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L I I? P .1 R Y

NiiCllxsau Q Late
University

 

ABSTRACT

EFFECT OF INDOMETHACIN (A PROSTAGLANDIN INHIBITOR)
ON THE OVULATORY CYCLE, TOTAL PLASMA CALCIUM
AND EGG SHELL THICKNESS IN
THE DOMESTIC LAYING HEN
(GALLUS DOMESTICUS)

By

Randall William Hammond

Indomethacin (Indocin), l-(p-chlorobenzoyl)-5-methoxy—
2-methylimdole-3-acetic acid, a prostaglandin inhibitor,
was administered to Single Combed White Leghorn laying hens
to determine the effect of this drug on the reproductive
cycle and total plasma calciuma

Hens, 18 to 20 months of age, with a 3 to 6 egg laying
sequence, with 1 skip day and no history of soft-shelled
or thin-shelled eggs, were individually caged and fed a

commercial layer ration and water ad libitum. They were

 

subjected to a 17.5 hr light and 6.5 hr dark photoperiod
(lights off 2300 to 0530 hr), with times of oviposition
being recorded.

Oral administration of indomethacin (50 mg/kg BW).
given at 2300 hr the evening of the skip day in Experi-
ment 1, significantly delayed the time of oviposition of

the first egg in the sequence (5.0 to 17.5 hr), when 7 hens

Randall William Hammond
were used as their own controls (p < 0.01). The second
egg was prematurely expelled (p < 0.01), but no signifi-
cant effect on time of oviposition of the third egg was
observed. Luteinizing hormone (LH) release or ovulation
was apparently not blocked by indomethacin as a second egg
was laid by all birds. However, 2 of 7 birds did not lay
a third egg.

The absorption and clearance of indomethacin from the
plasma were determined at 50 and 100 mg/kg BW dosages, in
Experiment 2. Indomethacin levels were determined by a
spectrophotofluorometric technique. The drug was present
in the plasma 1 hr after administration, reached maximum
levels at 2 hr, steadily dropped to very low levels up to
18 hr and was cleared from plasma at 32 hr. The disappear-
ance curves for the two-dose levels were found to be
parallel up to 18 hr, after statistical analysis by split-
plot method, and a dose-response effect was evident.

Luteinizing hormone was injected on the morning of
the last egg in the sequence, in several experiments, in
which each bird was its own control. This LH injection
added another egg to the sequence, and before and after
drug administration responses were observed.

Indomethacin (50 mg/kg BW), 1 hr before LH injection,
caused a delay in oviposition (p < 0.01) of the last egg
by 9.7 to 42.2 hr (Experiments 3 and 4). Most of these

eggs had thicker egg shells, when compared to their own

Randall William Hammond
control. Premature expulsion of the LH-induced egg was
confounded with the amount of shell deposition that
occurred. However, eggs which were expelled at the con—
trol time (I 2 hr), were thinner than controls (p < 0.01).
Total plasma calcium was lowered by approximately 30% from
controls at 11 hr to at least 32 hr after indomethacin.

In Experiment 5, indomethacin (50 mg/kg BW) adminis-
tered 1 hr after the LH injection, did not block ovulation
in any of the 7 birds, as compared to their own control.

Two other experiments investigated the effect of indo-
methacin on the time of LH injection to the presence of a
soft-shelled egg in the shell gland. In Experiment 6, a
subcutaneous injection of prostaglandin F2(50,ug/kg BW)

7 hr after indomethacin, expelled the last egg just prior
to ovulation of the next follicle. In Experiment 7, PGF2“
was used to prematurely expel the last egg in both the
control and treatment period at 0900 to 1000 hr prior to

LH injection. Birds were digitally palpated for the time

of arrival of the soft-shelled egg after placebo or indo-
methacin at 15 min intervals. Following indomethacin admin—
istration, the time from LH injection to the appearance of

a soft—shelled egg in the shell gland tended to be reduced
(0.1 to 2.2 hr) in both experiments.

It is concluded that prostaglandins probably do not
have an essential role in the processes of LH release or
ovulation, as previously shown in mammals. However, these

substances appear to have a role in the process of ovipo-

sition and may play a role in shell formation.

EFFECT OF INDOMETHACIN (A PROSTAGLANDIN INHIBITOR)
ON THE 0VULATORY CYCLE, TOTAL PLASMA CALCIUM,
AND EGG SHELL THICKNESS IN
THE DOMESTIC LAYING HEN
(GALLUS DOMESTICUS)

By

Randall William Hammond

A THESIS

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

MASTER OF SCIENCE

Department of Poultry Science

1977

to Lance and Christina

ii

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to

all individuals who made this thesis possible:
Dr. R. K. Ringer - my major advisor who enabled me
to gain research and teaching experience;
Dr. H. C. Zindel as Chairman of the Poultry Science
Department who made available the funds and facilities
required for this research;
Dr. J. L. Gill and Terry Wing for their statistical
guidance;
Drs. T. H. Coleman and B. J. Marquez for being com-
mittee members and enabling me to gain teaching
experience;
Mr. Sulo Hulkonen for technical assistance and care
of animals used in this research;
My wife, Bernadette, whose love and loyal support have

made my academic dreams become reality.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES ................................ v
LIST OF FIGURES ............................... vii
INTRODUCTION .................................. 1
REVIEW OF LITERATURE .......................... 3
OBJECTIVES .................................... 24

MATERIAISANDMETHODS O...OOOOOOOOOOOOOOOOOO... 25

Experiment 1 ................................ 26
Experiment 2 ................................ 28
Experiment 3 ................................ 32
Experiment 4 ................................ 35
Experiment 5 ................................ 37
Experiment 6 ................................ 38

Experiment 7 ................................ 39
Rmmum ”.n.”.H.H.H.H.H.H.H.H.H.u. 41
DISCUSSION .................................... 77
SUMMARY ....................................... 93
BIBLIOGRAPHY .................................. 95

iv

Table

l.

10.

LIST OF TABLES

Percent recovery of indomethacin from
pOOled hen plasma-i OOOOOOOOOOOOOOOOOOOOOOO

Time of oviposition for the first 3 eggs
in a sequence after indomethacin admin—
istration (50 mg/kg BW) as compared to
the average time of oviposition in the
four preceding sequences (expressed as
differences) ............................

Split-plot analysis of variance for
plasma levels of indomethacin at two
orally administered levels ..............

Loglo transformed plasma indomethacin
(Mean I SE) and the 95% confidence inter-
val over time ...........................

Unadjusted plasma indomethacin levels
(Mg/ml) over time .......................

Difference in oviposition time of the
last egg of the sequence and an addi-
tional LH-induced egg (hr) after indo-
methacin (50 mg/kg BW) ..................

Egg shell thickness of the last egg in
the sequence and the LH-induced egg after
indomethacin (50 mg/kg BW) ..............

Total plasma calcium levels (mg%) after
indomethacin (50 mg/kg BW) ..............

Difference in oviposition time of the

last egg of the sequence and an additional
LH-induced egg (hr) after indomethacin

(50 mg/kg BW) ...........................

Egg shell thickness (inches) for the last
egg in the sequence and the LH-induced
egg after indomethacin (50 mg/kg BW) ....

V

Page

31

42

45

47

48

54

55

58

6O

61

Table

ll.

12.

13.

14.

15.

16.

17.

18.

19.

LIST OF TABLES (cont'd)

Page

Total plasma calcium levels (mg%) after
oral dosing with indomethacin
(50 mg/kg BW) OOOOOOOOOOOOOOOOOOOOOOOOOO 63

Oviposition time of the last egg of the
sequence and an additional LH—induced

egg (hr) after indomethacin

(50 mg/kg BW) .......................... 64

Egg shell thickness (inches) for the

last egg in the sequence and the LH-

induced egg after indomethacin

(50 mg/kg BW) OOOOOOOOOOOOOOOOOOOO0.0... 66

Oviposition time of the last egg in
laying sequence and the LH-induced egg
after indomethacin (50 mg/kg BW) ....... 68

Time from LH injection to time of soft-
Shelled egg in the shell gland ......... 69

Egg shell thickness (inches) after PGF20L
and indomethacin (50 mg/kg BW) ......... 71

Time from LH injection to the time of
soft-shelled egg in the shell gland
after indomethacin (50 mg/kg BW) ....... 73

Time from soft-shelled egg in shell
gland and oviposition of the LH-induced

egg oooooooooooococo-00000000000000.coco 74

Egg Shell thickness (inches) for the

last egg in the sequence and the LH-

induced after indomethacin (50 mg/kg BW)

and PGF d.""°'°"'°"°’°"'°°'°"""' 75

Vi

LIST OF FIGURES

Figure Page

1. Experiment 2--Plasma indomethacin levels
over time, after a single oral dose ..... 49

vii

INTRODUCTION

In recent years, there has been a great deal of liter-
ature indicating a role for prostaglandins in several
aspects of mammalian reproduction. The majority of this
research deals with their smooth muscle stimulatory effect;
although these biologically active fatty acids may also
be acting as modulators of hormone actions.

Research suggesting a role for prostaglandins in the
reproductive physiology of the bird is limited to just a few
articles. These studies primarily deal with their possible
role in ovum transport and oviposition (egglaying). There
are no studies examining their possible role in gonadotropin
release or the process of ovulation in the bird.

In addition, there is evidence that prostaglandins may
play a role in calcium metabolism in mammals. Since the
regulation of calcium in the bird is extremely important
during the shell-forming phase of the ovulatory cycle, an
investigation of the possible role of these substances in
this phenomenon could be meaningful.

Several biologically active forms of prostaglandins
are available for experimental and clinical investigation,
as well as a large number of inhibitors and antagonists to

l

their biosynthesis.

In the following research, the prostaglandin synthe-
tase inhibitor indomethacin was administered orally to
laying hens to determine the effect of this drug on several
events in the laying cycle and on total plasma calcium.
Exogenous prostaglandin de was also administered to over-
come the indomethacin block of oviposition and as a tool
to determine the effect of this inhibitor on ovum trans-

port.

REVIEW OF LITERATURE

Indomethacin and Prostaglandins

 

Indomethacin (Indocin), l-(p-chlorobenzoyl)-5-
methoxy-2-methylindole-3-acetic acid, is one of the non-
steroidal anti-inflammatory drugs which has been shown to
block the synthesis of prostaglandins (Vane, 1971). This
experimental evidence offered support to the concept that
the therapeutic effects of aspirin-like drugs could be
ascribed to such properties. Smith and Lands (1971)
reported on the mechanism of this inhibition and have
shown that indomethacin inhibits the deoxygenase enzyme
of the sheep vesicular system in a time-dependent,
concentration-dependent manner, irreversibly blocking the
full activity of the synthetase system. Their work demon-
strated that the block in biosynthesis occurs at a very
early biochemical step, and thereby, shuts off the forma—
tion of all products--including all prostaglandins. The
discovery that indomethacin and many other inhibitors
interrupt the biosynthetic pathway at a specific point,
has allowed them to be applied as useful tools to eluci-
date the physiological actions of prostaglandins.

