ASCORBIC ACID AND
THE REPRODUCTIVE. CYCLE;
ASCORBIC Acm AND
THE esmous emu-2 '

Thesis for the Degree .of M. S.
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ABSTRACT
ASCORBIC ACID AND THE REPRODUCTIVE CYCLE:
ASCORBIC ACID AND THE ESTROUS CYCLE
BY

Young Hee Ha

Serum ascorbic acid has been reported to increase
about the middle of estrus in Cows (Phillips gt al., 1941).
This suggests that ascorbic acid might have an important
role in the ovarian cycle, and may be related to ovulation.
The purpose of this study was threefold: (l) to evulate
serum ascorbic acid changes around the time of ovulation
in cows, rats, rabbits and sows. (2) to determine serum
ascorbic acid changes after hormone treatment with human
chorionic gonadotropin, follicle stimulating hormone and

prostaglandin F a. (3) to determine whether there is any

2
relationship between serum ascorbic acid changes and
ovarian ascorbic acid.

Serum ascorbic acid in all Species studied did not
show a significant difference around the time of ovulation,
compared to other stages of the estrous cycle. There was
some specie variation, however serum ascorbic acid concen-
tration in all animals was very constant throughout the
estrous cycle. This suggests that serum ascorbic acid is

not a good indicator of time of ovulation, and probably is

not directly related to ovulation.

Young Hee Ha

Treatment of rabbits with follicle stimulating hormone
did not affect serum ascorbic acid changes, however ovarian
ascorbic acid decreased. Prostaglandin F20 at levels of
30 mg injected into hysterectomized cows increased serum
ascorbic acid levels within 10 hours. Serum progesterone
changes were not correlated with the ascorbic acid changes.
The significance of this mechanism is not understood,
however, ascorbic acid may be related to the steroid
metabolism, possibly, estrogen. Ascorbic acid in folli-
cular fluid of the cow ovary was measured. Follicles which

<

were in the developing stages (diameter = 1.0 cm) had

higher ascorbic acid concentration than mature follicles

Iv

(diameter _ 1.0 cm). Although follicular fluid had
relatively high concentrations of ascorbic acid that did

not influence serum ascorbic acid on a quantitative basis.

ASCORBIC ACID AND THE REPRODUCTIVE CYCLE:

ASCORBIC ACID AND THE ESTROUS CYCLE

BY

Young Hee Ha

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1974

Q. '7? ACKNOWLEDGMENTS

The author would like to express her sincere
appreciation to:
Dr. Olaf Mickelsen
for his guidance and help throught this study.
Dr. Jack Britt
for his help and suggestion throughout this study.
Drs. Rachel Schemmel, Modesto Yang
for their advice and encouragement.
Drs. Elwyn Miller, Richard Duklow, Masauki Takahashi
for their aid and suggestions.
Mr. R. Kittok, Mr. D. Harrison and Mr. J. Stelflerg
for their technical assistance.
Miss E. Schlenker, Mr. N. Shier
for their kind aid and suggestion.
And like to extend her gratitude to
Her parents and her brother, Mr. and Mrs. Ha

for their encouragement and love.

ii

Chapter
I

II

III

IV

V

TABLE OF CONTENTS

INTRODUCTION 0 I I O O O O O O O O C O O O 0

REVIEW OF LITERATURE . . . . . . . . . . . .

Ascorbic Acid and Ovarian Cycle. . . . . .
Ascorbic Acid and Gonadotropic Hormone . .
Ascorbic Acid and Ascorbic Acid Deficiency
Related to the Reproductive Process. . .
Ascorbic Acid and Steroidogenesis. . . . .
Gonadotropic Hormone and the Estrous
Cycle. . . . . . . . . . . . . .
Objectives of the Present Study. . . . . .

MATERIALS AND METHODS. . . . . . . . . . . .

First Trial: Rats . . . . . . . . . . . .
Second Trial: Rabbits . . . . . . . . . .
Third Trial: Sows . . . . . . . . . . .
Fourth Trial: Cows (Holstein) .
Fifth Trial: Ascorbic Acid and Follicular
Fluids of Cow Ovaries (Holstein) . . . .
Methods for Detecting Estrus . . . . . . .
Chemical Analysis of Ascorbic Acid . . . .
Radioimmuno Assay of Progesterone. . . . .

RESULTS AND DISCUSSION . . . . . . . . . . .

Serum Ascorbic Acid and Estrous Cycle

in Rats. . . . . . . . . . . . . . . . .
Ascorbic Acid Levels in Serum and Ovaries

After Gonadotropin Treatment . . . . . .
Serum Ascorbic Acid During Estrus in

Sows . . . . . . . . . . . . . . . . .
Ascorbic Acid Changes in Cows. . . . . . .
Relationship Between Serum Ascorbic Acid

and Ovarian Ascorbic Acid. . . . . . . .
Calculation. . . . . . . . . . . . . . . .

SUMMRY O O O 0 O O O O O I O O O O O O O O I

BIBLIOGRAPHY . . . . . . . . . . . . . . . .

iii

Page

0)

\DO‘ U109

25
28

29
32

38
39

42

43

LISTS OF TABLES

Table Page

1 Serum ascorbic acid levels in rats during

the estrous cycle . . . . . . . . . . . . . . 26
2 Serum ascorbic acid levels in rats during

estrus. . . . . . . . . . . . . . . . . . . . 27
3 Ascorbic acid levels of the serum and

ovaries of rabbits. . . . . . . . . . . . . . 30
4 Serum ascorbic acid levels in sows. . . . . . 31
5 Ascorbic acid concentration in follicular

fluid of cows (Holstein). . . . . . . . . . . 40
6 Ascorbic acid concentration in large and

small follicles . . . . . . . . . . . . . . . 40

iv

LIST OF FI GURES

Figure Page
1 L-ascorbic acid and L-dehydroascorbic
aCid O O O O O O O O O O O O O O O O O O O O O 7
2 Interrelationship between the pituitary, the

corpus luteum, the uterus and hypothalamus . . l4

3 Protocol of rabbit experiment. . . . . . . . . l8
4 Changes in the vaginal cell types found in

vaginal smears during the estrous cycle in

the rat. I O O O O O Q C O O O O O O O 0 O O O 21
5 Serum ascorbic acid levels in cows just

before and after estrus. . . . . . . . . . . . 33
6 Serum progesterone levels in cows before and

after estrus O D O O O O O O O O O O O O D O O 34

7 Serum ascorbic acid level after PGFZa
injection into hysterectomized cows. . . . . . 36

CHAPTER I

INTRODUCTION

Estrus and ovulation are the major events of the
ovarian cycle. Furthermore, study of the ovarian cycle
can lead to a better understanding of reproduction and
thus may be useful in the areas of population control and
treatment of infertility.

