebs—Ringer bicarbonate glu-
cose medium. The adrenal glands of all the rats in the
assay groups were handled similarly so that each sector on
the filter paper contained one quarter of an adrenal from
each of the animals in the group. During all these prepara-
tory steps the Petri dishes were kept over an ice bath.

The quartered adrenal segments were weighed on a micro-
torsion balance to the nearest 0.1 mg and placed in 25 ml
breakers containing 3.0 ml of Krebs—Ringer bicarbonate glu-
cose medium. The flasks were placed in a Dubnoff metabolic

incubator, flushed with angasphase of 95% oxygen - 5% carbon

21

dioxide and incubated for 45 minutes. The Dubnoff apparatus
was operated at 37°C, the agitating cam was set to operate
at 120 agitations per minute and the 95% oxygen - 5% carbon
dioxide flow was adjusted to 400 to 450 cc per minute.
During this preincubation period the assay samples were pre—
pared. Solutions of protein fractions were made up to a
constant volume with Krebs—Ringer bicarbonate glucose
medium. Plasma was assayed directly. After preincubation
the flasks were removed from the Dubnoff apparatus and the
preincubation media removed by aspiration and discarded.
Duplicate flasks containing 5 mls of assay material were
arranged in the following manner. Flasks No l and No 5
contained 5 mls of Krebs-Ringer bicarbonate glucose medium
only and served as controls for the assay. Flasks No 2 and
No 6 contained 5 mls of the protein solution being assayed
and ACTH standards of 2 and 4 mU of oxycellulose purified
bovine ACTH (Armour Lot 216—177-3). Flasks No 3 and No 7
and No 4 and No 8 were made up with 5 m1 duplicates of the
plasma-protein solutions being assayed. Appropriate blanks
of assay medium and Krebs-Ringer bicarbonate glucose me-
dium were also incubated with each assay. The flasks were
then returned to the Dubnoff incubator, flushed with 95%

oxygen - 5% carbon dioxide and incubated for 90 minutes

22

under the same environmental conditions as during preincu—
bation.

After incubation the flasks were removed and two 2.0
m1 aliquots of the incubation medium were pipetted into 12
m1 glass stoppered centrifuge tubes containing 4.0 mls of
methylene chloride purified according to the following
scheme: methylene chloride was redistilled, washed with an
equal volume of distilled water, dried by passing through
anhydrous sodium sulfate, and decolorized by agitating 20
minutes with charcoal. The solvent was stored over sodium
hydroxide pellets overnight, redistilled into foil-covered
bottles and stored under refrigeration. Prepared in this
manner the solvent remains usable after long periods of
storage.

The medium was extracted with methylene chloride by
shaking slowly for 2 minutes. After centrifugation, in a
clinical centrifuge for 5 minutes, the aqueous phase was
removed and discarded. The solvent was washed with 0.5 ml
of ice cold 0.1 N sodium hydroxide by vigorous shaking for
30 secoan. After 3 minutes centrifugation, the aqueous
phase was discarded and 3.0 mls of the methylene chloride
were pipetted into clean test tubes. Care was taken not to

transfer any of the aqueous phase into the clean tubes.

23

The methylene chloride was evaporated under a slow stream
of air in a water bath at 40°C. The sides of the test
tubes were washed down once with fresh methylene chloride
to concentrate the residue in the bottom of the tubes.
Glucocorticoid production of the incubated rat adrenal
gland quarters was quantitated by the Blue Tetrazolium
method of Elliott el al. (1954). The methylene chloride
residue was dissolved in 0.2 ml absolute ethanol (Gold Seal
Alcohol USP). To aid in the dissoIhtion of the residue the
tubes were dipped several times into flasks of boiling
water, ice cold water, and water at room temperature. After
dissolution, 0.15 ml of blue tetrazolium solution (30 mgs
blue tetrazolium in 20 mls absolute ethanol) was carefully
pipetted into the test tubes. Following mixing, 0.15 ml
of tetramethyl ammonium hydroxide solution (0.3 ml tetrae
methyl ammonium hydroxide and 9.7 mls absolute ethanol) was
added. The tubes were mixed, stoppered and placed in a
water bath at 30°C for 20 minutes to complete the color
development. At the end of 20 minutes, 1.5 mls absolute
ethanol was added, the tubes were agitated and their op—
tical densities read on a Coleman Junior Spectrophotometer

at 510 mu. Total alpha-ketol content was quantitated by

24

comparison with optical densities of known amounts of cor—
ticosterone carried through the same assay procedure.

Adrenocorticotropic activity was expressed in terms of
increased corticosterone production per 100 mgs incubated
rat adrenal tissue. Adrenocorticotropic activity was
quantitated by comparison of the increased corticosterone
production by the plasma protein fraction to the increased
corticosterone production in the standard ACTH incubation
flasks.

The methods utilized for plasma glucocorticoid analysis
were those used by Romanoff (1961) for plasma steroid hor-
mone analysis. Twenty mls of fresh or freshly thawed plasma
and about 0.01 uc of 4-C14-hydrocortisone (less than .05 pg
hydrocortisone) were pipetted into 250 ml erlenmeyer flasks.
The plasma was extracted with 10 volumes of acetone:
ethanol (1:1) with the aid of a magnetic stirrer. The sol—
vent protein mixture was warmed in a water bath at 50°C for
6 minutes after which the precipitated protein was separated
from the solvents by filtering through Whatman No 541 filter
paper with the aid of vacuum. The acetone:ethanol extract
was taken to dryness on a flash evaporator under reduced
pressure. The residue was dissolved in 7 mls of 5% olive

oil in n-hexane and transferred to a silica gel defatting

25

column (2 grams Davison silica gel, mesh size 100—200).

The column was made up with a n—hexane slurry and condi-
tioned by passing 15 mls n-hexane through it before the
extract was added. The residue from the plasma extract

was washed on the column with 4 mls n—hexane. The lipids
and other low polar hydrocarbons were eluted from the
column with 35 mls of 5% ether:benzene which were discarded.
The corticosteroids were then eluted from the column with
30 mls of 4% ethanol:ethy1 acetate. The ethanol:ethyl
acetate was taken to dryness under reduced pressure and

the residue dissolved in 60 mls n-hexane which was parti-
tioned 4 times with 20 mls 70% methanol. Most of the re-
maining lipids (which were not removed from the column) and
low polar C19 and C21 steroids remain in the n-hexane phase.
The 70% methanol phases containing the glucocorticoids were
combined and reduced to dryness on a flash evaporator. The
residue was next dissolved in 40 mls of cold methylene
chloride and washed with 10 mls of cold 0.1 N sodium hy-
droxide to remove estrogens and other acidic compounds.