Prostaglandins are 20-carbon carboxylic acids which

3

H
are formed enzymatically from polyunsaturated fatty acids.
Pike (1972) has reviewed the chemistry of these biologically
active fatty acids and many of the methods used in synthe-
sizing them. In this discussion, he points out that these
substances are found in the tissues of many species through-
out the animal kingdom--coral to man.

Possible Role of Prostaglandins in Several
Aspects of Mammalian Reproduction

 

 

The role of prostaglandins in gonadotropin secretion
has been postulated. Labhsetwar (1970) administered high
doses of prostaglandin F2d(PGF2d) to pregnant rats and
found a several fold increase in the amount of stored
luteinizing hormone (LH) in the pituitary. Further work

(Labhsetwar, 1971, 1972) showed that PGF induced ovula-

2d
tion in pregnant rats and hamsterS--the latter study Show—
ing a Significant increase in LH in the blood after
administration. 0rcyzk and Behrman (1972) provided addi-
tional evidence for this possible role when they reported
the blockage of ovulation by aspirin and indomethacin in
the rat. In addition, Carlson et a1. (1973) also observed
an increase in serum LH after intracarotid infusion of
PGF26(6.ug/hr) during the luteal phase of cycling ewes.

In contrast, Batta et al. (1974) presented data that sug-
gest PGF20L , when given intracerebrally to unanesthetisized

female rats, partially inhibits ovulation, whereas PGE,

does not exert any effect. It is apparent from this work

5

as well as other examples, that the prostaglandins'
actions vary greatly between Species. The exact physi-
ological role of prostaglandins in gonadotropin release,
if any, is not clear (Goldberg et al., 1975). Some of
these effects may be pharmacological and not physiological.
Armstrong and Grinwich (1972) concluded that pros-
taglandins were also involved in the process of ovulation,
in that orally fed indomethacin was able to block the
action of LH at the level of the ovary in the rat.
Tsafriri et a1. (1972, 1973) confirmed this block by indo-
methacin, showing that this drug does not interfere with
LH release, but interferes with a late phase of the ovu-
latory cycle, in that it prevents follicular rupture and
not ovum maturation in the rat. This indomethacin-induced
ovulatory block was later shown to occur in mice (Lau
et al.,l974). Grinwich et a1. (1972) further demonstrated
that LH-induced steroidogenesis was not interfered with
in this indomethacin-blocked follicular rupture in the
rabbit. More recently, prostaglandins E and F2d have
been shown to be involved with, and essential to, the
normal process of ovulation in the rat and rabbit (Arm-
strong et al., 1974; Marsh et al., 1974). Marsh et a1.
(1974) reported that both LH and cyclic AMP increased
prostaglandin synthesis in rabbit Graffian follicles in

vitro.

6

One of the first, and possibly the most important
biological activity of prostaglandins to be observed was
the stimulation of smooth muscle (Khrzrok and Lieb, 1930).
Bygdeman (1964) studied this activity further on human
uterine smooth muscle, and this pharmacological principle
is still being evaluated. Subsequent studies elucidating
the role of prostaglandins in mammalian uterus and oviduct
are indeed voluminous (for reviews see: Southern, 1972;
Goldberg et al., 1975; Karim, 1975). The exact mechanism
of action of prostaglandin smooth muscle stimulation is
unknown. However, Carsten (1972, 1973) suggests that
prostaglandins may be involved in the regulation of intra-
cellular calcium transport in causing their effect.

In 1974, Csapo proposed that the mechanism of action
of prostaglandins in inducing abortion (clinically or
spontaneously) involves the immediately induced contraction
of the myometrium followed by progesterone withdrawal,
due to their luteolytic effect. Phariss and wyngarden
(1969) were the first to report the luteolytic activity
of prostaglandins (PGF2d.) in pseudopregnant rats. This
phenomenon was later to be reported for the guinea pig
(Blatchley and Donovan, 1969), rabbit (Phariss, 1970),
sheep (McCracken et al., 1970), and hamster (Gutknecht
et al., 1971; Labhsetwar, 1971). McCracken et a1. (1971)

demonstrated that tritiated PGF may be transferred

26.

7

directly from the uterine vein into the ovarian artery,
which lie in close apposition in the sheep, and travel

to the corpus luteum, where interference with progesterone
synthesis occurs. As a result, the progesterone effect in
maintaining a quiescent state of the uterus is overcome
(Csapo, 1956). Hansel et a1. (1973) demonstrated that
estrogens are luteolytic in cows and ewes, and that this
group of hormones is capable of stimulating prostaglandin
synthesis in the uteri of cows and guinea pigs. In the
sheep the onset of labor was preceded by an increased level
of estrogen, and Caldwell et a1. (1972) have shown that
estradiol infusion into pregnant women at term produced

an increase in uterine activity, but this was not sufficient
to achieve delivery. During the 4 hr infusion periods,
there were no changes in the plasma levels of progesterone
or PGF. There are undoubtedly many between-species varia-
tions as to the effects of these biologically-active fatty
acids.

Labhsetwar (1975) has discussed the possible role of
prostaglandins in parturition and the general uterine
physiology of laboratory animals, and suggests that data
obtained by using various approaches strongly implicates
prostaglandins in these areas. Ham et a1. (1975) suggest
that estrogen controls prostaglandin synthesis by regula-

ting the prostaglandin synthetase complex in the uterus of

ovariectomized rats.

Karim and Hillier (1975) summarized the evidence
implicating prostaglandins in the physiology of labor
and spontaneous abortion in humans as being based on:

”(a) Presence of prostaglandins in maternal blood obtained
during labour; (b) Presence in intrauterine tissues and
fluids; (0) The ability to stimulate pregnant human
uterus; and (d) Delay in onset of labour and prolongation
of active labour by prostaglandin antagonists and synthe-
sis inhibitors." Because of these implications and
findings, exogenous prostaglandins (particularly PGF2d_
and PGE1 ) are being used clinically as abortifacients,
to terminate early and late stage pregnancies, as well

as to enhance normal labor at term.

There appears to be a relationship between prosta-
glandins and oxytocin in their abortifacient effect. The
uterotonic effect of oxytocin in humans is negligible
during the first half of pregnancy, but it is markedly
enhanced after exposure of the myometrium to prostaglan-
dins (Brummer, 1971; Embry, 1971). A review of this
relationship by Karim and Amy (1975) points out that
prostaglandins and oxytocin appear to work synergistically
when given to women in the second trimester of pregnancy.
Sharma and Fithatrick (1974) demonstrated that relatively
large doses of oxytocin (400-10,000 IU) administered as

bolus intravenous injections, will elicit elevated

9
posterior vena caval PGF in estrogen-primed anestrous
sheep, whereas, there was no effect on the untreated
anestrous control animals. Roberts et a1. (1974) have
Shown that the manipulation of the uterus in the Sheep
also caused PGF release. From these results, it is not
clear, whether the effect of oxytocin in increasing PGF
is a metabolic action or a stimulatory effect due to
uterine contractions. The exact relationship between
oxytocin and prostaglandins remains unclear. However,
Novy et a1. (1974) showed that indomethacin prolonged
normal gestation in the rhesus monkey, but the uterus was
still responsive to exogenous oxytocin.

Prostaglandins, with their potent smooth muscle
stimulation activity, are also believed to be important
in the mammalian oviduct. Spilman and Harper (1974) have
reviewed the relationship of prostaglandins to oviduct
motility and suggest a physiological role for prosta-
glandins in the passage of fertilized ova through the
oviduct. In discussing this subject, Goldberg and Ram-
well (1975) state: "In all species studied, PGE inhibited
the spontaneous contractions of the fallopian tube while
PGF stimulated tubal contractility." Prostaglandin
treatment was either shown to retard or accelerate ova
transport depending on individual Species. This stim-
ulatory or inhibitory effect of prostaglandins may also

implicate them as having a functional role in Sperm

10
motility and implantation. Lau et a1. (1973) showed
evidence for a role of endogenous prostaglandins in
normal tubal—transport of ova, in that administration
of indomethacin (225,ug/animal) to mice on day 2 of preg-
nancy, prevented implantation.

In summary, prostaglandins have been implicated as
having a possible physiological role in mammalian repro-
duction by acting as hormone modulators and/or smooth
muscle stimulators. The actions of these biologically
active fatty acids may be at the level of the hypothalamus
and pituitary (gonadotropin synthesis and release); the
ovary (necessary for ovulation and luteolysis); the ovi-
duct (causing motility in sperm and ovum transport); and
the uterus (regulation of uterine contractions throughout
the reproductive cycle, particularly during spontaneous
abortion and normal parturition).

Possible Role of Prostaglandins in

Several Aspects of Avian
Reproduction

 

 

 

Historically, the presence of endogenous prostaglan—
dins and the actions of exogenous prostaglandins in avian
species were limited to the domestic fowl (for review see
Horton, 1971). Early studies revealed the presence of
PGE2 and PGFQQ' in the central nervous system of adUlt
fowl, and the administration of exogenous prostaglandins

would cause such effects as sedation or temporary paralysis

11
in the chick. Prostaglandins were also shown to cause
vasodilation and vasoconstriction in the cardiovascular
system of the chick, presumably through their smooth
muscle stimulatory effect on the blood vessels. More
recently, Christ and Van Dorp (1972) demonstrated the
presence of the prostaglandin synthetase enzyme system in
the ovaries and oviduct of the hen and the seminal ducts
and testis in the cock, as well as in many other non—
reproductive avian tissues.

Hertelendy (1972) was the first investigator to
report the smooth muscle stimulatory effect of prostaglan-
dins in the reproductive system of birds. This study
showed that intrauterine injection of prostaglandins El,
E2, Al, A2, F1 and F2d. all induced premature egglaying
(oviposition) in the coturnix (Japanese) quail. Of these
prostaglandins, PGEl, was the most potent--in very small
amounts (5 ng per bird). This research also demonstrated
that the avian uterus was capable of synthesizing endo—
genous prostaglandins in that endogenous precursors
(arachdonic acid and phospholipase A) were able to cause
premature oviposition-—although the increase in PG'S was
not measured directly. The action of these precursors was
blocked by intrauterine injections of indomethacin (0.1 mg),
whereas twice this amount of inhibitor did not block the
actions of exogenous PGEl. Hertelendy (1973) extended

this work to Show that there is a possible relationship of

l2
oxytocin and prostaglandins in oviposition, by reporting
that oxytocin-induced oviposition was blocked by indo-
methacin and 5, 8, ll, 14 -eicosatetraynoic acid (another
inhibitor of prostaglandin synthesis). However, these
two inhibitors were unable to block the actions of PGE1,
in expulsion of the uterine contents, thereby suggesting
a role for prostaglandins in spontaneous oviposition.
Dibutyryl cyclic AMP, linoleic acid and human seminal
plasma (shown to have large amounts of prostaglandins)
were also shown to be capable of inducing oviposition in
the quail, while administration of fowl, turkey, bull,
boar, and stallion seminal plasma were ineffective agents
(Hertelendy, 1974).