Interest in the role of ascorbic acid in the repro-
ductive process was primarily brought about by the low or
trace amount of ascorbic acid in fresh semen of low
fertility bulls (Phillips gt al., 1940). It was shown that
ascorbic acid injections to these animals improved the
reproductive success of sterile and partially sterile
bulls. Study of the ascorbic acid role in the female
reproductive cycle was initiated. According to Phillips
25 31. (1941), plasma ascorbic acid levels were higher
during estrus than at other stages of the cycle in cows.
These investigators stated that it is possible to detect
poor breeding cows because of their abnormally low plasma
ascorbic acid levels. Subcutaneous ascorbic acid therapy
to these sterile cows resulted in 60% becoming fertile.
Recently, Valjuskin (1971) reported that injection of
1.6 gm of vitamin C into Swedish cows increased two-fold

the number of animals which came into heat.

The ascorbic acid changes related to the ovarian cycle
have been reported not only for domestic animals, but also
in humans (Mickelsen 25 11., 1943) and other laboratory
animals (Hoch-Ligeti and Bourne, 1948 and Parlow, 1958).

In rats, the ovary shows a significantly lower level of
ascorbic acid at estrus than at diestrus (Hoch-Ligeti and
Bourne, 1948). Other suggestive evidence that the ascorbic
acid was involved in the ovarian cycle is that ascorbic
acid is also related to the gonadotropic hormones which
control the ovarian cycle. According to Parlow (1958)
luteinizing hormone causes a significant decrease of
ovarian ascorbic acid in gonadotropin pretreated rats.
Although the physiological significance of this reaction

is not well understood, ascorbic acid might be an important

factor in steroid metabolism.

CHAPTER II

LITERATURE REVIEW

Ascorbic Acid and Ovarian Cycle

It has long been recognized that females show regular
cyclic sexual changes that are under endocrine control.
The cyclic alteration consists of many biological changes
such as hormone release and other biochemical variance.
The serum ascorbic acid changes related to this ovarian
cycle were reported by several investigators in rats
(Astrada and Laura, 1966 and Deane, 1952). The changes in
serum ascorbic acid during the estrous or menstrual cycle
perhaps affect puberty since females older than thirteen
years of age have higher levels of serum ascorbic acid
than males at the same dietary intake (Dodd, 1969). This
sex difference suggests that there may be some relation-
ship between ascorbic acid and the ovarian cycle.
Evidence suggesting a relation between ascorbic acid and
the ovarian cycle was reported in the 1940's by Phillips
33 21. (1941) who observed that during estrus, plasma
ascorbic acid level in cows was increased. The data
presented by Phillips et 31. (1941) also suggested an‘
increase in the serum level of ascorbic acid when ovulation

was thought to occur. Mickelsen gt a1. (1943) observed

that some women had increased fasting plasma ascorbic acid
levels at the midpoint of the menstrual cycle. More
recently Rivers and Devine (1972) reported that plasma
ascorbic acid levels increased the estrogen phases of the
cycle in women. In humans, ovulation occurs in the middle
of the cycle with the peak in plasma vitamin C levels
appearing at that time. Mickelsen gt gt. (1943) and Loh
and Wilson (1971) indicated that urinary excretion of
ascorbic acid reached a peak when ovulation occurred in
women. These observations suggest that serum ascorbic acid
may be related to ovulation, although the exact mechanism
is not understood.

In rats, ovarian ascorbic acid concentration is
significantly lower during estrus than during diestrus or
metestrus (Hoch—Ligeti and Bourne, 1948 and Mills and
Schwartz, 1959). In 1966, changes of ovarian ascorbic acid
were quantified by Astrada and Laura who reported that
the minimal ascorbic acid level in the ovary at estrus
was 5610.6 mg per 100 g of tissue, while the diestrus
ovary contained 69.3il.l mg per 100 g of tissue. The
ovary is known to have a very high concentration of ascor-
bic acid and the ovarian cycle may reflect the effect of
this cyclic variation of ovarian ascorbic acid. Although
the significance of these cyclic changes and the exact
role of ascorbic acid on the ovarian cycle is not fully
understood, this evidence suggests that the ovarian cycle

has a definite effect on the ascorbic acid.

Ascorbic Acid and Gonadotropic Hormone

A number of observations suggest a possible relation-
ship between ascorbic acid and the hormones involved in
the regulation of the estrous cycle. In 1949, Claesson
gt gt. described that injection of pregnant mare's serum
(PMS) to rabbits resulted in a decrease in ovarian ascorbic
acid concentration. Ovarian ascorbic acid depletion by
luteinizing hormone has been found in several laboratory
animals such as rats (Deane, 1952 and Mills and Schwartz,
1959), and guinea pigs (Reid and Sykers, 1945). Parlow
(1958) pointed out that the significant depletion of
ovarian ascorbic acid in rats was due to luteinizing
hormone and utilized this reaction as a bioassay technique
for that hormone. Goldstein and Sturgis (1961), using
histochemical methods, observed that ovarian ascorbic acid
depletion by luteinizing hormone seemed to involve the
corpus luteum of the rat's ovary. Ascorbic acid is widely
distributed in the ovary, but the corpus luteum concen-
tration is relatively high. By in vitro techniques,
inhibition of ascorbic acid uptake by gonadotropin was
examined by Stansfield and Flint (1967). He found that
luteinizing hormone significantly decreased the uptake of
ascorbic acid from the incubation medium. Addition of
pregnenolone and progesterone also significantly inhibited
ascorbic acid uptake by the tissue. He suggested that the
very low level of ascorbic acid concentration in the ovary

might be due to the inhibition of ascorbic acid uptake

rather than depletion of ascorbic acid from the ovary.
Recently, Loh and Wilson (1971) observed that urinary
secretion of ascorbic acid was increased in the middle of
the menstrual cycle in women. This urinary secretion of
ascorbic acid had the same pattern as that of luteinizing
hormone secretion. Evidence suggests that ascorbic acid
may be related to the steroids.