The methylene chloride was evaporated to dryness under a
slow stream of air in a water bath at 40°C. The residue
was then concentrated in a conical centrifuge tube and

transferred to Whatman No 1 filter paper prepared as follows:

26

(Eighteen cm strips of Whatman No 1 filter paper were
divided into six 3 cm lanes by removing about a 2 to 3 mm
strip from between the lanes. Twenty strips cut in this
manner were washed by descending chromatography in a glass
chromatography tank with the following solvents: 1000 mls
ethanolic ammonia (60 ml conc. ammonium hydroxide); 500 mls
water; 1000 mls 2 N acetic acid:water; 500 mls 50% ethanol:
water; and 1000 mls ether. The washed strips were then
dried and stored in aluminum foil until used). The paper
strips with the separate lanes containing the plasma ex-
tract residues, blank strips and strips containing hydro-
cortisone and corticosterone standards were placed in glass
chromatography tanks previously prepared with 75% methanol:
toluene and allowed to equilibrate for about 2 hours. The
chromatography tanks were contained in a chromatography
cabinet maintained at an environmental temperature of 37°C.
After equilibration, the toluene mobile phase was added and
allowed to flow over the papers by the descending chromato-
graphic technique for 2 to 3 hours. At the end of chromato—
graphy, the papers were removed and allowed to dry after
marking the position of the solvent front. The hydrocorti-
sone and corticosterone areas on the standard strips were

located by dipping the dried strips into a blue tetrazolium

27

solution (2:1 solution of 0.2 gm blue tetrazolium in 15 mls
methanol diluted to 100 mls with water and a 10% solution
of sodium hydroxide in methanolzwater (4:1) ). The hydro-
cortisone and corticosterone areas of the standard strips
were used to locate the respective zones on the plasma ex-
tract strips. The 3 to 5 cm portions of the paper con-
taining the hormones were eluted with 6 mls absolute ethanol
by allowing the ethanol to flow over the strips into clean
test tubes from 22 gauge needles mounted on syringe barrels.
The ethanol extract was reduced to dryness under a slow
stream of air in a water bath at 40°C. The extract was
washed to the bottom of the test tubes by washing the sides
of the tubes with additional absolute ethanol.
Corticosterone was quantitated by the blue tetrazolium
method of Elliott (1954) using the same procedure as was
used in the adrenocorticotropic assay. Hydrocortisone was
quantitated by dissolving the residue in 2 mls of phenyl-
hydrazine hydrochloride ethanolic sulfuric acid solution
(65 mgs phenylhydrazine hydrochloride dissolved in 100 mls
62% sulfuric acidzabsolute ethanol (2:1) ). The solution
was allowed to stand overnight at room temperature and the
optical densities were read at 410 mu in a Colemen Junior

Spectrophotometer. Amounts of hormone were quantitated by

28

comparison with the optical densities of known amounts of
hydrocortisone and corticosterone carried through the assay
procedures. Just before colorimetric assay of hydrocorti-
sone, the residue was dissolved in absolute ethanol and
1/20 of its volume was planchetted and its radioactivity
counted on a Nuclear Measurements Corporation gas-flow
proportional counter. Recovery of hydrocortisone radio-
activity was used to correct for loss of both hydrocorti-
sone and corticosterone in the technique.

Solvents used in the glucocorticoid assay procedure

were prepared as follows:

toluene -- redistilled, washed with concentrated sul-
furic acid until the acid no longer becomes discolored,
washed with water, dried by passing through anhydrous

sodium sulfate and redistilled again.

methanol and ethanol used for plasma extraction -—
redistilled from solution containing 0.5 gm 2-4-di—
nitrophenylhydrazine and 10 mls concentrated sulfuric

acid per 2 liters.

absolute ethanol -- Gold Seal Alcohol USP (no prepara—

tion) ether -— Merck USP (no preparation)

29

N—hexane, ethyl acetate, benzene, acetone and methylene
chloride -- freshly redistilled. (Except where pre—

viously noted.)

RESULTS

Plasma Adrenocorticotropic Activity

Attempts to assay adrenocorticotropic activity from 5
mls of plasma extracted with petroleum ether to remove
plasma corticosteroids were not satisfactory. The in—
crease in corticosterone production from the incubated rat
adrenal sections was not of sufficient magnitude to give
a clear increase over corticosterone secretion from the
adrenal quarters incubated with Krebs-Ringer bicarbonate
glucose medium alone.

The first attempt to increase the concentration of
adrenocorticotropin in the assay solution was by lypholi-
zation of plasma and assay of a 5 ml solution containing
10 milliliter equivalents of lypholized plasma dissolved
in Krebs—Ringer bicarbonate glucose medium. Again the in—
crease in corticosterone secretion from the incubated rat
adrenal quarters was not sufficient for reliable assay.

The next attempt to increase adrenocorticotropic activ-
ity in the assay medium was by cold ethanol fractionation
of plasma. Plasma adrenocorticotropic activity was con-
sistently found in fractions II + III and IV - I. Since

fraction II + III contained considerable amounts of protein

30

31

(average about 4.1 grams/100 mls plasma), and increased
protein concentrations reduce the effectiveness of ACTH,
this fraction was subjected to stepwise addition of its

6 to 25% ethanol concentration in an attempt to eliminate
some of the protein from the assay medium without losing
adrenocorticotropic activity. After stepwise separation,
the 12 to 25 % ethanol portion of fraction II + III con—
tained the adrenocorticotropic activity and the protein
concentration of the assay fraction was reduced from 4.1
to 2.1 grams/100 mls plasma. Fraction IV - I contained

a protein concentration averaging about 0.8 grams/100 mls
plasma. Thirty to fifty milliliter equivalents of the 12
to 25% ethanol portion of fraction II + III and fraction
IV + I were used for assay of the plasma adrenocorticotropic
activities presented in this study.

Since increased amounts of protein in the assay sample
depressed the dose-response relationship expected from rat
adrenal quaters incubated with exogenous ACTH, the ACTH
standards for each assay were incubated in a portion of
the protein solutions which were being assayed.

Corticosterone secretion by the assay rat adrenal tis-
sue during ACTH stimulation exhibited a linear relationship

for small amounts of ACTH (Figure 1). However, since the

32

increased corticosterone secretion stimulated by exogenous
ACTH varied significantly between assays, it was necessary
to include ACTH standards with each assay to accurately
quantitate the adrenocorticotropic activity in the protein
fraction being assayed.