The hypothesis that prostaglandins have a role in
oviposition was later extended to the domestic hen (Her-
telendy et al., 1974). Prostaglandins El’ E2, and F2°L
as well as their fatty acid precursor-~arachidonic acid—-
all induced premature oviposition. Further observations
showed that the hen was more responsive to PGEl 1-3 hours
before the expected time of oviposition (hard-shelled egg
in the shell gland) than 15-18 hours from this time (soft-
shelled egg in shall gland) (Hertelendy et al., 1975).
This effect, however, could not be attributed to lowered
levels of the steroid hormone progesterone, as has been
hypothesized in mammals, as the circulating levels of

this hormone in the plasma were higher when there was a

13
hard-shelled egg in the uterus.

A possible role of prostaglandins in the physiology
of the avian oviduct has also been suggested. Wechsung
and Houvenaghel (1975 a, b) reported that PGF2Q and
PGE2 had a stimulatory effect on the muscle tone of

circular uterine strips and that PGF2 causes contractions

d
of circular smooth muscle strips from other oviduct seg-
ments. These researchers later reported that PGF2¢pro—
voked contractions of longitudinal tissue strips from
uterine, vaginal and isthmus regions of the chicken ovi-
duct (Wechsung and Houvenaghel, 1976). No response was
induced in longitudinal tissue strips from the magnum
and infundibulum. In contrast, PGE2 induced contractions
of circular and longitudinal uterine strips and circular
strips from the isthmus and infundibulum. PGE2 caused
relaxation in both the circular and longitudinal vaginal
strips. Circular magnum strips contracted to low doses

of PGE and relaxed to high levels, however, there was

2
no response to this prostaglandin in longitudinal muscle
strips from the isthmus, magnum and infundibulum. .22
ziyg experiments by these researchers (Wechsung and
Houvenaghel, 1975c) showed that intravenously administered
PGng and PGE2 induced a dose-dependent pressure increase

in the magnum and isthmus portion of the oviduct, in

anesthetized laying hens as measured by a water-filled

l4

balloon and pressure transducer. It was noted that by
using PGF2d_ and oxytocin as alternate intravenous injec-
tion--on a molar basis--that oxytocin was approximately
1.5 times more potent than PGde in the blood. In addi-
tion to the above investigations, Verma et a1. (1976)
tested the effect of PGE1, on oviduct segments from
infundibulum, magnum, uterus, uterovaginal junction and
the vagina of actively laying hens at preoviposition time
by in vitro and in vivo methods. This study reported a
maximum stimulatory response (contraction) from.musc1e
strips from the infundibulum and a complete relaxation
was recorded from vaginal strips. At the level of the
uterovaginal junction, there was a difference in in vitro
and in vivo studies--a mild prolonged inhibitory response
in vitro and an increase in intraluminal pressure (con-
traction) in vivo. From this study, these researchers
hypothesized that PGEl causes contraction of the uterus,
while at the same time causes relaxation of the uterovag-
inal junction during normal oviposition.

Talo and Kekalainen (1976) approached the question
of how ovum transport in the oviduct occurs by recording
the electric activity of Japanese quail oviducts in vitro.
In this study, six flexible suction electrodes were
placed at various intervals along the oviduct and the
electric activity was recorded as the ovum moved through

this area. These researchers concluded that the oviduct

15
promotes its own transport in that as it progresses
through the oviduct, it causes excitation of the smooth
musculature. It was hypothesized that distension caused
the ovum to initiate a positive feedback cycle in which
the rate of transport is determined by maximum activity
achieved behind the ovum and the steepness of the fre-
quency gradient. Excitation was believed to be caused
by the direct effects of mechanical distension and it was
suggested that prostaglandins may be involved in this
process in that stretch has been shown to enhance pros-
taglandin synthesis and release (Poyser et al., 1971).
It Should be pointed out, however, that prostaglandin
levels have not been measured in the avian oviduct during
transport.

The above research suggests that prostaglandins may
play a physiological role in ovum.transport and oviposition
in the bird, however, no studies have investigated the
possible role of these biolOgically-active fatty acids in
gonadotropin release or the complex process of ovulation
in these species. In addition, there have been no studies
reporting the effect of inhibitors of prostaglandin syn-
thesis on ovum transport (through the oviduct), or the
effect of these compounds on other aspects of the ovula-

tory cycle.

16
Prostaglandins and Calcium Metabolism

Another area of possible involvement of prostaglandins
in the bird and the mammal is calcium and bone metabolism.
This subject was reviewed by Dietrich and Raisz (1975).
Briefly, it has been demonstrated that a wide variety of
prostaglandins are capable of stimulating bone resorption
in tissue culture, as determined by the release of pre-
viously incorporated 45Ca from fetal rat long bones. PGE2
was found to be the most potent in its action. The morpho-
logical effects of prostaglandins on bone were qualita—
tively similar to those produced by other resorbing agents
(parathyroid hormone, active metabolites of vitamin D and
osteoclast activating factor). Though the effects of
prostaglandins on bone formation are not consistent, it
appears that PGEl and PGFld. stimulate, and possibly others
are involved in amino acid uptake and protein synthesis.
However, PGE2 actually decreased collagen synthesis in.
vitro, suggesting that some prostaglandins may inhibit
bone formation. This work suggested a hypercalcemic effect
of prostaglandins. This was later shown to be the case
in that prolonged intravenous infusion of PGE2 was able to
cause hypercalcemia in the rat. Administration of indo-
methacin to hypercalcemic mice and rabbits decreased serum
calcium and PGE2, as well as the PGE2 content of bone
tumors. These reviewers conclude that prostaglandins may

play a role in bone resorption and formation, as well as

17
in pathological changes in bone even though their physio-
logical role is unknown.

Considering the extremely high turnover rate of cal-
cium in the domestic laying hen and the importance of
calcium to egg shell formation (see following review of
reproductive cycle of the hen), this researcher decided
that plasma calcium levels should be determined after

indomethacin administration.

The Laying Cycle of the Domestic Hen

 

The "laying cycle" or "ovulatory cycle" of the domes-
tic hen has been reviewed quite extensively (see General
Bibliography). This description will deal with the general
aspects of this cycle, with Specific attention given to
those areas that will be dealt with in the following
research. Only specific reference will be made to publi-
cations whose detail are Considered to be important to
this study.

The "laying cycle” of the hen is relatively short in
duration, lasting about 25-26 hr from the time of ovulation
to the time of ovipositing (laying) of the egg. Hens will
lay one or more eggs on consecutive days (sequence or
clutch) and then end this daily phenomenon with one or
more "skip" or "pause" days (on which no egg is laid),
and then start a new sequence.

The endocrine control of the laying cycle is fairly

18

well understood, even though some aspects of it are still
unclear. Egg production begins when a hen reaches sexual
maturity and follicular growth begins~—apparently due
primarily to the release of follicle stimulating hormone
(FSH) from the anterior pituitary lobe. There is a grada-
tion in the maturation of these developing follicles in
that several of varying sizes exist at one time and grow
at different rates. The "egg formation phase" begins
with the ovulation of the largest follicle (in most cases),
which appears to be due to luteinizing hormone (LH)-~being
released from the anterior pituitary some 6 to 8 hr prior
to this event. Follicular rupture is preceded by the
formation of the "avascular-looking” stigmal area and
apparent reduction in blood flow. It is possible that
this sudden change in this preovulatory follicle is par-
tially caused by prostaglandins via their smooth muscle
stimulatory capabilities or other mechanisms, in that they
have been previously shown to be necessary in the process
of follicular rupture in several mammals (as discussed
above).

Immediately after ovulation, the ovum is engulfed
by the infundibulum (funnel) and spends the following
periods of time in each portion of the oviduct (average
figures from Warren and Scott, 1935): infundibulum,
0.3 hr; magnum, 2.9 hr; and isthmus, 1.5 hr. The average

time reported by these researchers, for ovum transport down

19
the oviduct from the funnel to the Shell gland, was
4.4 hr. After reaching the Shell gland, the egg remains
there during the shell forming process for a period of
approximately 20.8 hr. These time periods would vary,
depending on the length of the egg sequence, and other
variations due to individual birds.

During the ovum's journey down the oviduct, the
various layers of albumen are laid down in the magnum
portion; the two shell membranes are formed and some water
is added in the isthmic region; and in the shell gland
(uterus), water and salts are added to the albumen before
the deposition of the shell over the entire unit. In
addition, the chalazae (paired twisted strands of albumen
attached at opposite poles of the yolk, and parallel to
the long axis) are formed by the mechanical twisting
and segregation of the mucin fibers from the inner layer
of albumen (General Bibliography, Sturkie, 1975). Appar-
ently, the egg is rotated on its long axis in the isthmus
and uterus to produce these strands.

The shell forming phase, during the egg‘s long dura-
tion in the uterus, is extremely complex and a great deal
of information is known concerning it (see General Bibli-
ography, Sturkie (1975)--for reviews and major articles).
During this time, calcium carbonate deposition is the

prevailing event. This deposition gradually increases

20

during the first 5 hr to approximately 300 mg/hr and this
rate remains constant until about 2 hr before oviposition.
Because of this high rate of deposition and the shell
gland inability to store significant amounts of calcium,
this ion must be constantly extracted from the blood--
being derived from both the protein-bound and the diffu-
sible calcium of the blood (Simkiss, 1967). Hodges (1970)
showed that arteriovenous gradients in blood calcium can
be detected between the sciatic artery and the inferior
oviducal vein (draining part of shell gland) at approxi-
mately 2 hr after an egg enters the Shell gland. This
gradient ranged from 2-4 mg% between the fifth and the
sixteenth hr of shell formation, and then decreased grad—
ually. Sturkie (General Bibliography, 1975) points out
that the process of shell formation withdraws an average
of 100-150 mg calcium/hr from the blood of the laying hen,
and that unless the concentration of calcium in the blood
of the laying hen, and that unless the concentration of
calcium in the blood were not continuously replenished
through intestinal absorption and mobilization from bone,
this concentration would be zero within 8-18 minutes.