Observations suggest that ascorbic acid has an
augmentative effect on gonadotropic hormone. Di Cio and
Schteingart (1942) noted that the combination of gonado-
tropic hormone and ascorbic acid produced heavier female
genital organs compared to injection of the hormone alone
in rats. Reid and Sykers (1945) reported that the combina-
tion of ascorbic acid and gonadotropin injected into
guinea pigs produced higher ovarian weights than when
gonadotropic hormone alone was injected. This augmentative
effect of ascorbic acid on the gonadotrpic hormone appears
to further suggest that some relationship exists between

ascorbic acid and gonadotropic hormones.

Ascorbic Acid and Ascorbic Acid Deficiency Related to the

 

Reproductive Process

 

Zilva in 1923, did much of the early work on the
isolation of L—ascorbic acid and established the general
chemical properties of this vitamin. Dehydroascorbic acid
(Figure 1) is formed from the L-ascorbic acid by the

oxidative removal of two equivalents of hydrogen (Sebrell

and Harris, 1967). Although the distribution of this form
in the body is different from L-ascorbic acid, it has the

same biological activity.

 

Figure 1. L-ascorbic acid L-dehydroascorbic acid
HO [on 0 0
in / i,H I
HO- -H() \0 0 o
| Ho-c-H
CHZOH |
CHZOH

A recent study by Horning gt gt. (1972) suggests that
L-ascorbic acid uptake by the adrenals and ovaries is
faster than that of dehydroascorbic acid. The reason is
that L-ascorbic acid transport is an energy dependent
process while the dehydroascorbic acid is transported by
passive diffusion. The energy dependent uptake is mainly
due to the structural specificity of the enzymes involved
in this mechanism. The outstanding property of ascorbic
acid is largely due to the ease with which it may be
oxidized and reversibly reduced.

Ascorbic acid metabolism differs slightly depending
on the species involved. For those animals that are not
dependent on dietary sources, L-ascorbic acid is
synthesized from six carbon precursors such as glucose,
fructose, galactose and mannose (Sebrell and Harris, 1967).

The synthesis of ascorbic acid differs in species, but most

of the synthesis takes place in the liver. In animals
including men, monkeys and guinea pigs, there is no
oxidative enzyme which converts L-gulonolactone to L-
ascorbic acid. Therefore these animals must depend on an
exogenous source of ascorbic acid (King, 1967). Burns

gt gt. (1958) and Martin and Mecca (1961) found that only
very small amounts of ascorbic acid are absorbed from the
upper part of the small intestine of rats. Guinea pigs,
on the other hand, exhibited active transport of the
ascorbic acid from the duodenum to the blood. Another
difference occurred in the potential of the kidney to
oxidize ascorbic acid to carbon dioxide. The kidney
homogenates of rats, rapidly oxidized ascorbic acid to
carbon dioxide in vitro, whereas, the kidneys of guinea
pigs were much less active in this mechanism.

The most important functions of ascorbic acid in the
body is its role in hydroxylation and oxidation-reduction
reactions. In the biochemical system, ascorbic acid plays
a role in electron transport, the metabolism of tyrosine,
collagen formation and perhaps in the reproductive process.

Many workers reported that a deficiency of ascorbic
acid affects the female reproductive system. Greenblatt
(1953) and Pena (1954) reported that ascorbic acid
deficiency caused habitual abortion or still birth and‘
frequently premature birth in women. They suggest that this
might be due to capillary fragility of the placenta or

vascular disturbances throughout the body because of the

abnormality of collagen hydroxylation. Another
possibility involves the abnormality of steroid hormone
synthesis, which might be related to ascorbic acid. Javet
(1954) reported that ascorbic acid deficiency in pregnant
women resulted in frequent signs of decidual hemorrhage
and spontaneous abortion and these patients had very low
serum ascorbic acid levels.

Kraemmer gt gt. (1933) showed that female guinea pigs
receiving a scorbutigenic diet supplemented with less than
3 cc of orange juice per animal per day failed to give
birth to live young. The ovaries from these animals showed
degeneration of graafian follicles and a lack of normal
development of new follicles. Ovaries of guinea pigs
dying from scurvy contained many degenerating follicles and

there were no new corpora lutea or no new follicles.

Ascorbic Acid and Steroidogenesis

 

Since the original description by Szent-Gy5rgy of the
presence of a strong reducing substance in the adrenal
cortex and pituitary, ascorbic acid was thought to be
involved in steroidogenesis. Sayers gt gt. (1944) described
changes of ascorbic acid and cholesterol concentrations in
the adrenal cortex after ACTH treatment in rats; this was
related to the secretion of steroids in the adrenal cortex.
Nathani and Nath (1972) also observed that ACTH signifi-
cantly increased the total blood ascorbic acid. In spite

of these suggestions that ascorbic acid may play a primary

10

role in adrenal steroidogenesis, the direct mechanism
whereby ascorbic acid is involved in these reactions has
not been established. ACTH added to an in vitro system
caused significant decreases in the ascorbic acid and
cholesterol concentration in the adrenal glands and
increased adrenal steroid synthesis (Kitabchi, 1967a).
This suggests that the depletion of ascorbic acid from the
adrenals removes hydroxylase inhibition thus permitting
increased adrenal steroidogenesis. In 1951, Banerjee and
Deb found that in scorbutic guinia pigs, the adrenal
ascorbic acid and cholesterol content were decreased and
the total cholesterol in the body of guinea pigs was much
increased (Banerjee and Singh, 1958). Cholesterol is the
most important precursor in steroid synthesis.