Levels of plasma adrenocorticotropic activity from the
bulbs utilized in this study are tabulated in Table 1.
Average adrenocorticotropic activities from all bleedings
for each bull are plotted in Figure 2. With the exception
of instances when trouble was encountered in obtaining
blood samples from obviously stressed animals, plasma adre-
nocorticotropic activity was low (below 2.0 mU/100 mls
plasma) in all bulls under 2 years of age. Adrenocortico-
trophic activity could not be detected in several blood
samples. On the other hand, blood samples from all bulls
above 3 years of age contained substantial amounts of
adrenocorticotropic activity. The highest value recorded
was 15.4 mU/100 mls plasma from Iowana, the 14 year old
bull. It is apparent that (Figure 2) plasma adrenocortico-
tropic activity is higher in the older bulls. Data from
the 6 year old Angus bull (6.9 mU adrenocorticotropic
activity/100 mls plasma) and No 118-A, the one month old

Hereford bull (1.0 mU adrenocorticotropic activity/100 mls

33

plasma at one month of age) were in agreement with data
obtained from dairy bulls. Average levels of adrenocortico-
tropic activity from all bulls beyond 3 years of age were
significantly higher (P < 0.01) than the plasma adrenocor-
ticotropic activities from bulls under 2 years of age. The
formula for the best straight line plotted from these data
(Figure 2) was Y' = 1.65 + 0.57x with a standard deviation
of 12.0.

Adrenocorticotropic activity from No 118-A at 3 days
of age (4.7 mU/lOO mls plasma) was significantly higher than
the activities from bulls 1 month to 2 years of age.

Although the accumulated values of plasma adrenocortico-
tropic activity from cows were not as voluminous or con-
clusive as the data from bulls, the activity from the 7
year old Holstein Friesian cow (9.6 mU/lOO mls plasma) and
the 3 year old Hereford cow (2.3 mU/lOO mls plasma) were
certainly within expected ranges if these data were in-
cluded with those accumulated from work among the bulls.

Difficulties were encountered in collecting blood sam-
ples from the Hereford bull, No 209, (Table 1) both before
and after castration. These difficulties must be con-
sidered in evaluating the rather high levels of plasma

adrenocorticotropic activity from both samples (9.0 and 12.5

34

mU/100 mls plasma).

Although both fractions II + III and IV — I both con-
tained substantial amounts of adrenocorticotropic activity
in most plasma samples, it was apparent (Table 1) that the
relative amounts of adrenocorticotropic activity in the
respective fractions varied considerably from sample to
sample. Since plasma adrenocorticotropic activity in hu-
man plasma is associated with both the gamma globulin and
beta lipoprotein fractions and these proteins are distri-
buted in both fractions II + III and IV — I in somewhat
variable quantities, it is not surprising that the adre-
nocorticotropic activities of the respective fractions
exhibit the observed alterations. These differences prob—
ably reflect slight alterations in fractionation procedure
rather than real changes in protein assocation of adreno-
corticotropin in plasma. Portions of all the protein frac-
tions assayed for adrenocorticotropic activity were ex-
tracted to determine whether glucocorticoid hormones were
associated with the protein. No glucocorticoid hormones
were detected from any of the protein samples assayed in-
dicating the plasma glucocorticoids remained in the super—

natent after precipitation of fraction IV - I.

 

35

Plasma Glucocorticoids

Recoveries of 4—C14—cortisol added to bull plasma
ranged from 40 to 70%. Recoveries were substantially in-
creased by the addition of 4% ethanol to the ethyl acetate
used to elute the corticosteroids from the silica gel de-
fatting column.

Plasma glucocorticoid values from the bulls are pre-
sented in tabular form in Table 3 and their averages are
plotted in graphic form in Figure 3. Glucocorticoid values
from the cows are included in Table 2. The plasma cortisol
level from the bull calves No 118—A at 3 days of age (4.4
ug%) and the level from 703—B at 2 months of age (2.5 ug%)
were considerably lower than average cortisol values from
the older bulls. Above 2 months of age, plasma cortisol
levels from the bulls ranged from 6 to 9 ug/lOO mls plasma and
did not demonstrate a consistent alteration with increasing
age or differences in plasma adrenocorticotropic activity.
Average corticosterone values, on the other hand, ranged
from 6.6 to 13.9 ug/lOO mls plasma and the levels were not
as consistent for individual blood samples as were the
cortisol data. Although there was an indication of de—
creased levels of corticosterone in the plasma from the

older bulls, this difference was not significant. Levels

36

of glucocorticoids from the cow plasma (Table 2) were com-

parable to values for plasma glucocorticoids in the bulls.

Hematology:

Table 4 contains the average for the hematocrit, total
red blood cell count and total and differential leucocyte
count from each animal in the study. These data are pre—
sented graphically in Figures 4, 5, 6 and 7.

Red blood cell counts ranged from 11.64 x 106 cells
per cubic millimeter in No68543 the 4 month old bull, to
6.84 x 106 cells per cubic millimeter in Iowana, the 14
year old bull (Figure 4). Bulls below 3 years of age had
increased numbers of red blood cells when compared to the
older animals.

Total leucocyte counts (Figure 5) also decreased with
age and ranged from 14,550 cells per cubic millimeter in
No 703-B, a two month old bull calf, to 3,150 cells per
cubic millimeter in Iowana, the 14 year old bull. Total
red blood cells and leucocyte counts from the cows were
not different from the data collected from the bulls.

Uncorrected hematocrit values from the various aged
bulls ranged from 38 to 51% and did not exhibit any con-

sistent alterations among the various aged animals.

37

Percent blood eosinophils (Figure 6) from the bulls
ranged from 0.0 to 11.3% of total blood leucocytes. Al-
though changes in percent eosinophils were not directly
related to aging, it was apparent the lower values pre-
dominently represent younger bulls while the higher per-
centages represent the older animals. Percent blood
eosinophils from the cows (3% from No 118 and 5% from No
575) and the Hereford steer (1% for No 209) were not dif-
ferent from the data from bulls.

Figure 7 illustrates the average percent lymphocyte
and neutrophil counts in the various aged bulls. Among
bulls from 1 month to 3 years of age, blood neutrophils
ranged from 15 to 39% of the total leucocytes, and in all
instances were substantially lower than the percentages
of blood lymphocytes, which ranged from 49 to 77% of the
blood leucocytes. The ratios of lymphocytes to neutrophils
were not consistent in bulls beyond 3 years of age. Seven
of the bulls beyond 3 years of age had greater percentages
of neutrophils than lymphocytes and 7 bulls had higher
percentages of lymphocytes than neutrophils. The average
percentage of blood neutrophils was 44% of the total blood
leucocytes and the average percentage of blood lymphocytes

was 46% of the total blood leucocytes, in the bulls 3 years

38

of age and older. From these data there were no apparent
trends in percent concentration of these two cell types in
the older bulls during aging.

Hematological alterations noted in No 118-A, the new—
born Hereford bull, were very marked in the blood sample
taken at 1 month of age compared to the sample taken at 3
days of age. At 3 days of age the packed cell volume was
26%» the lowest hematocrit recorded. At 1 month of age
the hematocrit was 39%, a value within the range recorded
from the older cattle. Total red blood cell count in-
creased from 6.86 x 106 cells per cubic millimeter, from
the 3 day old sample (which again was one of the lowest
values recorded from any of the bulls), to 10.16 x 106 cells
per cubic millimeter at 1 month of age, and the total
leucocyte count increased from 5,111 to 12,700 cells per
cubic millimeter of blood during the same time span. The
ratio between lymphocytes and neutrophils was also markedly
altered during the first month after birth. At 3 days of
age the lymphocytes made up only 14% of the total leuco-
cytes and by 1 month of age they had increased to 49% of
the total white blood cells. The percentage of blood
lymphocytes in the 3 day old animal was far lower than

data recorded from any aged animal. On the other hand,

39

neutrophil percentage decreased from 77 to 39% of the total
blood leucocytes during the first month of post-natal life.