Taylor (1972) suggests that requirements for calcium
may be a limiting factor in egg production. In this
review, he points out that there may be a link between

calcium metabolism and ovulation. This is supported by

21

the fact that pullets fed a diet deficient in calcium
(0.2%) ceased laying after five or six eggs had been pro-
duced and had atretic follicles whereas control birds
given the same diet and injected with chicken pituitary
extract laid an egg a day for at least 5 days after the
first injection and their ovaries contained many growing
follicles (Taylor et al., 1962). Taylor (1972) suggests
that these results are due to a lowered ionic calcium
in the plasma causing a reduction in gonadotropin secre-
tion and reduced follicular growth, which in turn, would
reduce the rate of estrogen secretion and lower egg pro-
duction by lowering the rate of yolk synthesis in the
liver. In addition, this reviewer states that the neces-
sity to secrete large amounts of calcium for shell forma-
tion in some way limits egg production. In defense of
this position, he cites experiments by Lake and Gilbert
(1964) in which a surgical thread was placed in the shell
gland of laying hens. These researchers showed that this
operation caused the bird to lay thin or soft shelled eggs
due to premature expulsion from the shell gland and to
ovulate at a higher rate than the unoperated control.

The above research is interesting in that the reason
for the premature oviposition and subsequent higher rate
in ovulation may be directly or indirectly related to

prostaglandin synthesis. In the mammalian uterus,

22
Eckstein (1970) suggests that the presence of the foreign
intrauterine contraceptive devices (IUCD) causes
increased uterine and tubal motility via an increase
in prostaglandin synthesis. If this were the case in
the birds with the surgical thread in the uterus, then
prostaglandins may be the possible mechanism of the pre-
mature expulsion. In addition, if prostaglandins are
involved with calcium mobilization in birds, as previously
suggested in mammals, then via this mechanism an increase
in ovulation rate could come about. Another possibility
is the direct effect that prostaglandins may have an
ovulation in the bird. However, in addition to Lake and
Gilbert's (1964) experiments, Koga (1965) showed that
insertion of a glass catheter in the shell gland resulted
in premature eXpulsion of eggs, but in contrast a cessa-
tion of lay was the outcome. To date, a role for pros-
taglandins in regulating these events has not been estab-
lished.

The immediate causes of uterine contraction and ovi-
position are not completely understood. However, most
reviewers agree that the posterior pituitary hormone
arginine vasotocin may be the initiator of oviposition.
In addition, as previously discussed, prostaglandins may
play an important role as an intermediate or work syner-

gistically with vasotocin to induce oviposition. Other

23
agents such as acetylcholine, nembutal, and uterine dis-
tension may also cause premature oviposition, but it

appears that these effects are indirect.

OBJECTIVES

The purpose of this study was to investigate the
effect of the prostaglandin synthesis inhibitor indo-
methacin on total plasma calcium and the following
reproductive phenomena of the Single Combed White Leg—
horn (S.C.W.L.) laying hen:

l) Ovulation - as determined by the presence or
absence of a soft-shelled egg in the shell
gland after LH injection

2) Ovum transport - indirectly, by determining
the time from LH injection to the presence of
a soft-shelled egg in the Shell gland

3) Oviposition

4) Egg Shell thickness.

24

MATERIALS AND METHODS

General Procedures and Comments

 

The following experiments were conducted in a sequen-
tial manner in order to elucidate a possible role for
prostaglandins in the physiological regulation of several
reproductive events in the laying cycle of the hen.

Since levels of prostaglandins were not measured directly,
the amounts of inhibitor (indomethacin) were adjusted

to levels tolerated by the animals and those demonstrating
a dramatic delay in time of oviposition in the test
animals.

Seventy Single Combed White Leghorn (S.C.W.L.) laying
hens of a commercial strain, 18 to 20 months of age, were
randomly assigned to treatment groups. The hens were
housed individually in wire cages within three—tiered
portable laying battery units within a windowless room
with constant temperature (22 I 1°C) and forced air cir-
culation. All hens had a 3 to 6 egg laying sequence, with
a 1 skip day and no history of soft-Shelled or thin-
shelled eggs. A commercial layer ration and water were

given ad libitum. A 17.5 hr light and 6.5 hr dark photo-

 

period was maintained in which lights were automatically

25

26
turned off at 2300 hr and were turned back on at 0530 hr.
Under this lighting schedule, most eggs were laid between
1000 and 1600 hr. Times of oviposition were recorded
individually at 30 min intervals between 0900 and 1700 hr
each day. These records were started at least one month
prior to the beginning of the treatment periods.

Experimental Design and Procedures
of Individual Experiments

 

 

Experiment 1

 

Rationale

 

This experiment was conducted in order to test the
effect of indomethacin on the ovulatory cycle and normal
sequence of laying hens. Previous research has shown
that this drug will delay oviposition. Time of ovipo-
sition, effect on subsequent ovulations and general char-
acteristics of the eggs after a single bolus of the drug
were recorded.

Procedure

 

Indomethacin (Merck, Sharp and Dome, Rahway, N.J.)
was administered orally (50 mg/kg body weight (BW) in
gelatin capsules (No. 0, Eli Lilly and Company, Indian-
apolis, Indiana) to 7 randomly assigned hens at 2300 hr
(just before lights-out) on the evening of their skip day.
This was done by extending the bird's neck, placing the
capsule into the mouth, and flushing the bolus down the

esophagus with water. All birds were palpated manually

27
to confirm the presence of a hard-shelled egg in the
shell gland at this time. The subsequent effects on the
reproductive cycle were then monitored for the first 3
eggs in the following laying sequence. The effect of the
drug on ovulation was determined by digital palpation--
i.e., the presence of a soft-shelled or hard-shelled egg
in the shell gland after oviposition of the first egg in
the sequence was noted. In addition, the effect of indo-
methacin on the time of oviposition of the first 3 eggs
in the present sequence was compared to the average ovi—
position time of these eggs in the 4 previous egg
sequences, respectively. The time of oviposition was
monitored at 30 min intervals during the light hr only,
in order that no disruption of the photoperiods of these
birds and other birds within this room would occur. Those
eggs (soft or hard-shelled), which were found the follow-
ing morning, were recorded as being laid at 0215 hr (mid-
way through the dark period). When lights came on at
0530 hr, all the treatment birds were palpated digitally
for the presence of a hard-shelled or soft-shelled egg
in the shell gland and this information was recorded.
General observations of the effect of the drug on the

birds and the eggs laid were noted.

28
Statistical Analysis

 

Student's t-test for paired observations was used
for the statistical analysis of data. The fact that
previous research (Hertelendy, 1972, 1973) and the
experimental results showed a delayed-oviposition effect

of the drug, a one-tailed test was used.

Experiment 2

 

Rationale

 

AS will be Shown subsequently, the results from
Experiment 1 indicated that considerable variation in
response to orally fed indomethacin existed between indi-
vidual birds. In an attempt to reduce this variation,
feed was removed from the birds several hr before the
bolus was given. Levels of indomethacin in the plasma
were also determined over time, in order to establish
a dose-response.

In addition, the administration of 100 mg/kg BW
dose of indomethacin to one treatment group was decided
upon, because the 50 mg/kg level in Experiment 1 had
failed to block ovulation of the second egg of the
sequence, and in 5 of 7 birds did not block the ovulation
of the third egg of the sequence. It was felt that a
higher level of the drug may cause blockage of gonado-
trOpin release and/or ovulation, as previously shown in

mammals.

29

Part 1: Determination of Plasma Indomethacin by
Serial Bleeding

 

Procedure

 

Two groups of 7 laying hens each, were given a Single
bolus of indomethacin (as previously described in Experi-
ment 1) at levels of 50 mg/kg BW and 100 mg/kg BW,
respectively, at approximately 1100 hr. Feed was removed
from the birds just before lights-out of the previous
evening (2300 hr) and returned one hr after drug treat-
ment.* Ten heparinized blood samples (4 ml each) were
then taken from the wing vein of each bird at the follow-
ing time periods after drug administration: 0 (just prior
to dosing--used for control), 1, 2, 4, 6, 8, 10, 18, 24,
and 32 hr. The collected heparinized blood was immediately
centrifuged (2600 g) in a refrigerated centrifuge for
10 min. The plasma was then pipetted into 12 x 75 mm
stoppered test tubes and refrigerated at 4°C until the
time of assay for indomethacin content--within 72 hr of
collection.

The plasma samples were assayed by a slightly modi-
fied spectrophotofluorometric technique previously
described by Hucker et al. (1966). This procedure is

as follows:

 

*This procedure was used in all subsequent experi-
ments, in both the control and treatment periods.

l)

3)

5)

30
Two 1 ml samples of plasma were pipetted into
two 40 ml glass—stOppered centrifuge tubes con-
taining 2 ml of 0.5 M citrate buffer (pH 5.0) each.
To each tube, 25 ml of heptane containing 3%
isoamyl alcohol were added and the tubes were
shaken for 15 min.
The tubes were then centrifuged for 10 min at
2600 g and 20 ml of the organic phase (upper
portion) were transferred into two 40 ml centri-
fuge tubes containing 5 ml of 0.1 N Na OH.
The tubes were Shaken for 5 min and centrifuged
at 3000 g for 15 min.
The upper organic phase was then carefully aspir-
ated, and fractions of the bottom aqueous phase
were transferred to a quartz cuvette for fluores-
cence reading, in triplicate. These readings
were then averaged for final determination of
levels of indomethacin.
The fluorescence was measured in an Aminco-Bowman
spectrophotofluorometer (activation maximum,
295 mAL; fluorescence maximum, 385 m); uncorrected).
Standards (1 to 10 Alg/ml) were made alkaline
(0.1N NaOH) and read at the beginning, middle,
and end of each recording period. These values
were averaged and a standard curve plotted for
each group of samples that were read at the same

interval.

31
8) Recovery from pooled laying hen plasma with addi-
tions of indomethacin up to 50 Img/ml and refrig-
erated for 72 hr was 95.10 t .65% (Means i S.E.)—-

see Table 1.

 

 

Table 1. Percent recovery of indomethacin from pooled
hen plasma.
Amount Added (Ms/ml) 5 10 25 50
Sample No. l 92.00 96.67 97.80 94.42
2 90.80 100.22 93.04 93.94
3 92.00 96.67 94.64 93.94
4 98.80 99.36 91.44 92.20
5 96.20 99.36 94.64 93.94

 

Analysis of Data

 

Plasma indomethacin levels that were taken by repeat

measurement were analyzed by a split-plot method pre-

viously described by Gill and Hafs (1971).

correct for heterogeneous variance and non-normal distri-

In order to

bution, the individual plasma indomethacin values up

through the 18 hr were transformed to their loglo equiv-

alent (Gill, 1977) and analyzed by a computer using a

Split-plot analysis of variance program.

32

Part 2: Determination of Plasma Indomethacin After
One SampIe Bleeding

 

 

Procedure

 

Serial bleeding and possible hemodilution may have
had an effect on the levels of indomethacin in the blood
over time. In order to test for this possible effect,
two more randomly assigned groups of 7 birds each were
given a Single bolus of 100 mg/kg BW of indomethacin per
bird, after taking a control sample of blood (4 ml) from
the wing vein. An additional 4 m1 sample was drawn from
one group at 8 hr and the other group at 32 hr post treat-
ment. These blood samples were treated in the same
fashion as those in Part 1 of Experiment 2 in determining
the indomethacin levels.