The ovary synthesized a class of steroids like the
adrenals which have a common basic structure. The bio-
synthetic step, via cholesterol to pregnenolone are common
for all steroids. In 1968, DeNicola gt gt. studied the
effect of ACTH on adrenal ascorbic acid and its relation to
steroid synthesis in rats. He observed that l7-B-estradiol
and progesterone also inhibited the transport of ascorbic
acid into the adrenal in vitro. In the presence of
progesterone and estrogen in the incubation medium, adrenal
corticoid synthesis was increased. In manners to that.
adrenal ascorbic acid was depleted by ACTH, ovarian
ascorbic acid in a number of species, i.e., rats (Parlow,

1958), guinea pigs (Reid and Sykers, 1945) and rabbits

11

(Claesson gt gt., 1949) was reported to be depleted by LH.
A specific effect of LH on the ovary seems to increase
steroid hormone, especially production of progesterone.
However, it is not clearly understood whether ascorbic
acid depletion by LH is also related to sex steroid
synthesis.

In 1970, Khatamov studied the effect of sex steroids
on the content of ascorbic acid in the internal organs.
With progesterone injection ascorbic acid content in all
internal organs decreased significantly, and estrogen
adm1nistration further decreased ascorbic acid in these
organs. The interrelationship between adrenal steroido-
genesis and gonadal steroidogenesis is not fully understood.
However, there may be some mechanism for maintaining

equilibrium between these two sites of steroid synthesis.

Gonadotropic Hormone and the Estrous Cycle

 

The estrous cycle is a series of physiological events
involving the pituitary and ovary which provide for
follicular growth and ovulation. Either fertilization and
zygote maintenance occurs or the process starts over again.

The estrous cycle in cows, as in other species, is
controlled by pituitary gonadotropic hormones. The
ovarian-pituitary relationship has long been recognized.
The luteinizing hormone, seems to be the major hormone
responsible for triggering ovulation, corpus luteum growth

and progesterone secretion (Baird, 1972). The onset of

12

preovulatory maturation of the follicle is marked by the
sudden and dramatic rise in the release of LH from the
pituitary, the so-called LH surge. In cows LH surges
occurred 3-6 hours after the onset of estrus (Henricks

and Dickey, 1970). Schams and Karg (1969) using a radio-
immunoassay reported that serum LH reached peak 15-20 hours
before ovulation. In the cow, ovulation occurs 25-27 hours
after the onset of estrus. This LH seems to be produced

by the positive feed back of estradiol on the hypothalamus,
causing a discharge of LH releasing factor. This releasing
factor is transported to the anterior pituitary via the
portal blood system.

Cows are unique, among all species, in that the
pituitary contains relatively high levels of LH and low
levels of FSH, however FSH also contributes to the growth
and development of follicles from the antral to the
preovulatory stage. A significant drop in FSH and LH
content of the pituitary occurred in cows in 1-8 hours
after the onset of estrus (Rakha and Robertson, 1965).

This indicated that FSH as well as LH plays a role in the
initiation of ovulation in cattle. Progesterone is one
of the most important gonadotropic hormons controlling
estrous cycle. Plasma progesterone levels declined
precipitously between day 16-19 of the estrous cycle,
while plasma LH levels rose rapidly in cows. After serum
progesterone levels decreased to levels of 1 ng or less,

ovulation occurred (Hansel and Trimberger, 1952).

13

Following the growth of the corpus luteum, progesterone
secretion begins to increase, and remains relatively high
during the luteal phase until the next ovulation and estrus
stage occurs.

Recently, PGF a showed a luteolytic effect on the

2
corpus luteum regression which resulted in a decreased
progesterone synthesis. Progesterone stimulates the

20. This PGan

is transported to the ovary and destroys the corpus luteum

endometrium of the uterus to produce PGF

thereby decreasing progesterone synthesis. As a result
of the falling progesterone levels, a graafian follicle
begins to secrete a large amount of estrogen, which

triggers a surge of LH from the pituitary, and another

cycle begins.

14

Figure 2. Interrelationship between the pituitary, the
corpus luteum, the uterus and hypothalamus.
(From: Reproductive Hormones, IN Hormones in
Reproduction, Baird, D. T., 1972, p. 56)

 

Stimulus
Various CNS +
Input
\ QYPOTHALAMUEI

 

Portal Blood System
(release Releasing Factor)
LH-RF

Prolactin-RF

IEITUITARYI
/ \

Blocks positive response Pituitary Gonadotropins
of Hypothalamus to LH+FSH for Ovulation
Estrogen and so inhibits LH+Prolactin for Luteal
LH surge Maintenance

 

l,’0xytosin

’
I
’
I
’

—é’”’PGF a, Destroy CL* Follicles-Estrogen

-------2- --------------- ‘7- Ovulation

Stimulate Endometrium Corpus Luteum-
to synthesize PGFZa //// Progesterone

//
\ ‘\ //

\‘J
Progesterone

*CL - Corpus luteum

15

Objectives of the Present Study

The objectives of this study are:

1. To determine whether serum ascorbic acid changes around
the time of ovulation in rats, sows, rabbits and cows.

2. To determine whether ovarian ascorbic acid depletion
at estrus may directly influence the serum ascorbic
acid.

3. To determine whether there is any relationship between
the changes of serum ascorbic acid and progesterone in
cows.

With the above mentioned objectives in mind, we chose
rats, rabbits, sows and cows as our experimental subjects.
These animals can synthesize ascorbic acid. Thus, serum
ascorbic acid levels are less influenced by dietary sources
than is true of guinea pigs and other animals dependent
on the diet for this vitamin. Since measurement of the
concentration of ascorbic acid in blood is a good tool
that can be easily used in endocrinology, the experiments
were mainly designed to determine the changes in the serum
ascorbic acid.