One month after castration, a 5 month old Hereford bull,
No 209, exhibited several hematological changes. The total
blood leucocyte count increased from 8,700 to 13,200 cells
per cubic millimeter. The percent concentrations of blood
lymphocytes and neutrophils changed from 53% lymphocytes
and 40%)neutrophils before castration to 44% lymphocytes
and 50%.neutrophils following castration.

The hematological values from the cows did not exhibit
significant differences when compared to comparable aged

bulls.

Plasma Proteins

Total plasma proteins (Table 5 and Figure 8) ranged
from 5.1 grams percent from No 118-A, the 3 day old bull
calf, to 8.4 grams percent in the older bulls. Plasma
protein increased with increasing age in bulls up to 3
years of age, but did not show consistent alterations be-
tween the various aged animals beyond 3 years of age. 0n
the other hand, the percentages of plasma gamma globulins
and albumins were different in bulls beyond 3 years of age

(Table 6 and Figure 9). Plasma albumin ranged from 34.8%

40

of the total plasma protein from Iowana, the 14 year old
bull to 59.5%.from Climax, the 3 year old bull. As illustrated
in Figure 9, plasma albumin tended to decrease with increasing
age in the bulls. However, plasma gamma globulins, in
general, increased with age of the bulls. Gamma globulin
concentrations ranged from 18.0% of the total plasma pro-
tein from Climax to 46% of the total plasma protein from
Madcap, a 13 year old bull. The formulas for the best
straight lines for gamma globulins and albumin from these
data were, Y' = 20.5 + 1.17x with a standard deviation of
5.3 for plasma gamma globulins and Y' = 57.35 - 1.23x with
a standard deviation of 5.9 for plasma albumin.

After castration total plasma proteins from No 209 in-

creased from 6.4 to 7.9 grams per 100 mls plasma.

41

TAB

PLASMA ADRENOCORTICOTR

 

 

mU/lOO m1
Name Age lst sample 2nd
yr. mo. II+III IV-I Total II+III
Iowana 14-0 3.0 2.4 15.4 0.4
Beauty 13-1 2.4 3.8 6.2 4.8
Madcap 12-10 3.2 6.4 9.6 5.6
Bell 12-6 3.0 0.0 3.0 8.8
Vic 11-6 7.8 4.8 12.6 7.4
Chieftain 10-9 2.8 2.6 5.4 0.8
Symbol 10-5 0.6 9.2 9.8 7.4
Duke 9-6 3.6 6.8 10.4 3.4
Birch 9-4 0.2 3.4 3.6 6.1
Ben 9-4 6.4 4.8 11.2 5.6
Roburke 6-2 1.6 4.2 5.8 3.9
Pure Gold 5-0 2.2 6.6 8.8 4.6
Explorer 4-7 2.0 5.6 7.6 5.0
Climax 2-10 0.0 4.0 4.0 0.0
Imperial 2-9 3.0 3.8 6.8 3.0
685-A 1-4 1.0 2.8 3.8 0.1
643 1-3 0.6 0.0 0.6 0.7
685-B 0—4 3.6 7.0 10.6* 0.0
703-A 0—2 0.0 1.4 1.4 0.0
703-B 0-2 0.0 0.0 0.0 0.0
Angus Bull 6-0 1 2 5.7 6 9
118—A 3 da. 3.7 1.0 4.7
0-1 1.0 0.0 1.0
209** 0.5 6 3 2.7 9.0* 6.7

 

*Difficulty encountered in collecting the blood sample
**Castrated 1 mo. before second sample

42
II+III IV—I Total Average

Total

IV-I

 

 

OPIC ACTIVITY IN BULLS
5 plasma
sample

LE

69188931331352807170 9
”.5969387596762620100 6

*
1 7 76 97
8 7 31 20
4. 9 0.0 .1.3
11 0 0.0 1.0
7. 8 ./,o 9.4
6 ,6 2.1. 1.0

85556447043710717400
75808 3776581600100

2.
7

47972603984110700400

7 0.2.1.1 nunv111.111.1113nvn.1.n.0

l
O

12.5*

5.8

 

43

TABLE 2

PLASMA ADRENOCORTICOTROPIC ACTIVITY AND GLUCOCORTICOID

HORMONE LEVELS IN COWS

 

mU/100 mls plasma ug/lOO mls plasma

 

A
Number rgemo adrenocorticotrophin glucocorticoids
Y ' ° II+III IV-I Total Cmp B Cmp F
118 3-0 1.4 7.9 8.4
600 7-0
575 9-0 . . . 13.4 8.6

 

44

TAB

PLASMA GLUCOCORT
(Hg/100 mls

 

 

N Age lst sample 2n
ame

yr. mo. Cmp B Cmp F Cmp B
Iowana 14-0 14.0 6.3 6.8
Beauty 13-1 11.0 8.3 7.4
Madcap 12-10 8.0 6.6 10.8
Bell 12-6 12.5 4.9 9.4
Vic 11-6 10.3 5.4 10.5
Chieftain 10—9 13.5 6.2 11.5
Symbol 10-5 11.2 6.5 11.4
Duke 946 12.3 5.0 10.3
Birch 994 10.8 7.4 7.8
Ben 9-4 6.2 5.6 6.9
Roburka 6-2 15.6 9.0 12.1
Pure Gold 5-0 11.5 7.8 14.6
Explorer 4-7 7.0 6.9 4.6
Climax 2-10 14.7 6.9 7.4
Imperial 2-9 12.4 7.0 14.5
685-A 1-4 13.7 9.3 8.9
643 1-3 10.0 5.4 5.7
685-B 0—4 6.0 6.3
703-A 0—2 15.6 8.2 5.8
703-B 0-2 11.2 2.5 4.5
Angus Bull 6—0 8.4 6.1
118-A da. 8.5 4.4
118-A 0-1 11.0 8.0
209 after castration 7.9 10.8

 

45

LE

ICOIDS IN BULLS

. plasma)

 

Averages

Cmp B

3rd sample

Cmp B

d sample

Cmp F

Cmp F

Cmp F

 

9285

7776

4.2.4.0.
0.9.9.1

4191

see.
9688

11.4
12.5

13.5

qaqoavq,1_4

196338

12.0

13.5
13.5

4315418

69nu.7.—/.6r0.

.8714595
.6777664

12.8
3
1
7
11.0
0
7

16.3
15.7

9484255
..D.84955r0.