Analysis of Data

 

The values of indomethacin in plasma at 8 hr and
32 hr post—treatment for this experiment were compared
to the two respective time period levels of the serially-
bled birds in Part 1 of Experiment 2 by a t-test for

differences (Gill, 1977).

Experiment 3

 

Rationale

 

From the results of Experiment 1, as will be shown
subsequently, it was noted that LH release or the sub-
sequent ovulation of the second ovum in the sequence was

not blocked by indomethacin. For the third ovum of the

33

sequence, 2 of 7 birds did not ovulate. This may indi-
cate an effect of the drug on gonadotropin release or an
effect on maturation of smaller follicles, or a failure
of the oviduct to receive the ovum. The first egg of

the sequence appeared to have a very thick Shell after
its delayed period in the shell gland, whereas the second
and third eggs seemed to vary greatly in their egg Shell
thickness, as well as their time in the shell gland.

In order to avoid a possible effect of indomethacin
on gonadotropin release, luteinizing hormone (LH) was
injected on the morning of the day of the last egg of
the hen's laying sequence, and thereby adding an addi-
tional egg onto the sequence. Total plasma calcium and
egg shell thickness was also measured.

Procedure

 

Seven randomly assigned birds were subjected to a
control period in which each bird was used as its own
control, allowed to complete 1 or 2 normal sequences,
and was then given indomethacin during the day of the
last egg of this latter sequence. In the control period,
each bird was given a placebo (empty gelatin capsule)
between 0900 and 1000 hr followed by an intravenous
(i.v.) injection of LH (Bovine-NIH-B8) into the wing
vein, at the dosage of 60 ng/kg BW--l hr after placebo.

The treatment period was conducted in the same

34
fashion as in the control period, except that the birds
were given a 50 mg/kg BW dose of drug in a gelatin capsule
instead of an empty capsule. Ovulation of the next ovum
would occur approximately 8 hr after the LH injection--
approximately 9 hr after drug administration.

The day of the last egg was determined by past laying
records, and the presence of the egg in the shell gland
was determined by digital palpation. The time of ovipo-
sition of the last egg of the sequence and LH induced egg
were determined. These eggs were broken open, any internal
abnormalities were noted, and the egg shells were rinsed
clean of all albumen with cold water and allowed to dry
for 48 hr at room temperature. The egg shell thickness
was then measured, including the shell and shell membranes,
with an Ames Thickness Measure and recorded to the nearest
ten thousands of an inch.

One ml heparinized blood samples were taken from the
wing vein at 11, 24, and 32 hr after placebo. These
samples were centrifuged at 2600 g in a refrigerated cen-
trifuge and the plasma was transferred to 12 x 75 mm glass
test tubes with plastic stoppers which were frozen (-5°C)
until analysis for total calcium--within 72 hr. The total
calcium content was measured by a Corning Calcium Analyzer,
Model 940, in duplicate, and the average value was

recorded in mg%.

35

The same procedures as above were followed in the
treatment period, with a 50 mg/kg BW dosage of indome-
thacin being administered 1 hr before the LH injection.

The time from the LH injection to the presence of
a soft-shelled egg in the shell gland was determined
by digital palpation, at 15 min intervals, during the
control period and when the presence of hard-shelled egg
did not prohibit the timing of this event in the treat-
ment period.

Statistical Analysis

 

Each bird was used as its own control in this experi-
ment. A t-test for paired comparisons was used for com-
paring differences in oviposition times, egg shell thick—
ness, and total plasma calcium levels in the treatment

period as compared to the control period.

Experiment 4

 

Rationale

 

In Experiment 3, several of the last eggs in the
sequence were expelled during the dark hours (lights-out),
as were a few of the soft-shelled LH-induced eggs. The
times of these expulsions were determined by duplicating
Experiment 3, and placing the hens in an adjacent room
during both the control and treatment periods. This was
done so that the birds could be monitored for time of
oviposition without disruption of the other birds in

their previous environment.

36

Procedure

 

Seven randomly selected hens were subjected to the
same treatment as the hens in Experiment 3 and the same
parameters were measured. However, this group was removed
from their light-controlled caged environment and placed
in new cages in another room during the dark cycle of
the control and treatment periods and returned to their
former cages after each period. These birds were then
monitored for the time of oviposition at 30 min intervals
throughout the dark portion of the photoperiod. The egg
Shell thickness of the last egg and LH-induced (additional)
egg in both the control and treatment period were measured
and compared, as in Experiment 3. Blood samples were
taken for total plasma calcium determination at 11, 24,
and 32 hr after placebo or indomethacin treatment, as pre-
viously described in Experiment 3.

Statistical Analysis

The t-test for paired comparisons was used for compar-
ing differences in oviposition times, egg shell thickness,
and total plasma calcium levels in the treatment period
as compared to the control period, as was done in Experi—

ment 3.

37

Experiment 5

 

Rationale

 

The previous experiments demonstrated no block in
ovulation when indomethacin was given before LH release
or LH injection. It is possible that LH is able to over-
come any negative effect that indomethacin may have via
reduction in prostaglandin synthesis. Indomethacin may
also be clearing from the hen's tissues and blood before
the ovulation process. Administering indomethacin after
LH injection and closer to the time of ovulation may
enhance a block of ovulation.

Procedure

 

Seven randomly selected hens were injected with LH
(60 Rig/kg BW) between 0900 and 1000 hr and were given a
placebo (empty gelatin capsule), 1 hr afterwards on the
day of the last egg in the sequence. The birds were then
monitored for effect on the subsequent ovulation induction
and time of oviposition.

In the treatment period, indomethacin (50 mg/kg BW)
was given in place of the empty capsule after each bird
had completed one or two sequences after the control

period.

38

Experiment 6

 

Rationale

 

In the previous experiments, indomethacin did not
block LH-induced action when administered either 1 hr
before or 1 hr after the LH injection. Because of the
delay of the last egg in the sequence, it was impossible
to determine the time from LH injection to the presence
of a soft-shelled egg in this organ. In this experiment,
PGF2d_ (The Upjohn Company, Kalamazoo, Michigan) was used
as a tool to induce oviposition of the last egg in the
sequence, 6 hr after LH injection or 7 hr after indomethacin
administration so that the effect of the time from LH to
ovulation and time for ovum transport could be determined.

Procedure

 

Seven birds randomly assigned to this treatment group
were used as their own controls, given placebo in the
control period and indomethacin (50 mg/kg BW) in the treat-
ment period between 0900 and 1000 hr on the day of the last
egg of the sequence. One or 2 egglaying sequences lapsed
between the control and treatment periods. Luteinizing
hormone was given 1 hr after oral placebo/indomethacin
treatment. Saline in the control period and PGFZQ.

(5O ,Mg/kg BW) was administered subcutaneously in the treat-
ment period, 6 hr after the LH injection or 7 hr after the
placebo/indomethacin treatment. This procedure induced

oviposition of the last egg of the sequence just before the

39

ovulation of the LH-induced ovum in the treatment period.
The birds were then palpated at 15 min intervals from

9 hr after LH administration to the time of a soft-shelled
egg in the Shell gland. These times were recorded for the
control and treatment period and compared statistically
by paired-t analysis, as were egg shell thickness differ-

ences .

Experiment47

 

Rationale

 

AS will be presented, results from Experiment 6 sug—
gest that indomethacin given 8 hr before ovulation (assum-
ing 7 hr from LH injection to ovulation), had little effect
on the time from LH injection to the entrance of a soft-
shelled egg into the shell gland, even though there was a
trend toward a reduction of this time after indomethacin.
Indomethacin levels in the plasma at this time are, however,
quite low (4 to 6ng/ml plasma during transport period),
as determined in Experiment 2.

Procedure

 

In this present experiment, the last egg of the
sequence was expelled from the shell gland (6-7 hr pre—
maturely) between 0900 and 1000 hr on the day of the last
egg of the sequence in both the control and treatment
periods by an S0 injection of PGF2& (501Lg/kg BW). Lutein-

izing hormone was then injected 1 hr after the premature

40
expulsion (60,ag/kg BW), followed by placebo/indomethacin
orally 3 hr after this injection. According to the plasma
indomethacin data in Experiment 2, this would subject each
bird to levels of approximately 30 ug/ml of indomethacin
in the plasma just prior to ovulation and during the ovum
transport period (5aug/ml as ovum reaches the uterus).
The birds were palpated at 15 min intervals beginning 8 hr
after the LH injection for the presence of a soft-shelled
egg in the Shell gland and these times were recorded and

compared between the control and treatment period.

RESULTS

Experiment 1

 

The data collected in Experiment 1 are tabulated in
Table 2. Analysis of the data by the t-test for paired
comparisons showed that time of oviposition of the first
egg in the sequence of all birds was significantly dif-
ferent (delayed) from the average time of oviposition
of the first egg of the four previous sequences (p < 0.01),
whereas the second and third eggs of the same treatment
sequence were not significantly delayed (p > 0.2) when
compared to their respective average oviposition times
of four previous sequences. In fact, 5 of 7 of the
second eggs in the post—treatment sequence were prematurely
expelled from the uterus, ranging from 6.9 to 15 hr
(p < 0.01), whereas, 2 of 5 of the third eggs in the
post-treatment sequence were prematurely expelled by 1.2
and 10.2 hr.

Six of the second eggs in this sequence were expelled
as soft-Shelled eggs; 5 of these prematurely and l was
delayed by 7.5 hr (Bird No. 6) from the average time of
the previous four sequences of this bird for this egg in
the sequence.

The second egg of the treatment sequence of Bird

41

42

Table 2. Time of oviposition for the first 3 eggs in a
sequence after indomethacin administration
(50 mg/kg BW) as compared to the average time
of oviposition in the four preceding sequences
(expressed as differences).

 

 

 

 

. — — b —
Bird No. C1-Cl (hr)a 02-02 (hr) 03-03 (hr)
1 + 11.8 — 9.2 + 2.5
2 + 12.3 - 9.2 + 0.8
3 + 12.9 - 6.9 + 3.1
4 + 21.8 + 20.1 N.D.

5 + 5.0 - 7.8 - 10.2
6 + 17.5 + 7.5 N.D.
7 + 11.7 - 15.0 - 1.2
Significance p < 0.01 N.S. N.S.

 

aDifference in oviposition time of indomethacin
treated egg sequence (C1 = first egg; C2 = second egg;
and C3 = third egg) and the average time of oviposition

of eggs of the four preceding sequences, i.e., Cl’ Cé
and C5, respectively.

bPrematurely expelled (p < 0.01).

N.D. - No data--no egg laid and no soft-shelled egg
after oviposition of second egg in sequence.