The experiments were designed to:

1. Determine the serum ascorbic acid levels during estrus
including that immediately before and after estrus.

2. Determine ovarian ascorbic acid concentrations and the

follicular fluid ascorbic acid concentrations.

CHAPTER III

MATERIALS AND METHODS

First Trial: Rats

 

Twenty Osborne Mendel female rats weighing about
200 g were housed singly in metal cages, and were given
water and fed a natural grain ration ad libitum. Stages
of the estrous cycle were determined three times a day for
each rat, by vaginal smear and chloride content of vaginal
washings. About 30 pl of deionized water was put into the
vagina of the rats, left for approximately two seconds and
then sucked out with an eyedropper. This material was
placed on silver chromate paper, which developed a white
spot. A blood sample was secured each day; the rats were
anesthetized with ether and blood was collected by heart
puncture. As soon as the blood was collected, the tubes
were placed in ice and brought to the laboratory. Serum
ascorbic acid were analyzed by a slightly modified
Schwartz and Williams method (1955) for total ascorbic

acid. Hematocrits were also determined for each animal.

Second Trial: Rabbits

 

Twenty-five New Zealand White rabbits weighing about

3.5 Kg were separately caged. Water was always available

16

17

and laboratory chow diet was fed ad libitum. Experiments
were designed following the protocol shown in Figure 3.
Rabbits were divided into five groups of five animals.

The rabbits in four groups received follicle stimulating
hormone and human chorionic gonadotropin treatment while
the rabbits in the fifth group were controls. At the
beginning of the experiment, the animals in the first group
were sacrificed immediately by cervical dislocation, blood
was collected by heart puncture and the ovaries removed.
The remaining animals were injected intramuscularly twice
a day, with FSH at levels that were about 0.4 mg per
rabbit per day. Seventy-two hours after the first FSH
injection, 100 i.u. of HCG was given intravenously, and
rabbits were killed at different time intervals after HCG
injection; 30 minutes, 2 hours and 4 hours. All ovaries
were trimmed and weighed separately. Each pair of ovaries
was ground in a glass homogenizer with 10 m1 of 5% of
trichloroaceticaufixlsolution and the homogenates were used

for ascorbic acid determinations.

Third Trial: Sows

 

Five sows with normal estrous cycles were selected
and estrus was detected by the behavior of the animals.
During estrus and immediately before and after estrus,-
blood was collected from the anterior vena cava. Blood
samples were usually taken about 8 o'clock in the morning.

Blood samples continued to be taken after estrus every 24

18

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mm ON AN .2.¢ oouNH
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ma on Ha .z.¢ omum
mm on HA DUE .2.¢ m Nb MBIH Iw
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19

hours for one week. On the day when ovulation was scheduled
to occur, a second sample of blood was collected about one
o'clock in the afternoon. The tubes containing the blood
were placed in an ice chamber and brought to the laboratory

for immediate analysis.

Fourth Trial: Cows (Holstein)

 

Five cows with normal cycles were selected and fed a
grain, hay and corn silage mixture ad libitum. Estrus was
detected by ovarian palpation and behavior of the cows.
Blood progesterone was analyzed to more accurately detect
the time of ovulation.

Around estrus a series of blood samples were collected
from these cows by jugular vena puncture at 12 hour
intervals for a period of one week. Blood samples were
usually taken at 7:00 a.m. and 5:00 p.m.

Three cows were hysterectomized and 30 mg of PGF 0

2
was injected intramuscularly in order to destroy the
corpora lutea. A series of blood samples were taken by
jugular vena puncture at different time intervals for five
days; blood samples were taken at 10 minute intervals for
the first hour after PGF2a injection, at 30 minute inter-
vals for the following three hours, at 6 hour intervals
for the remaining 24 hours and at 2 hour intervals for the

next 3 days. Blood serum from these cows was used for

ascorbic acid and progesterone analysis.

20

Fifth Trial: Ascorbic Acid and Follicular Fluids of Cow

Ovaries (Holstein)

 

Fresh ovaries were collected from cows at slaughter
house and the approximate stage of the cycle was predicted
by the ovarian morphology and stage of pregnancy.
Follicular fluid from each ovary was mixed with 10%
trichloroacetic acid (Follicular fluidzTCA = 5:3 ratio) to
stabilize the ascorbic acid and precipitate any protein

present. Follicles from another group of similar ovaries

<

were divided into two groups; small follicles (diameter
1.0 cm) and large ones (diameter 2 1.0 cm). The follicular
fluid from each group of follicles in one ovary was combined
and mixed with 10% of TCA as previously described. This

solution of follicular fluid and TCA was used for the

ascorbic acid analysis.

Methods for Detecting Estrus

 

Epithelial changes in the vagina were used to deter-
mine the time of ovulation in rats. The vaginal smear
exhibits cyclic changes during the estrous cycle which
can be differentiated on the basis of strong staining
reactions (Erikson, 1963). These changes are influenced
by estrogen and progesterone. This method is based upon
the fact that under the influence of estrogen, cornifica-
tion is induced in the outer epithelial layer of the
vagina and can be observed in a smear. During proestrus,

the epithelial cells are large with round nuclei; later in

21

estrus the cells consist only of large squamous epithelia
without nuclei. In metestrus, these cells become infilter-
ated with leucocytes, and in diestrus, only leucocytes can

be seen in the smear (Figure 4).

Figure 4. Changes in the vaginal cell types found in
vaginal smears during the estrous cycle in the
rat.

em 0 5% 9%:
o 0 I; ‘if
3% {QR up $902..