8.4

 

TABL

HEMATOLOGY IN

 

 

Average
Age RBC3
2

Name yr. mo. HCt- WBC x 106
Iowana 14-0 3,150 6.84
Beauty 13-1 42% 4,810 8.54
Madcap 12—10 5,700 9.38
Bell 12-6 5,750 8.21
Vic 11—6 41% 4,680 7-34
Chieftain 10-9 4,800 8.04
Symbol 10-5 50% 4,800 9.20
Duke 9-6 43% 6,720 6.98
Birch 9-4 38% 4,225 8.01
Ben 9-4 39% 6,150 7.02
Roburke 6-2 41% 6,270 7.55
Pure Gold 5-0 48% 6,400 7.86
Explorer 4-7 6,070 10.35
Climax 2—10 8,850 10.05
Imperial 2-9 6,225 9.20
685—A 1-4 51% 7,720 10-88
643 1—3 40% 8,850 7.58
685-B 0-4 10,775 11.64
703-A 0-2 46% 13,200 10.96
703-B 0—2 39% 14,550 11.53
Angus Bull 6-0 43% 7,200 9.50
118-A 3 da. 26% 5,111 6.86
0-1 39% 12,700 10.32
209 0—5 47% 8,700 9.63
(after castration) 13,200 10.16

Cows
118 3-0 45% 5,900 8.56
575 9-0 9,200 7.05

 

lUncorrected packed cell volume

2Total leucocyte count per cubic millimeter blood

3Total red blood cell count per cubic millimeter blood

47

E 4

BULLS AND COWS

 

 

 

Values
Leucocyte Differential Count
Mono. Lymph. Neut. Eosin. Baso.
5 42 47 6 0
4 42 52 2 0
4 34 60 2 0
7 48 41 4 0
1 59 24 5 0
6 51 40 3 l
6 52 38 4 0
1 40 47 11 l
3 50 43 5 0
7 38 41 9 0
3 50 2) 4 0
8 48 38 7 l
5 40 48 5 l
5 61 31 3 0
5 68 34 3 0
4 72 21 3 0
5 73 21 2 l
5 77 15 0 l
5 66 28 l 1
4 60 35 1 1
3 28 69 0 0
8 14 77 0 l
9 49 39 1 2
5 53 40 2 0
5 44 50 0 1
4 57 35 3 l
2 63 30 0

 

48

TABLE 5

TOTAL PLASMA PROTEINS IN BULLS AND COWS

 

 

grams%
Age lst 2nd 3rd
Name Average
yr. mo. sample sample sample

Iowana 14-0 7.6 7.4 7.5
Beauty 13-1 8.2 7.7 8.0
Madcap 12-10 8.6 8.2 8.4
Bell 12-6 8.1 8.2 8.1
Vic 11-6 8.2 8.7 8.3 8.4
Chieftain 10-9 7.9 7.7 7.8
Symbol 10-5 8.5 8.4 8.3 8.4
Duke 9-6 7.2 7.7 7.5
Birch 9-4 6.9 7.6 7.2
Ben 9-4 8.2 8.1 8.1
Roburke 6-2 7.6 8.5 8.0
Pure Gold 5-0 7.4 7.9 7.6
Explorer 4—7 7.7 7.9 7.9
Climax 2-10 7.4 7.4 7.6 7.5
Imperial 2-9 7.2 8.0 7.6
685-A 1-4 6.8 6.4 6.6
643 1-3 6.0 6.6 6.9 6.5
685-B 0-4 5.7 6.0 6.2 6.0
703-A 0-2 6.4 6.2 6.3
703-B 0-2 5.7 6.2 6.0
Angus Bull 6-0 7.7 7.7
118-A 3 day 5.1
118-A 0-1 6.2 6.2
209 0-5 6.4

after castration 7 9
Cows.
118 3-0 7.8
600 7-0 7.5
575 9-0 8.1

49

TABLE

6

PLASMA PROTEIN COMPONENTS BY ELECTROPHORESISQ/

%.Of total plasma protein

 

 

 

 

Name Age Globulins .

yr. mo. Gamma Beta Alpha Ablumin
Iowana 14-0 36.1 15.7. 13.4 34.8
Beauty 13-1 32.1 15.2 10.3 42.4
Madcap 12-10 46.0 11.0 7.0 36.0
Bell 12-6 33.7 10.9 9.7 45.7
Vic 11-6 35.0 15.0 9.6 40.4
Chieftain 10-9 30.8 10.4 10.0 48.8
Symbol 10-5 38.0 12.4 7.8 41.8
Duke 9-6 26.3 13.2 8.9 51.6
Birch 9-4 24.5 19.7 9.1 46.7
Ben 9-4 31.4 11.4 9.3 47.9
Roburke 6-2 26.4 11.1 7.4 55.1
Pure Gold 5-0 34.3 9.0 9.7 47.0
Explorer 4-7 22.0 12.0 9.0 57.0
Climax 2-10 18.0 14.5 8.0 59.5
Imperial 2—9 30.4 16.0 14.4 39.2
11/

Spinco, Durrum arrangement, veronal buffer, pH 8.6

TISSUE

fl, cameos-mos: seesaw/loo m9 spasm

 

nouns l
RESPONSE OF' INCUBATED RAT ADRENAL TISSUE

 

TO ACTH
/
l/
I
’z
z”
I
[’0'
”
’
’ze’
/
’z
i .3 I; T
mu ACTH

noun: a
PLASMA ACTH WITH AGING

 

 

 

501.

 

 

FIGURE 3
PLASMA CORTICOIDS WITH AGING
no.0 I o . coa'nsm.
. O ICORTICOSTERONE
I40 * O
i e
. . o
e
3 Ian . '
O O 0 .
_ e
I 00.0 .
8 1, 3 ° .
\ .9 O o O O O
. g a O O o
x . .
00 ° °
‘5 i 5 S 3 5 5 9 8 6 .6 {u (2 Ta :2

AGE (mas)

53

neon: 4
no RED BLOOD CELLS WITH AGING
0°
ll.O-o O
p O
no.0) °

RED aLooo ccusxuo‘
. O
a '.
O
O

.“
e
U

 

 

57

 

noun 5
“000 TOTAL BLOOD LEWOCYTES WITH AOIW
e
asses
guess-
noses-
? .....
g "00- T o e
{I 0000- ° 90 °
.O- OO
0- «00- on e
‘000 l l l I I j l I l
u s s 4 s s 1 n It Is

 

515’

 

rm 6
BLOOD EOSINOPHILS
WITH AGING
IS;
’ O
8IO’ O
3 P o
D. O
5- e o o o o 0 o
L" ' . °°
° T i» 3 Z 3 f i 3 ~ 3 :6 {I {2 n3 .2

AGE (YEARS) ‘

°/o WBC

70

20

 

 

56..