43

No. 4 was dramatically delayed by 20.1 hr. Despite this
longer period in the shell gland, this egg was very thin-
shelled and possessed an indentation in the side of the
egg, giving this region a flat-type surface. An egg with
this shape is sometimes referred to as a "truncated” egg.
This indentation occurs when a soft-shelled egg enters
the Shell gland at the same time that a hard-shelled egg
is present. The pressure of the uterine musculature upon
this new soft-Shelled egg causes an indentation in the
Shell membranes and the shell is laid down with the egg
in this position with this Shape solidifying, giving the
Shell an indented appearance. The second egg of the other
birds of this treatment group, with the exception of Bird
No. 5, also had a "truncated" appearance, even though
there was little or no calcification of the Shell on these
eggs at the time of their expulsion. The lack of this
irregular shape in the second egg of Bird No. 5 was prob-
ably due to the relatively short period of delay in ovi-
position of the first egg (5.0 hr). This normal shape
indicated that the soft-shelled egg had not arrived or
just arrived at the shell gland before the expulsion of
the first egg (new soft-shelled egg in shell gland 5-6 hr
after normal expulsion of the previous egg).

There was no ovulation or the ovum was not picked up
by the fimbriae ofthe oviduct for the formation of the

third egg in the treatment sequence in Birds No. 4 and 6.

44

These two birds also experienced a delay in oviposition
time of the second egg of 20.1 hr and 7.5 hr respectively.
The previous sequences of these 2 birds were 4 to 5 eggs
in length.

The first, second, and third hard—shelled eggs in
the treatment sequence were collected and broken out to
check for any obvious visual abnormalities. The first
eggs, which were held in the shell gland for an extended
period of time, appeared to have very thick and chalky-
looking shells. The second "truncated" eggs appeared to
have relatively thin-shells, as compared to the first egg
of this sequence and eggs of previous sequences. The
third eggs of the sequence (total of 5) appeared to be
normal as to shell-thickness, with the exception of Bird
No. 5's egg, which was quite thin--probab1y due to its
premature expulsion of 10.2 hr.

There were no abnormalities found in the interior of
these eggs when they were broken out into Petri dishes

and examined with the unaided eye.

Experiment 2

 

Part 1

The analysis of variance data are presented in
Table 3. The f-Statistic for treatments indicates that
the levels of indomethacin in the plasma for the

50 mg/kg BW and 100 mg/kg BW are significantly different

45

Table 3. Split-plot analysis of variance for plasma
levels of indomethacin at two orally admin-
istered levels.l

 

 

 

Source of Sum of Mean f-
Variation d.f. Squares Squares Statistic
Treatment 1 1.978 1.978 8.447*
(level of drug) _
Animals Within 12 2.811 0.234

Treatments

Time 6 24.85 4.142 34.23**
Treatment X Time 6 0.499 0.083 0.688
Residual Error 72 8.711 0.1210

Total 97 38-85

 

1Values are plasma indomethacin expressed in lo

g10
of,ug/m1 of plasma.

-X-
Significant at p 0.01.

-X-*
_Significant at p < 0.01.

46

(p 21.01). In addition, the two levels of drug are dif—
ferent in the plasma at the different sampling times
(p < 0.01). However, there is no interaction between
treatments and time, indicating that the curves for the
two levels are essentially parallel and different at most
points between the curves. Presented in Table 4 are the
loglO transformed plasma indomethacin values for the two
treatment levels as determined over time. The mean
values over time for the 100 mg/kg BW level of drug
ranged from 1.51 to 57.54 ug/ml plasma, whereas, the
50 mg/kg BW level range was from 1.10 to 38.011Mg/ml
plasma.

The original unadjusted data is shown in Table 5
and represented graphically in Figure 1. These mean I
standard error (SE) values for the 100 mg/kg BW values
ranged from 0.79 I 0.25 to 62.4 I 9.79,ug/ml plasma
(mean I SE) whereas, the 50 mg/kg BW dose values ranged
from 0.44 i 0.14 rig/ml to 48.0 I 28.3 pkg/ml (Table 5).
Both plasma levels of indomethacin peaked at 2 hr after
administration and both levels were at very low levels
in the plasma 18 hr after oral dosing and almost com-
pletely cleared at 32 hr.

The 95% confidence interval of the overall mean
values (Table 4) are 0.97 I 0.15 (loglo) for the
50 mg/kg BW dose of indomethacin with a lower limit of

6.6l.ug/ml and an upper limit of 13.18 ug/ml. This same

Table 4. LoglO transformed plasma indomethacin levels
SE) and the 95% confidence interval
over time.

(Mean

47

 

 

Time After
Administration
(hr)

1O

18

 

Marginals--
(overall mean
I SE)

95% confidence
interval of
overall mean
values

antilog values

A |-—J Aw AU‘I Aw

6.61, 13.18

12.

Levels of Indomethacin (mg/kg BW)

100

.46 I 0.18

(28.84)

.76 i 0.18

(57.54)

.64 I 0.18

(43.65)

.53 I 0.18

(33.88)

.30 I 0.18

(19.95)

.94 I 0.18

(8.71)

.18 I 0.18

(1.51)

.26 I 0.07

(18.20)

.26 I 0.15
.11, 1.41

88, 25.70

 

lUpper values in each cell are loglo transformed

mean I SE values and the lower value is the antilog of

the mean value (pg/ml plasma).

48

Table 5. Unadjusted plasma indomethacin levels (Mg/ml)
over time.

 

 

Time After
Administration Levels of Indomethacin (mg/kg BW)

(hr) 29. £99

1 35.3 I 9.63a 36.7 I 7.87
2 48.0 I 28.30 62.4 I 9.79
4 30.0 I 9.33 47.1 I 7.40
6 18.8 I 5.62 35.6 I 4.87
8 6.9 I 1.02 21.6 I 3.02
10 4.2 I 0.99 10.9 I 1.78
18 1.6 I 0.42 1.9 I 0.46
24 0.8 I 0.26 1.2 I 0.42
32 0.4 I 0.14 0.7 I 0.25

 

aMean I SE.

49

Figure 1. Experiment 2--Plasma indomethacin levels
over time, after a Single oral dose.

 

$505 NEE.

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O O O O O
to <1- m N -—
(um/5d) NIOVHIBWOONI vwsvwo Nvaw

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51
confidence interval for the 100 mg/kg BW dose is
1.26 I 0.15 (loglo) with a lower limit 12.88 .ug/ml and
an upper limit of 25.701ug/ml.

Although the birds were subjected to treatment
without regard to the presence or absence of a soft—
Ishelled or hard-shelled egg in the shell gland, 3 birds
of 14 given the 100 mg/kg BW oral dose with a hard—shelled
egg in the shell gland at the time of dosing, laid very
thin-shelled "truncated” eggs after the expulsion of the
first delayed egg. When broken open, the interior of
these eggs were normal looking, with the exception of
there being no highly twisted chalazae at each pole of
the yolks. This was not noted in "truncated" eggs from
the birds given the 50 mg/kg BW dose.

Fifteen of the 21 birds given the 100 mg/kg dose
of indomethacin died between 50 and 80 hr after treatment
(4 of the serially-bled and 11 of the birds that were
bled twice). For this reason, this level of drug was
not used in any subsequent experiments.

Just prior to death, a 1 m1 sample of heparinized
blood was taken from the wing vein of 2 of the birds that
were bled twice. These samples were centrifuged in a
refrigerated centrifuge (2600 g) for 10 min and total
plasma calcium was determined on a Corning Calcium Anal-
yzer Model 940. The average values of duplicate reading

for these 2 birds were 6.92 and 8.53% (normal values of

52
the laying hen being 20-30 mg% (Sturkie, 1976).

Soon after death, these 15 birds were autopsied.
Upon examination, the internal organs and supporting
tissue were Spotted with an off-white granular precipi-
tate, which was presumed to be uric acid, indicating
kidney dysfunction. Indomethacin may have profound
effects on the gastrointestinal tract and the kidney in
that the prostaglandins, whose synthesis is inhibited by
indomethacin have been shown to be necessary in the
process of digestion via their smooth-muscle stimulating
effect and have a role in normal kidney function. In
addition, follicular atresia was evident throughout the
ovaries of all 15 of these birds.

It was noted that at 2 to 4 hr or longer after oral
dosing at both the 50 and 100 mg/kg BW levels, that the
crops of the birds were greatly engorged with feed and
water and that the amount of excreta deposited beneath
the cages was markedly reduced as compared to pre-treatment
observations.

Part 2

The t-test for differences revealed that there was
no significant differences between the serially-bled and
the birds that were bled either 8 or 32 hr after dosing
at the 100 mg/kg BW level. The values for the serially-
bled birds were 21.6 I 3.02,ug/ml plasma (Mean I SE) at

the 8 hr bleeding and 0.7 I 0.26,ug/ml at the 32 hr bleed-
ing, as compared to 20.2 I 5.24,ug/ml at 8 hr and

53
0.5 I .31 ug/ml at 32 hr bleeding for the two groups that

were bled at these time periods respectively, after a

control bleeding.

Experiment 3

 

In Table 6 are Shown the differences in the times
of oviposition after indomethacin treatment. The last
egg in the sequence was significantly delayed (p < 0.01)
when compared to the average time of the last egg of the
four previous sequences (CT'-CT). This time of delay,
when compared to the time of oral indomethacin adminis-
tration ranged from 16.5 to 28.3 hr. With the exception
of l bird (No. 6 - 28.3 hr), all times of delayed ovi-
position from the time of drug were from 16 to 18 hr.
The clearance curve in Experiment 2 (Figure 1) indicates
that this is the approximate time that the drug had
dropped to very low levels in the plasma. The LH-induced
egg, which was added on to the sequence in both the con-
trol and treatment period was expelled prematurely
(p < 0.05) in 6 of 7 birds in the treatment phase
(CI'—CI). Three of these birds expelled these soft-shelled
eggs with or Shortly after the delayed hard-shelled eggs
during the dark portion of the photoperiod. AS in Experi-
ment 1, the eggs found at 0530 hr (lights-on) were esti-
mated to be expelled at 0215 hr. Therefore, the time
from drug administration to the time of expulsion of the

last delayed egg and the LH-induced egg were recorded as

54

 

 

 

 

 

Table 6. Difference in oviposition time of the last egg
of the sequence and an additional LH-induced
egg (hr) after indomethacin (50 mg/kg BW).

Bird No. (CT'-CT) (CT'-TO) (CI'-CI) (CI'-TO)

1 + 10.0 + 16.5 — 17.1 + 16.5
2 + 9.8 + 16.7 - 17.7 + 16.7
3 + 11.4 + 17.5 + 1.2 + 34.5
4 + 11.4 + 16.6 - 1.0 + 34.1
5 + 12.4 + 18.2 - 15.6 + 18.2
6 + 22.4 + 28.3 - 0.4 + 33.5
7 + 9.7 + 16.8 - 1.1 + 31.7
Significance p < 0.01 p < 0.05
CT - average time of last egg in four previous
sequences
0 '-time of last egg after indomethacin treatment

C
C
T

T
I
I
O

— time
'- time

- time

of LH-induced egg in control period
of LH-induced egg after indomethacin

of indomethacin administration

55

Table 7. Egg Shell thickness of the last egg in the
sequence and the LH-induced egg after indo-
methacin (50 mg/kg BW).