Proestrus Estrus Metestrus Diestrus

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ovulation can be estimated by the changes that occur
in the cervix. Cervical mucus undergoes cyclic changes in
physical and chemical properties. Characteristics physico-
chemical changes are induced by estrogen and progesterone
(Cohen, 1966). One of these involves changes in the
chloride concentration. The latter can be detected by
silver chromate paper. A simple spot test for detecting
ovulation by the chloride content of cervical mucus was
studied by McSweeney and Sbarra (1964) and modified by
Hardy gt gt. (1970). Silver chromate paper for this purpose
was made from Whatman filterpaper immersed serially in
solutions of silver nitrate (0.275 M) and potassium v
chromate (0.175 M); when dried at room temperature avoiding

the sun light, the paper was brown in color. Vaginal mucus

22

from a series of rats was put on this paper. During
ovulation or estrus, the mucus produces a white precipitate
(white spot) in this paper the size of the spot being pro-
portional to the chloride concentration of the cervical
mucus. This high chloride concentration of mucus (0.5%
and above) could easily be differentiated from low
concentration (0.4% and less). On the other hand, during
the anovulatory cycle, no positive Spot was seen on this
silver chromate paper. For the rat, the combination of
the phenomena, vaginal smear and positive chloride test on
the silver chromate paper, was regarded as the time of
estrus and ovulation.
Rabbits do not have a cycle, but require a mating
stimulus for ovulation and corpus luteum formation.
Estrus usually occurs 9-12 hours after stimulation produced
by copulation or gonadotropic hormone. This is due to
stimulatory release of a hypothalamic neuro hormone which
stimulates the anterior pituitary to release ovulatory
gonadotropic hormone (Desjardins gt gt., 1967). For this
reason FSH or PMS and HCG cn: LH are widely used for
inducing ovulation in rabbits. For this study, FSH and
HCG injections were used for inducing ovulation in rabbits.
For cows, estrus was detected by ovarian palpation and
estrus behavior. This phenomena occurs under the influence
of steroids, especially estrogen. .The plasma progesterone
levels begin to drop very rapidly on day 17 of the estrous

cycle (21 day cycles in cows). During estrus, serum

23

progesterone levels are so low, they are almost non-
detectable. After ovulation, progesterone starts to
increase and high levels are maintained during the luteal
phase. In cows, PGan induced the regression of corpus
luteum and the synchronization of estrus. Hafs gt gt.

(1972) observed that about 74:3 hours after the PGF 0

2
injection, estrus begins to occur and a plasma LH peak
appeared 60il5 hours later. He suggested that intravenous
administration of PGan would appear to be an extremely
efficient method of inducing ovulation or synchronizing

estrus in cows.

Chemical Analysis of Ascorbic Acid

 

The amount of ascorbic acid in serum and ovaries was
determined by a slightly modified Schwartz and Williams
method (1955) for total ascorbic acid.

One ml of each sample (serum or homogenates of
ovaries) was placed in a centrifuge tube, to which was
'added 0.6 m1 of 5% TCA solution in order to precipitate
serum protein. The mixture was centrifuged at 3000 rpm
for 10 minutes; 0.3 m1 supernate was used for ascorbic acid
analysis.

Reagents: Ascorbic acid (Merck) 1 mg% solution
5% Trichloroacetic acid (TCA)
0.2% 2,6,-Dichlorophenolindophenol
Sg Thiourea dissolved in 95 g of 5% TCA

Zg 2,4-Dinitrophenyl hydrazine dissolved in 98 g

24

of 65% H2504

Phospho-hydrochloric acid (85.8% H3PO4:37% HCl =
1:3)
Instead of metaphosphoric acid, we used the 5% TCA
solution. The optical density of each sample was determined

by means of a Beckman Spectrophotometer (DB), with wave

length set for 530 mu.

Radioimmuno Assay of Progesterone

 

Progesterone of cow's serum was analysized by radio-

immunoassay, which is described by Kittok and Louis (1973).

CHAPTER IV

RESULTS AND DISCUSSION

Serum Ascorbic Acid and Estrous Cycle in Rats

 

Serum ascorbic acid levels at estrus when compared to
the other stages of the cycle indicated no significant
changes of ascorbic acid during estrus in rats (p<0.05,
Table 1). Serum ascorbic acid levels at the time of
ovulation are shown in Table 2 as compared to the rest of
the estrus stage. According to Yassman and Taymor (1970),
ovulation occurs in rats around 2 A.M. early in estrus.
For that reason blood samples were taken at specified
intervals during the 24 hours following the appearance of
proestrus in rats. Using Yassman's criteria, it would
appear that the serum ascorbic acid levels at the time of
ovulation were not different from those at the estrus
stage. Serum ascorbic acid levels in estrus also showed
no significant changes from those in the diestrus stage
(p<0.05).

There was a certain amount of variation in the serum
ascorbic acid level of the rats throughout the entire
series. This slightly complicates the interpretation of
the data. Part of that fluctuation may be associated with

the stress imposed on the animals by collecting blood from

25

26

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27

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mvv.o mmm.o wwm.o mmv.o H
msuumm msuumm Acoflumas>ov monummoum .oz Dam

monumw >Humm mama
.E.m oH .mmlm .E.m w .mmlm .E.m m .wmnm .E.m OH .nmnm mumo

 

 

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.N magma

28

the heart following general anaesthesia. That stress may
be involved in this procedure is suggested by the fact
that when a series of blood samples were collected within
a period of a day, the serum ascorbic acid level tended to
increase serially. However, 12 hours after the first
sample was collected, the serum ascorbic acid tended to
return to the level existing prior to the start of the
blood collection period.

Although there are no statistically significant
differences in serum ascorbic acid levels at various
stages of the estrous cycle, the highest levels occurred
more frequently on the day when the rat was in estrus.

The final answers to the question of possible variations
in the ascorbic acid levels of rats' blood during the
estrus cycle will have to await the development of a
technique for securing blood which impose no trauma on the
animal.

The ovarian ascorbic acid levels in estrus showed
significantly lower than diestrus. This significant
depletion of ovarian ascorbic acid in estrus confirmed the

previous report (Hoch-Ligeti and Bourne, 1948).