 

rm: 1
BLOOD LYMPHOCYTES AND NEUTROPHILS
' WITH AGING
o-uumoevus
'e o-uumumuu
a.
I . o
O O o
e e
o O .O .
8 .
' o 0. 0°
0 o
o O
B .
O
. oo
0
“;£‘.‘ ' ‘ ‘ .3 .1 (.3.

C 7 G O
AGE(YEARS)

 

rm 6
TOTAL PLASMA PROTEINS WITH AGING
”p
o e e
8.0: 0 0 e e o 0
°0 ° 0 o 0
" O
5. "°’
8
GD 0

A A L A a A A A J

I2 3 4 3 e 7 e 9 IO M I2ISI4
AGE(YEARS)

 

 

8‘8
8 "I" 9
G ‘ILPUMI
co
“~‘° 0 Owing..."
5 5° T‘~4’£873 .MW
‘1' ‘0 o T‘z‘fi’x .
2 ‘\J
l. e ‘ e
‘
# 30 . g \"
I- . e 0 ‘K
B YIZGS-HJ'IX ‘
to --------- f----"'O--.-"-'.-':
3 . ' ,. ‘
0. IO
0 I ,
2 a .
‘ ° . 5 .
e - .
AGE (YEARS) '0 II I: “we

 

DISCUSSION

Clear indications of histologically detectable degen-
erative changes in the adrenal glands of many species of
animals with increasing age have already been discussed.
There is substantial indirect evidence implicating adreno-
cortical insufficiency in cattle. Cupps et a1. (1954 and
1956) and Saarinen and Shaw (1950) reported gross and
histological degenerative changes to be associated with
ketosis in cows and reproductive difficulties in bulls. It
has been clearly established that changes in adrenal phys-
iology are related to these conditions. In another study
Shaw (1947) noted the similarities between the clinical
symptoms of bovine ketosis and Addison's disease in humans.
In the same report Shaw noted a more marked hyperglycemic
effect from injected ACTH in normal cows as compared to
ketotic cows, further evidence that the adrenals of ketotic
cows are not functionally normal. From this information,
it would seem likely that cattle may be more prone to ad-
renal disorders than other mammalian species.

Sayers (1961) indicated that the normal titer of adreno-
corticotropic hormone from normal human subjects was less

than 0.5 mU/100 mls plasma. An extensive study of plasma

59

60

adrenocorticotropic hormone levels from normal subjects and
patients with endocrine disorders was made by Miyake et a1.
(1962) using the corticosterone levels in rat adrenal venous
plasma as a bioassay for ACTH activity (Lipscomb and Nelson,
1959). These workers could not detect adrenocorticotropic
hormone in the plasma of normal subjects, patients with
congenital adrenal hyperplasia, or Cushing's syndrome. 0n
the other hand, patients suffering from Addison's disease
had high titers of plasma adrenocorticotropic activity
(greater than 40 mU/100 mls plasma).

The adrenal glands of senile humans were not as re-
sponsive to exogenous ACTH as the adrenals from younger
subjects (Moncloa et a1., 1963). The loss of diurnal varia—
tions of plasma adrenal corticoids in elderly human patients
(Pincus et a1., 1954) also indicates that the adrenal glands
from these aged subjects may require increased amounts of
plasma adrenocorticotropic hormone to achieve normal phys-
iological secretory rates Of adrenal corticoids.

Attempts to estimate adrenocorticotropic activity in
bovine plasma are to this writer's knowledge original to
this laboratOry. Nellor (1958a) reported qualitative de-
tection of gonadotropic hormone activities and quantitative

detection of thyrotropic, growth promoting and

61

adrenocorticotropic activities in the plasma of young and
aged bulls. Plasma adrenocorticotropic activity was esti-
mated by induced lipid redistribution in the hypophysecto-
mized rat adrenal gland (Simpson et a1., 1943). Plasma
adrenocorticotropic activities estimated by this method of
assay (Nellor, 1958b) ranged from 1.0 to 56.0 IU ACTH ac-
tivity/100 mls plasma in the young to aged animals. A sub-
sequent stduy concerned with the separation Of growth pro-
moting and adrenocorticotropic activities in the plasma of
a limited number of bulls (Sutton, 1960) supported the
concept of increased plasma adrenocorticotropic activity
with aging. Adrenocorticotropic activities in this study
as estimated by Sayers (1948) adrenal ascorbic acid de-
pletion in hypophysectomized rats, increased from less
than 0.02 IU in a 2 year old bull to from 0.03 to 0.08 IU
ACTH activity/100 mls plasma in aged bulls.

The results of the present study, utilizing ig_yi££g_
corticosteroid production by the rat adrenal as an esti-
mate of adrenocorticotropic activities confirm the concept
of increased plasma adrenocorticotropic activities among
Older bulls. This increase has now been established by

three methods of estimating adrenocorticotropin.

62

The plasma adrenocorticotropic activities recorded from
non-stressed bulls from 1 month to 2 years of age were com-
parable with the 1ow Levels of activity reported from
normal human subjects. 0n the other hand, the plasma
adrenocorticotropic activities measured in bulls above 3
years of age were substantially higher than the accepted
ranges for adrenocorticotropic activity from normal human
plasma. It is clear that plasma adrenocorticotropic ac-
tivity increases with increasing age in cattle. However,
this increase may not occur in a strictly linear fashion.
The data collected from the limited number of bulls from
3 to 9 years of age indicate that plasma adrenocorticotropic
activity may increase rapidly from 3 to 6 years of age fol-
lowed by a more moderate increase in subsequent years. The
levels of adrenocorticotropic activity present in the plasma
from the Older bulls would be described clinically as Ad-
disonian if they were found in humans.

The unusually high values Of adrenocorticotropic ac-
tivities detected by Nellor (1958b) and Sutton (1960) are
not supported by those reported in this study. It must be
pOinted out that the lipid redistribution assay and the

adrenal ascorbic acid depletion method as employed are

63

semi-quantitative. Both Of these assay techniques are
substantially less sensitive to exogenous ACTH than is the
ig_vitro incubation of adrenal gland tissue method, used
in the present study. The extremely variable dose—response
relationship encountered with the ascorbic acid depletion
assay as employed by Sutton (1960) made an accurate quan-
titation of adrenocorticotropic activity very difficult.
The Saffran and Schally (1955) assay used in this study
is superior to the lipid redistribution and adrenal ascorbic
acid depletion methods in that: the assay is based on ad-
renal hormone production which is accepted as the most
important physiological action of adrenocorticotrOpin; the
response is measured by a chemically determinable and point
which is more easily quantitated; there is minimal animal
preparation necessary for the assay; eight incubation
flasks containing adrenal gland tissue of equal stimulatory
potential are available for each assay which allows each
assay to be standardized by blank and ACTH standard incu-
bations; a linear dose-response curve is Obtained at rela-
tively low levels of ACTH stimulation; and the use Of an
ig_vitro system permits more environmental control during

the assay period.