 

 

 

 

Bird No. Egg Shell Thickness (inches)
(CT"CT) (CI"CI)
1 + .0013 — .0139
2 + .0011 - .0112
3 + .0020 - .0063a
4 + .0007 - .0077a
5 - .0001 - .0103
6 - .0001 - .0091a
7 - .0002 _ ,oo5ha
Significance p < 0.05 p < 0.01
CT - last egg in sequence of control phase
CT! - last egg in sequence of treatment phase
CI - LH-induced egg of control phase
01' - LH—induced egg of treatment phase

a - Oviposition time was only slightly premature
or Slightly delayed as compared to control
period.

56
being the same time. All 3 of these soft-shelled eggs
had a Slightly "truncated" appearance, indicating that
they had spent some time in the shell gland with the
last egg of the sequence (blocked from expulsion from
the shell gland by indomethacin). The other 4 LH-
induced eggs also had this "truncated" appearance after
indomethacin treatment. All of the birds responded to
LH injection in both the control and treatment period,
as determined by the presence of a soft-shelled egg in
the shell gland. The time of the soft-shelled egg's
arrival to the Shell gland could not be determined due
to presence of the delayed hard-shelled egg, as was the
case during the control phase of this experiment. The
time from LH to soft-Shelled egg in the uterus ranged
from 12.3 to 13.4 hr (Mean I SE = 12.8 I 0.14 hr) in the
control period. Assuming that the time of transport
down the oviduct is 4.4 hr, as previously determined by
Warren and Scott (1935), then the average time from the
LH injection to the time of ovulation for these birds is
approximately 8.4 hr.

Egg Shell thickness data for the last egg in the
sequence and the additional LH-induced egg are shown in
Table 7. The difference in Shell thickness between the
last egg in the treatment and control phase (CT'-CT) are

significant at p < 0.05. Four of the last eggs in the

57
treatment phase had Slightly thicker egg shells, whereas

the 3 remaining eggs had slightly thinner egg shells.

The shell thickness of the LH—induced egg in the
treatment phase was significantly different (thinner)
from the control egg (CI'-CI). It Should be noted that
3 of these eggs were prematurely expelled by a short
interval (0.4 to 1.1 hr) and the other egg was laid 1.2 hr
later than the control egg. When these 4 eggs were com-
pared to their controls, they were also significantly
thinner (p < 0.01).

Total plasma calcium levels after indomethacin are
given in Table 8. The calcium levels were significantly
reduced (p < 0.01) at 11, 24, and 32 hr after oral dosing

with indomethacin.

Experiment 4

 

The last eggs in sequence were laid between 0200 and
0945 hr the following day (dark cycle to early lights-on)
--4 of these being laid during the dark cycle at 0200,
0230, 0400, and 0430 hr. The other 3 birds oviposited
their eggs at 0630, 0830, and 0930 hr. Bird No. 2 laid
a soft—shelled egg approximately 15 min prior to laying
the delayed last egg of the sequence. This bird was dig-
itally palpated after discovery of the unplumped soft-
shelled egg and the delayed last egg of the sequence was

felt in the Shell gland of the bird. This bird was checked

58

 

 

 

 

Table 8. Total plasma calcium levels (mg%) after indo-
methacin (50 mg/kg BW).
Bird No. Control After Indomethacinl
11a 24 32 11 24 32
1 24.71 21.92 22.04 13.40 11.71 12.79
2 26.40 25.16 28.59 22.31 21.58 21.56
3 22.56 22.13 21.10 19.36 17.81 20.01
4 17.53 17.10 18.42 10.59 8.40 9.73
5 31.52 24.92 29.31 19.15 20.02 20.22
6 21.22 20.99 24.61 15.13 16.74 16.40
7 25.22 27.84 29.43 22.71 18.43 21.56

 

lSignificantly lower at all time periods (p < 0.01)

as compared to controls.

aHours after placebo/indomethacin administration.

59
following a 15 min interval and the hard-shelled egg had

been oviposited.

The last eggs of the sequence were Significantly
delayed (p < 0.01) when compared to the mean value of
the last eggs of the four previous laying sequences
(Table 9)° The last egg of the sequence of Bird No. 6
was delayed by 42.2 hr. The same bird was the only bird
that did not respond to the LH injection (as determined
by failure of the presence of a soft-shelled egg in the
shell gland after the delayed egg was oviposited) and
went out of production for more than 30 days post-
treatment.

As previously shown in Experiment 3, the delayed
eggs were oviposited after the drug was shown to be in
low levels in the plasma (17-18 hr).

Egg shell thickness data for the last egg in the
sequence and LH-induced egg are shown in Table 10. After
the indomethacin treatment, the shells of the delayed
last eggs in the sequence were thicker than controls
(p < 0.1). All but 2 of these eggs were thicker than
the pre-treatment control (CT'-CT).

The LH-induced egg was significantly thinner after
indomethacin (p < 0.01) as compared to the control Shell
thickness (CI'-CI), even though 5 of these eggs were only

slightly prematurely expelled or delayed by a short period

60

Table 9. Difference in oviposition time of the last egg
of the sequence and an additional LH-induced
egg (hr) after indomethacin (50 mg/kg BW).

 

 

 

 

Bird No. Time of Oviposition
(CT"CT)* (CT"T0) (01"01) (Clt-TO)

1 + 12.8 + 20.4 + 0.2 + 34.0
2 + 12.7 + 20.1 — 13.2 + 19.8
3 + 10.8 + 17.6 + 2.3 + 36.8
4 + 11.2 + 17.1 + 0.2 + 33.2
5 + 14.5 + 24.5 - 1.6 + 32.6
6 + 42.2 + 47.2 N.D. N.D.
7 + 15.1 + 21.4 - 1.8 + 35.9

CT - average time of last egg in four previous

sequences

CT'- time of last egg after indomethacin treatment

CI - time of LH-induced egg in control period

CI'- time of LH-induced egg after indomethacin

TO - time of indomethacin administration

9(-

- time of oviposition significantly delayed
(p < 0-01)

N.D. - No data - no ovulation from the LH injection
during the treatment period.

61

 

 

 

 

Table 10. Egg Shell thickness (inches) for the last egg
in the sequence and the LH-induced egg after
indomethacin (50 mg/kg BW).

Bird No. Egg Shell Thickness (EST)

(CT"CT) (01"CI)
1 - .0004 - .0062a
2 + .0007 - .0119
3 .0000 - .0061a
4 + .0011 - .0019a
5 + .0003 - .0082a
6 + .0005 N.D.
7 + .0001 - .0034a

Significance p < 0.1' p < 0.01

CT - EST of last egg in sequence of control period

CT'- EST of last egg in sequence of treatment
period

CI - EST of LH-induced egg of control period

CI'- EST of LH—induced egg of treatment period

- Oviposition time was only slightly premature
or slightly delayed as compared to control
period.
N.D. - No data--no response to the LH-injection as

determined by no soft-Shelled egg in the
shell gland.

62
of time (Table 10 - (CI'-CI). When these 5 eggs were
compared to their controls they were also thinner Shelled
(p < 0.01).
The differences in total plasma calcium levels are
shown in Table 11 and demonstrate the same Significant
drop (p < 0.01) after indomethacin, as was the case in

Experiment 3.

Experiment 5

 

Indomethacin (50 mg/kg BW) did not block the ovula-
tion process when administered 1 hr after the LH injection.
All 7 of the birds responded to the LH injection, as
determined by the presence of a soft~shelled egg in the
shell gland after oviposition of the delayed last egg.

Table 12 shows the results of delay or change in
oviposition times for the last egg in the sequence and
the LH-induced additional egg. The time of expulsion of
the last egg in the sequence was significantly delayed
(p <0.01) when compared to the average time of expulsion
of the last egg of the four previous sequences (CT'-CT).
This egg was expelled from the Shell gland between 12.2
to 22.6 hr (Mean 2 15.8 hr) after drug administration—-

a time when drug is at low levels in the plasma, as was
shown in the two previous experiments. After indomethacin
treatment, all of the birds expelled the LH—induced egg

prematurely as compared to controls (CI'-CI) and from

Table 11.

63

Total plasma calcium levels (mg%) after oral
dosing with indomethacin (50 mg/kg BW).

 

 

Bird No.
113‘
1 31.05
2 26.20
3 24.51
4 21.38
5 23.78
6 18.07
7 19.54

Control

24

29.40
24.78
24.21
20.59
23.85
17.28
18.85

32

30.
24.
23.
21.
22
17.
19.

70
85
89
84

.52

84
72

After Indomethacin

11

19.98
17.30
16.23
l7.9l
16.18
14.95
15.72

24

14.74

9.34
15.41

8.11
18.54
13.91
12.84

1
32

18.65

5.48
14.10
10.54
19.21
11.27
17.74

 

lSignificantly lower at all time periods (p < 0.01)

as compared to controls.

23’Hours after placebo/indomethacin administration.

64

 

 

 

Table 12. Oviposition time of the last egg of the sequence
and an additional LH-induced egg (hr) after
indomethacin (50 mg/kg BW).

Bird No. Time of Oviposition (hr)

(CTI-CT) (CT"T0) (CI"CI) (CI"TO)
1* + 11.5 + 13.5 - 16.1 + 13.5a
2 + 11.2 + 15.7 - 3.1 + 30.0
3 + 10.4 + 15.6 - 16.2 + 15.68‘
A + 7.9 + 12.2 - 18.4 + 15.5a
5 + 9.3 + 15.4 - 0.2 + 32.4
6 + 15.4 + 22.6 - 8.9 + 23.0a
7 + 9.1 + 15.3 - 1.8 + 29.9

icance

 

*Bird died about 30 hr after indomethacin admin-
istration.

aSoft-shelled eggs expelled prior to or shortly after
the expulsion of the indomethacin delayed last egg.

CT

H000
OHHI-Ei

average time of last egg in four previous
sequences

time of last egg after indomethacin treatment
time of LH-induced egg in control period
time of LH-induced egg after indomethacin

time of indomethacin administration.

65
15.5 to 32.4 hr (Mean = 22.8) after the drug administra-

tion (CI'—T Four of the birds expelled the LH—induced

6)-
egg prior to, or shortly after the expulsion of the
indomethacin-delayed last egg of the sequence. The time
of expulsion of either of these eggs was estimated to be
0215 hr or midway through the dark cycle as in Experi-
ments 1 and 3.