Ascorbic Acid Levels in Serum and Ovaries After Gonado-

 

tropin Treatment

 

Rabbits do not have a regular cycle, but require a
mating stimulus for ovulation and corpus luteum formation

resulting in either pseudopregnancy, depending on whether

29

the mating was fertile. This is called induced ovulation.

Usually 10 hours after the gonadotropin stimulus, ovulation

occurs, induced by LH released from the anterior pituitary.

Gonadotropin treatment of the rabbits did not
significantly change the serum ascorbic acid levels com-
pared to the control group (p<0.05). Furthermore, serum
ascorbic acid levels about the time of ovulation did not
differ from those in the other stages of the cycle.
Ovarian weight increased after HCG injection compared to
control rabbits (Table 3).

In rabbits, ovarian ascorbic acid was also signifi-
cantly decreased after FSH and HCG injection, these data
agree with that from rats, which suggest LH causes the
ovarian ascorbic acid depletion (Parlow, 1958). In
rabbits, serum ascorbic acid level did not change at the

time of ovulation.

Serum Ascorbic Acid During Estrus in Sows

 

Table 4 shows the serum ascorbic acid levels during
the estrus cycle in five sows.

During estrus, serum ascorbic acid levels did not
change significantly as compared to the other stages of
the cycle (p<0.05).

In all of these animals including rats, rabbits and
sows no significant changes of serum ascorbic acid at the
time of ovulation have been observed. This may be due to

species differences in ascorbic acid metabolism or serum

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31

 

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32

ascorbic acid may not be directly related to ovulation. At
any rate, it does not appear to be a good parameter for

measuring the time of ovulation.

Serum Ascorbic Acid Changes in Cows

 

Serum ascorbic acid levels at the beginning of estrus
were significantly higher than later in this period.
However serum ascorbic acid levels throughout estrus were
not significantly increased above the other stages of the
cycle. Phillips EE.E£- (1941) reported that the plasma
ascorbic acid levels showed a peak in the middle of estrus
even though the increase was small.

Results of the present study showed that serum ascorbic
acid did not change significantly (p<0.05), during the
estrous cycle. Actually, the trend was toward a decrease
at the middle of estrus. Our data (Figure 5) show that
there are marked individual variations in plasma ascorbic
acid levels. For example, cow No. 935 showed very high
levels of serum ascorbic acid (about 6 times the "normal
level") about 40 hours before estrus.

Serum progesterone levels for most of the cows were
not highly related to serum ascorbic acid levels except in
cow No. 935. The progesterone levels of this cow tended
to be related to the changes in ascorbic acid concentration.

These results suggest that serum ascorbic acid levels
do not relate to ovulation. The observations from other

species including rats, rabbits and sows also confirmed

(mg/100ml serum)

ASCORBIC ACID

0.

 

33

 

O / Q

o ’0. V 9 S

E . 0......) 1225
: e... '0'. O....

 

--:h.4.fi.‘ . .1 1182
' I 0 L l J I a n o
r 1 ~Estrua ------ 1 2

Day before and after Estrus.

FIGURE 5 SERUM ASCORBIC ACID LEVELS IN COWS JUST BEFORE AND AFTER ESTRUS

Serum Progesterone Levels

( ng / m1 )

200

1.5

H
O

O
u:

34

FIGURE 6 SERUM PROCESTERONE LEVELS IN COWS BEFORE AND
AFTER ESTRUS

 

 

 

 

L
b
r
1225
’I — O -
P - ' *\'Q.. ...o"oo.. /.
" ‘5'. 8.0... ... 1182
lWXWV‘x‘”
n
i E" 1230}
o 1 a LA 1 it ' I I
- 1 pEacmo —-4 1

Day of Estrous Cycle

35

the fact that ascorbic acid does not change at the time of
ovulation. In other words, serum ascorbic acid is not a
good indicator of the ascorbic acid role in the reproduc-
tive process.

On the other hand, there appears to be some indication
of a relationship between serum ascorbic acid levels and
prostaglandin injection in cows. Within 10 hours after
PGF2a injection, serum ascorbic acid starts to increase,
reaching a peak which remains for only several hours
(Figure 7). With one exception, cow No. 801, the cows
showed secondary peaks of serum ascorbic acid 28 hours

following PGF a treatment. According to Hafs gt gt. (1972)

2

ovulation occurs about 74 hours after PGF a injection into

2
cows. During this period of time in which ovulation should
occur, serum ascorbic acid levels were very constant.

Serum progesterone levels were also measured and within one
hour after PGFZa injection, progesterone levels decreased
very rapidly and remained low for the three days following
estrus which was as long as that parameter was measured
(Figure 7). In cow No. 1188, serum ascorbic acid levels
were low throughout the experimental period, although she
also showed a second peak of ascorbic acid after PGFZG
treatment. She was very sick throughout the experimental
periods, which might explain the low serum ascorbic acid.

She also showed very low levels of progesterone throughout

this period.

36

 

 

 

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HYSTERECTOMIZED c
INJECTION INTO
Serum ASCORBIC ACID LEVEL AFTER PGFiA

Pigure.7

37

Prostaglandin is known to have luteolytic effects on
the corpus luteum and to decrease progesterone synthesis.
The significance of this phenomena and the exact mechanism
of the PGan and ascorbic acid is not clearly understood.
One possibility is that ascorbic acid may be related to
steroid synthesis. The role of ascorbic acid in sex
steroid synthesis is not clear; it is not known whether
ascorbic acid has the same effect on sex steroid synthesis
as it does on adrenal steroidogenesis. However, all
steroids share the same common pathway, i.e., the inter—
mediate from acetate to cholesterol, is pregnenolone.
Ascorbic acid is known to have an inhibitory affect on
the hydroxylase system in adrenal steroidogenesis. A
decreasing concentration of ascorbic acid in the adrenal
cortex is associated with an increased adrenal steroido—
genesis (Kitabchi, 1967b).