64

Levels of plasma cortisol recorded in this study were
some what higher than values of l7-hydroxycorticosteroids
in the plasma of cattle reported by Shaw et a1. (1960)
(non-stressed heifers and cows averaged from 3.09 to 4.68
ug 17-OHCS/100 mls plasma) and Robertson and Mixner (1956)
(values from milking, non-pregnant cows averaged 4.58ug
l7-OHCS/RX)mls plasma). However, the higher values recorded
in this study may in part be due to the correction for
hormone loss during the preparative steps before final
quantitation of the hormones by the addition of 4-C14-
cortisol to the plasma in the isotope dilution principle
as an internal standard.

The relative biological importance of cortisol vs. cor-
ticosterone in cattle has not been ascertained. Therefore,
it is important that both of these hormones be quantitated
for an adequate understanding of bovine glucocorticoid
physiology. It is of interest that the relative concen-
trations of corticosterone: cortisol reported in this study
(about 1-1/2 times as much corticosterone as cortisol) are
in agreement with the corticosterone: cortisol secretion

ratio from perfused bovine adrenals (Hechter, 1953) before

ACTH stimulation.

65,

If the unchanged plasma levels of corticosterone and
cortisol with increasing age are considered alone it could
be concluded that adrenal function does not alter as cattle
grow older. However, Gray et a1. (1961) pointed out that
in cases of adrenocortical insufficiency, the concentration
of free glucocorticoids in the plasma depended upon the
extent of destruction of the adrenal cortex. Therefore the
levels Of adrenocortical hormones in plasma from patients
with adrenocortical insufficiency of Addison's disease may
be extremely low or within the normal expected range.
Remnants of adrenal cortex from a patient with adrenal in-
sufficiency may be under continuous maximum ACTH stimulation
and are thus able to maintain near normal levels of plasma
glucocorticoids (Eik-Nes et a1., 1955). However, these
patients are not able to mobilize additional secretory ac-
tivity during times of stress as evidenced by lack of in-
creased glucocorticoid secretion during and after exogenous
ACTH infusion. Under the principle of feedback control of
pituitary-adrenal function, patients suffering from adreno-
cortical insufficiency would require additional amounts of
plasma adrenocorticotrOpic hormone in their attempt to
maintain physiologically normal amounts of circulating ad-

renal cortical hormones. From these data it would appear

66

logical that the increased levels of plasma adrenocortico-
tropic hormone may be required to maintain normal adrenal
cortical secretory activity as cattle grow older.

If the adrenals in the cattle studied in this report
were responding to increased plasma adrenocorticotropic
activity with increased glucocorticoid hormone production,
the increase should have been evident in increased levels
of circulating adrenal corticoids. It is also possible
that glucocorticoid hormone metabolism could increase
simultaneously with increased adrenal synthesis producing
no increase in levels of circulating glucocorticoids. How-
ever, Holcombe (1957) reported no increase in urinary
"reducing corticoids” in Older sheep and cattle Of either
sex. Additional evidence against increased adrenal cortical
secretion in this study was the high level of plasma adreno-
corticotropic activity. With the feedback mechanism of
pituitary-adrenal control functioning normally, the in-
creased plasma glucocorticoids would have been expected to
effect a decrease in the levels of plasma adrenocortico-
tropic hormone similar tO the undetected levels Of ACTH
activities reported from congenital adrenal hyperplasia

and Cushing's syndrome patients (Miyake et a1., 1962), both

67

of which involve high levels of circulating adrenal
glucocorticoids.

There also seemed to be marked differences in the re-
sponse to stress between young and Old bulls in this study.
When trouble was encountered in collecting blood samples
from No 685-B, No 643 and No 209, high levels of plasma
adrenocorticotropic hormone were encountered (10.6, 2.9,
9.0 and 12.5 mU/100 mls plasma). On the other hand when
sampling difficulties were encountered among the older
bulls, there was no apparent increase in plasma adreno-
corticotropic hormone compared to ther samples taken from
the same animals when no stresses from collecting the
samples were involved.

On the basis of this evidence, the physiological al-
teration indicated by the increased levels of plasma ad-
renocorticotropic hormone and normal levels of circulating
glucocorticoid hormones is interpreted to indicate either
an adrenocortical insufficiency in older cattle or they
are not as sensitive to adrenocorticotropic hormone. In
View of the normal plasma glucocorticoid levels this adrenal
insufficiency would be evident only in conditions of in-
creased stress, would occur in disease, emotional or en-

vironmental changes, or a further increase in age. While

68

none of the bulls in this study canlxzclassified as truly
adrenocortically insufficient, it is probable that the
condition would be evident in the terminal stages of this
physiological condition.

The high level of plasma adrenocorticotropic hormone
from NO 118-A at 3 days of age (4.7 mU/100 mls plasma) is
of considerable interest. Leeman (1963) studied the
corticosterone secretion from newborn rats. She found no
significant increase in plasma corticosterone after intra-
venous injection of ACTH in rats under 3 weeks Of age. On
this basis, the high level of plasma adrenocorticotropic
activity from N0 118-A at 3 days of age may indicate the
adrenal gland or the newborn calf is either unable to re-
spond normally to plasma ACTH, the relatively low level of
adrenal glucocorticoids in the plasma of the newborn calf
may not be of sufficient magnitude to trigger the feedback
mechanism controlling pituitary ACTH synthesis and release,
or the pituitary-adrenal regulatory systems of the newborn
calf may not be functionally mature at this age.

A reduction in corticosterone secretion into adrenal
venous blood from castrated male rats was demonstrated by
Kitay (1963). Corticosterone secretion was reduced in

spite of an increase in adrenal gland weights.

69

Administration of testosterone or high levels of ACTH
restored corticosterone secretion rates. Nowell and
Jones (1957) reported decreased pituitary content of ACTH
after castration of rats. They hypothesize that the de-
crease in pituitary content of ACTH reflects increased
secretion of ACTH by the pituitary. Although difficulty
was encountered in collecting blood samples before and
after castration from NO 209, the higher levels of adreno-
corticotropic activity encountered after castration may
be due to adrenal secretory abnormalities as described by
Kitay (1963).

Increased levels of red blood cells present in the
bulls below 3 years of age are consistent with changes in
red blood cell counts with age in cattle reported by
Schalm (1961). Schalm also reported decreased amounts of
all species Of blood leucocytes except eosinophils in
older cattle. Although total blood leucocyte counts did
tend to decrease in the dairy cow from 2 to 14 years of age,
the average value was 6,630 leucocytes per cubic millimeter
of blood which is considerably above the average value of
blood leucocytes from the bulls in this study of similar

age (5,190 leucocytes per cubic millimeter of blood).