Our results showing increasing serum ascorbic acid
after PGFZa may be related to steroid synthesis possibly
of estrogen. If ascorbic acid inhibits the hydroxylase
system which appears to be an essential step in estrogen

synthesis, then PGF a injection may possibly result in an

2
increased release of ascorbic acid from the ovaries
producing a transient increase in serum ascorbic acid.

One other possibility is that PGF a has stimulated the

2
anterior pituitary to release hormones especially ACTH.
It may be that under these circumstances, ACTH acts on

both the adrenal cortex and ovaries to release ascorbic

38

acid in manners analogous to a stress reaction. PGFZa
may also be related to the release of other anterior
pituitary hormones, e.g. thyroid and growth hormone. The
hormones might increase basal metabolic rate and carbo-

hydrate metabolism. So overall metabolic rate may increase

with a concomitant increase in ascorbic acid synthesis.

Relationship Between Serum Ascorbic Acid and Ovarian

 

Ascorbic Acid

 

Cows, like other mammals, have high concentrations of
ascorbic acid in the ovary. The most prominant changes in
the ovary before ovulation is follicular development and
the production of hormones that play a role in the growth
of the follicles. On day 17 of the cycle in cows,
progesterone levels start to decrease and maximum
follicular growth occurs at this time with increasing
estrogen synthesis. The significant increase of serum
ascorbic acid after PGan injection may be related to these
hormonal changes before ovulation. These suggest that the
ovary may be one of the possible sources of increased
serum ascorbic acid. To directly test this hypothesis,
ascorbic acid levels of the follicular fluid were measured.
Table 5 shows the results of the ascorbic acid levels in
follicular fluid of cows. During the stages of most.rapid
follicular fluid formation, there was a higher concentration

of ascorbic acid than in any other phase of the cycle; this

occurred about day 17 of the cycle. Such evidence

39

indirectly supports the hypothesis that ascorbic acid may
be involved in estrogen formation. Small follicles,
those in the growing stage, have higher ascorbic acid
concentrations in their fluid than the large ones; the
difference being about two-fold (Table 6).

Whether or not this high concentration of ascorbic
acid in follicular fluid can be a possible source of
increased serum ascorbic acid has been examined. Suppose
a cow's weight is about 1000 pounds, and that the gut fill
is about 75 pounds. Therefore the actual body weight of
cow is 925 pounds or 412 Kg. The plasma volume of cows is
about 3% of body weight or 12.36 Kg. The average serum
ascorbic acid level in cows during anestrus is 0.3 mg%.
Therefore, the total circulating plasma ascorbic acid is
37.1 mg. However the follicular fluid has only 2 mg% of
ascorbic acid. Consequently the ascorbic acid in
follicular fluid cannot influence the serum ascorbic acid
on a quantitative basis. This stems from the fact that
although follicular fluid has very high concentrations of

ascorbic acid, the organ itself is relatively small.

Calculation

 

Body weight : 1000 pounds or 450 Kg

Gut fill : 75 pounds or 38 Kg

Corrected body weight : 450 Kg - 38 Kg = 412 Kg
Plasma volume : 3% of body weight or

412 x 0.03

12.36 Kg (12360 9)

40

Table 5. Ascorbic acid concentration in follicular fluid
of cows (Holstein). All values in mg per 100 ml
of follicular fluid.

 

 

 

Physiological state Ascorbic acid level
mg/100 ml
Mid cycle: Day 10-12 1.58
Mid to late cycle: Day 17 3.27
Midcycle 0.70
50 days of pregnancy 1.33
8.5 months of pregnancy 2.89

 

Table 6. Ascorbic acid concentration in large and small
follicles. The ovaries were secured from cows
immediately after slaughter. All values in mg
ascorbic acid per 100 ml follicular fluid.

 

 

 

Physiological state Large follicles Small follicles
; 1.0 cm ; 1.0 cm

Day 6-8 of cycle 1.04 1.84

Day 10-12 of cycle 1.25 2.50

50-60 days of pregnancy 1.22 2.51

4-5 months of pregnancy 1.43 1.75
7-8 months of pregnancy -- 2.85

 

41

Plasma ascorbic acid : 0.3 mg%

0.3 X 12360
100

 

= 37.1 mg

Follicular fluid : 2 mg% of ascorbic acid

CHAPTER V

SUMMARY

Serum ascorbic acid did not show significant differ-
ences at the time of ovulation from the other stages of the
estrous cycle. Although there was some species variation,
serum ascorbic acid levels were very constant during the
estrous cycle. The result suggests that serum ascorbic
acid may not be directly related to ovulation.

PGFZG seems to be related to the ascorbic acid
metabolism in cows. Intramuscular injection of 30 mg of
PGan to hysterectomized cows was associated with a
significant increase in serum ascorbic acid may be related
to steroid synthesis, possibly estrogen.

Ascorbic acid concentration in follicular fluid is high
during the developing stages of the cycle which is also the
estrogen phases of the cycle. Although the follicle has a
relatively high concentration of ascorbic acid, there

appears to be no relationship between serum ascorbic acid

changes and ascorbic acid in follicular fluid.

42

BIBLIOGRAPHY

BIBLIOGRAPHY

Armstrong, D. T. and Black, D. L. (1966). Influence of
Luteinizing Hormone on Corpus Luteum Metabolism and
Progesterone Synthesis Throughout the Bovine Estrous
Cycle. Endocrinol. 1§:937.

Astrada, J. J. and Laura, C. (1966). Ovarian Ascorbic Acid
Concentration and Its Modifications During Sexual and
Reproductive Activity in the Rat. Acta Physiol. Lat-
Amer. t§:1.

Baird, D. T. (1972). Reproductive Hormones, IN: Hormones
tg Reproduction, Austin, C. R. and Short, R. V. (Ed.)
pp. 1-28, Cambridge University Press.

 

Bane, A. and Rajakoski, E. (1960). The Bovine Estrous
Cycle. Cornell Veterinarian §t:77.

Banerjee, S. and Deb, C. (1951). Effect of Scurvy on
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