70

The low percentage of lymphocytes (14% total
leucocytes) from No 118-A at 3 days of age was consistent
with data reported by Schalm (1961) for newborn calves.
Within the first weeks of life calves reach what are called
"normal" levels of blood lymphocytes and neutrophils
(lymphocytes 60 to 80% and neutrophils 8 to 35% Of the
total blood leucocytes). In contrast to the reduced per-
centages of lymphocytes and increased percentages of
neutrophils from aged bulls in these studies, Schalm re-
ported relatively constant concentrations Of blood lym-
phocytes and slightly reduced percentages of blood neu-
trophils as cows increase in age from 2 to 14 years. Since
these observations by Schalm are in agreement with data
collected from the cows in this study (No 118, the 3 year
old, had 57% lymphocytes and 35% neutrophils, and No 575,
the 9 year old, had 63% lymphocytes and 30% neutrophils),
there is an indication of sex induced variations in the
relative percentages of leucocytes in aged cattle.

The percentage changes in blood neutrophils and lym-
phocytes from NO 209 following castration are of great
interest. Before castration the 5 month old Hereford bull
had 53% lymphocytes and 40% of the blood leucocytes were

neutrophils. However, 1 month after castration, the

71

leucocyte species had been reversed to 44% lymphocytes and
50%.Of the blood leucocytes were neutrophils. This obser-
vation may be correlated with the alterations in percentage
of blood lymphocytes and neutrophils from the Older bulls.
The decrease in testicular humoral function that occurs with
age (Hollander and Hollander, 1958), may be involved in the
decrease in blood lymphocytes and increase in blood neu-
trophils Observed in the older bulls.

Although the increase in blood neutrophils and decrease
in blood lymphocytes could be related to increased adreno-
cortical hormone production (Daugherty and White, 1947),
this is unlikely since these changes were not observed in
like aged cows with similar levels of plasma adrenocortico-
tropic and glucocorticoid hormones and the other evidence
presented in this study implicating adrenal insufficiency
is too encompassing.

The increase in blood eosinophils with aging was not
consistent. However the average eosinophil count from
cattle beyond 3 years of age (5.1% of total blood leuco—
cytes) was substantially higher than the average eosinophil
count from cattle below 3 years Of age (1.6% of the total
blood leucocytes). This increase in blood eosinophils

with age can be interpreted as indicative of decreased

72

adrenocortical function (Hills et a1., 1948).

The reason for the increase in total plasma protein
up to about 3 years of age was not apparent. However, since
plasma protein levels show no further alteration beyond
this age, the attainment Of physical and/or reproductive
maturity may be involved. If this assumption is correct
the increased plasma protein could reflect an equilibrium
in protein anabolism in the mature bull. This hypothesis
of increased total plasma protein reflected from decreased
protein anabolism was supported by the increase in plasma
protein in No 209 from 6.4 grams percent before castration
to 7.9 grams percent after castration.

The apparent changes in percentages of plasma albumin
and gamma globulins in bulls beyond 3 years Of age are
consistent with the hypothesis of adrenal insufficiency
in the older cattle. The increase in gamma globulins and
decrease in albumin percentages are in agreement with ob-
servations from human subjects with adrenal insufficiency
(McCullagh and Lewis, 1960).

There were no alterations in hematocrit, cell counts,
or plasma protein concentrations that would indicate hemo-
concentration with aging, a factor that would effect af-
fective plasma distribution volume of the circulating

hormones.

73

The changes in concentration Of eosinophils, and rel-
ative alterations in percentages of plasma albumins and
gamma globulins are considered to be additional evidence
supporting the blood hormonal data implying adrenocortical
insufficiency in aging cattle.

Cattle may be a near ideal biological subject to study
pituitary-adrenal physiology, especially for those condi-
tions involving adrenocortical insufficiency. Their size
and ease of handling without Obvious stress allow the in-
vestigator to collect large volumes of blood at frequent
intervals, and to conduct experiments not physically

feasible to consider in the common laboratory animals.

S UMMARY

Variations in pituitary—adrenal function in cattle,
in relation to age, were investigated in 26 beef and dairy
cattle ranging in age from 3 days to 14 years by the simul-
taneous biological assay of plasma adrenocorticotropic
hormone and the chemical assay of the glucocorticoid hor-
mones, cortisol and corticosterone.

Plasma to be used for adrenocorticotropic hormone as-
say was fractionated into its major protein components in
order to increase the relative concentration of the adreno-
corticotropic hormone in the protein sample being assayed.
Adrenocorticotropic activity was estimated by the increase
in corticostermone production of rat adrenal gland after
incubation with the protein fractions containing the adreno-
corticotropic activity. Levels of bovine plasma cortisol
and corticosterone were chemically determined after several
purification steps followed by isolation of the hormones
by paper chromatography. The blood humoral data were sup-
plemented by measurement of blood hematocrits, total red
and White cell counts, leucocyte differential counts, total
plasma protein levels, and relative amount of the plasma

protein components following electrophoretic separation.

74

75

Adrenocorticotropic activity in bulls below 2 years of

age was low (average values ranged from 0 to 2 mU ACTH

activity/100 mls plasma) and in many cases undetectable.
Beyond 2 years of age, plasma adrenocorticotropic activity
was significantly increased. Cattle above 3 years of age
had substantial amounts of plasma adrenocorticotropic ac-
tivity (average values ranged from 3.9 to 11.6 mU IKTTH
activity/100 mls plasma).

Levels of plasma cortisol (6 to 9 ug/lOO mls plasma)
and corticosterone (6.6 to 13.9 ug/lOO mls plasma) did not
exhibit any consistent alterations related to the increased
plasma adrenocorticotropic activity.

A newborn calf also exhibited a high level of plasma
adrenocorticotropic activity with normal levels of circu-
lating glucocorticoids.

Although total blood leucocytes decreased with increas-
ing age, the percentages of blood eosinophils were higher
in the older cattle. There were also increased percentages
of blood lymphocytes in bulls beyond 3 years of age. Total
red blood cell counts were higher in blood from bulls up
to 3 years of age but there were no changes in red cell

counts in bulls beyond 3 years of age.

76

Total plasma protein increased with increasing age in
bulls up to 3 years of age. Although total plasma protein
were not different in cattle beyond 3 years of age, the
relative percentages of plasma albumin tended to decrease
while plasma gamma globulins showed a slight increase with
increasing age.

The increased levels of plasma adrenocorticotropic
hormone with increasing age occurring without a simultan-
eous increase in levels of plasma glucocorticoids is in-
terpreted as indicating that cattle undergo either a
progressive adrenocortical insufficiency or become in—
sensitive to ACTH with increasing age. The concomitant
increases of adrenocorticotropin secretion are believed to
be necessary in order to maintain blood levels Of gluco-
corticoids within the normal ranges. This interpretation
was supported by increased percentages Of blood eosinophils,
decreased percentages of plasma albumin and increased per-

centages of plasma gamma globulins from the older bulls.

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78

Dorfman, R. I. 1957. Biochemistry of the steroid hormones.
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