Differences in egg shell thickness in the control
and treatment period for the delayed last egg and the
LH-induced egg are tabulated in Table 13. The indomethacin-
delayed last egg was significantly thicker than the con-
trols (p < 0.05) even though 3 of these birds laid slightly
thinner eggs (.0001 to .0002 in.thinner). All of the
LH-induced eggs were prematurely expelled when compared
to controls. This premature expulsion was confounded by
the egg Shell thickness data (CI'-CI), which Showed that
these egg shells were significantly thinner (p < 0.01)
than controls. Three of these birds expelled these eggs
a short time before their control oviposition time (Birds
Nos. 2, 5, and 7 with 3.1, 0.2, and 1.8 hr premature ex-
pulsion, respectively). When these 3 eggs were compared
to their control, they were also thinner (p < 0.01).

Bird No. 1 died about 30 hr after indomethacin.treat-
ment. Despite this lethal dose, ovulation after the
LH injection in the treatment period was not blocked.

Autopsy revealed the same basic internal abnormalities

66

Table 13. Egg shell thickness (inches) for the last egg
in the sequence and the LH-induced egg after
indomethacin (50 mg/kg BW).

 

 

 

 

Bird No. Egg Shell Thickness (EST)
:_ 1.
(CT CT) (CI CI)
1 + .0018 - .0108
2 + .0011 - .0047
4 - .0002 - .0110
5 + .0012 - .0055
6 - .0001 - .0101
Significance p < 0.05 p < 0.01
CT — EST of last egg in control period
CT' - EST of last egg in treatment period
CI - EST of LH-induced egg in control period
CI' - EST of LH-induced egg in treatment period

67
that were noted in the 15 birds dying from the 100 mg/kg

BW dose in Experiment 2.

Experiment 6

 

In the control period, all 7 birds failed to respond
to the subcutaneous (SC) saline injection, whereas, all
birds expelled their eggs within 2.6 to 4.5 min after the
SC injection of PGFQG (Mean = 3.7 min). Table 14 Shows
that the indomethacin block of oviposition, shown in the
previous experiments, was over-ridden by the SC injection

of PGF The first column (CT'-CT) shows that the sig-

2d:
nificantly delayed egg (p < 0.01) was expelled by PGF20k
0.1 to 1.2 hr after the expulsion time of the control.

The second column (CI'-CI) indicates that 5 of 7 birds
prematurely expelled the LH-induced egg after indomethacin
treatment, despite the fact that there was no delayed egg
in the Shell gland (having been previously removed by

the PGF injection). These premature expulsions ranged

2m
from -O.4 to ~17.5 hr from the control period's expulsion
times. The 2 remaining birds slightly delayed their
expulsion time by 0.3 and 0.5 hr as compared to their
own control period.

All of the birds responded to the LH injection in
both the control and treatment periods, as indicated by

the presence of a soft-shelled egg in the shell gland.

Compared in Table 15 is the time from the LH injection to

68

Table 14. Oviposition time of the last egg in laying
sequence and the LH-induced egg after indo-
methacin (50 mg/kg BW).

 

 

 

 

Bird No. Oviposition Time (hr)
(CT"CT) (CIt-CI)
l - 0.6 + 003
2 - 0.1 - 10.8
3 - 1.0 - 17.4
4 - 0.7 - 17.5
5 - 0.2 - 0.4
6 - 0.4 + 0.5
7 - 102 -' 2.9
Significance p < 0.01 p < 0.01
CT — average time of last egg in four previous
sequences
CT'- time of PGng expelled egg from bird treated
W1th 1ndome ha01n
CI — time of LH-induced egg in control period
CI'- time of oviposition of LH-induced egg after

indomethacin

Table 15.

69

Time from LH injection to time of soft-Shelled
egg in the Shell gland.

 

 

Bird No.

NQU‘l-P'UONH

Time from LH injection to time of
soft-shelled egg in shell gland (hr)

 

Control (C) Treatment (T)_ C-T
12.6 12.7 + 0.1
11.5 13.3 + 2.2
11.5 13.3 - 1.8

9.1 8.4 + 0.7
9.8 10.2 - 0.4
12.0 11.8 + 0.2
11.1 10.8 + 0.3

 

70
the time of a soft-shelled egg in shell gland for both the
control and treatment period. The difference (C-T) in
times were not significant when analyzed by the t-test for
paired comparisons, even though 5 of 7 of the soft-shelled
eggs (after indomethacin) arrived at the Shell gland sooner
than the controls by 0.1 to 2.2 hr.

The egg shell thickness of the last egg of the sequence
and the LH-induced egg in both the control and treatment
period are tabulated as differences in Table 16. There is
no significant difference between the thickness of the
last egg in the sequence between the control and treatment
period (CT'-CT). The difference in these thicknesses
ranged from -.0038 to +.0012 in. However, there is a sig-
nificant difference in the LH-induced eggs in that the
PGF2¢ and indomethacin treated egg in significantly thinner
(p < 0.01). This thinness in egg shells appears to be
partially due to premature expulsion of these eggs. How-
ever, the reduction in total plasma calcium may be also
contributed to this phenomenon (as shown in Experiments 3

and 4).

Experiment 7

 

One of the 7 birds (No. 1) did not respond to the LH
injection in the treatment period as it had in the control
period two sequences prior to this time. The other 6

birds responded to the LH injection in both the control

71

Table 16. Egg shell thickness (inches) after PGF20L and
indomethacin (50 mg/kg BW).

 

 

 

 

Bird No. Egg Shell Thickness (EST)
(CT"CT) (CI'"CI)
l + .0012 - .0029
2 - .0038 - .0039
3 .0000 - .0101
4 + .0002 — .0113
5 - .0004 - .0016
6 - .0003 - .0031
7 + .0001 - .0044
Significance N.S. p < 0.01
CT - EST of last egg of sequence in control period

CT' EST of last egg of sequence in treatment
period

0
l

I EST of LH-induced egg in the control period

01' EST of LH-induced egg in the treatment period

72
and treatment period, even though indomethacin was in rela-
tively high levels in the plasma in the pre-ovulatory phase
and prior to and during the ovulation process. The expul-
sion of the last egg in the sequence by PGF2d in both the
control and the treatment period ranged between 2.6 to
5.2 min after the SC injection of this prostaglandin
(Mean I SE = 4.0 I .25 min).

After indomethacin, 5 of the 6 birds responding to the
LH injection demonstrated a Significant reduction in the
time period required for the LH to cause ovulation and for
this ovum to be transported to the shell gland (p < 0.05),
as compared to pre-treatment (control) time periods
(Table 17). These values ranged from 0.2 to 1.4 hr differ-
ence from controls. The other bird delayed the ovulation/
transport of the treatment egg 0.1 hr.

The LH-induced egg was prematurely expelled in 4 of 6
birds which responded to the LH injection in the treatment
period (Table 18). This premature expulsion of this egg
was not significant (p < 0.2) when all 6 times were compared
to the control by the t—test for paired comparisons. These
eggs were expelled 24.8 to 31.5 hr after indomethacin
treatment (Mean = 28.1 hr). Thus, considerably after the
time when indomethacin in the plasma is at very low levels
(18 hr). The time that these LH-induced eggs spent in the
shell gland was Slightly longer for controls than for the

treatment period (Mean = 23.3 and 22.0 hr, respectively)

73

Table 17. Time from LH injection to the time of soft-
Shelled egg in the shell gland after indo-
methacin (50 mg/kg BW).

 

 

 

Bird No. Time from LH injection to time of
soft—shelled egg in Shell gland (hr)

Control (C) Treatment (T) C-T*

1 11.5 N.D. N.D.

2 10.1 9.1 - 1.0

3 9.3 7.9 - 1.4

4 9.1 8.5 - 0.6

5 10.3 9.5 - 0.8

6 11.6 11.7 + 0.1

7 8.4 8.2 - 0.2

 

*Significant difference in LH to soft-shelled egg
time (p < 0.05)

N.D. - No data--bird did not respond to LH injection
in the treatment period.

74

 

 

 

 

 

 

 

Table 18. Time from soft-shelled egg in Shell gland and
oviposition of the LH-induced egg.
Bird Time (hr)
No.
:_ z- _ t- t _
(CI CI) (cI To) (cI SSI) (cI SSI ) (s0I SGI)
l N.D. N.D. N.D. N.D. N.D.
2 + 1.0 26.6 21.8 20.8 - 1.0
3 — 2.5 28.2 24.4 23.4 - 1.0
4 - 3.9 24.8 24.6 19.3 — 5.3
5 - 4.0 27.2 24.0 20.7 - 3.3
6 + 1.4 31.5 21.5 22.8 + 1.3
7 + 2.5 30.2 23.4 25.0 + 1.6
Sig. p < 0.2 p < 0.05
CI’ I - time of oviposition of LH-induced egg in
control and treatment period, respectively
SSI, SSI' - time of soft-shelled egg in shell gland for
control and treatment period, respectively
(from time of LH injection)
SGI’ SGI' - total time of the egg in the shell gland for
control and treatment period, respectively
TO - time of indomethacin administration
N.D. — No data-—bird did not respond to LH injection

in the treatment period.

75

Table 19. Egg shell thickness (inches) for the last egg
in the sequence and the LH-induced after indo-
methacin (50 mg/kg BW) and PGF2d .

 

 

 

 

Bird No. Egg Shell Thickness (EST)
(CT"CT) (CI"CI)
1 — .0030 N.D.
2 - .0227 - .0075
3 + .0003 - .0043
4 + .0013 - .0069
5 - .0003 — .0057
6 + .0003 - .0033
7 - .0003 - .0036
Significance N.S. p < 0.01
CT — EST of last egg in sequence in control period
after premature expulsion by PGFZM
CT! - EST of last egg in sequence in treatment
period after premature expulsion by PGF2«_
CI - EST of LH-induced egg in control period

01' — EST of LH-induced egg in treatment period

76
as shown in Table 18. Four of these eggs, after indo-
methacin, were in the shell gland for a shorter period
than their controls (SGI'-SGI).

Differences in egg shell thickness for the last egg
in the sequence and the LH-induced egg are presented in
Table 19. The last egg in both the control and treatment
period were prematurely expelled from the shell gland
by an injection of PGF2d , as previously described.
Column (CT'-CT) indicates that the differences in thick-
nesses between control and treatment as being different,
but not significantly so. The second column (CI'-CI)
indicates that the LH-induced egg was significantly
thinner (p < 0.01) even though only 5 of these birds
prematurely expelled these eggs by 0.2 to 1.4 hr.

DISCUSSION

Experiment 1

 

The results from Experiment 1 (Table 2) indicate that
the oral administration of indomethacin (50 mg/kg BW) to
laying hens, significantly delays the normal expulsion of
the first egg of the sequence, when the dose was given
11 to 12 hr prior to the normal time of this event. The
fact that indomethacin caused a delay in time of oviposi-
tion in the S.C.W.L. hen has been previously reported by
Hertelendy et a1. (1974). The fact that various prostaglan-
dins were able to overcome this prostaglandins inhibitor's
block of oviposition, suggests that prostaglandins may play
an active role in the normal process of oviposition.

The effect of indomethacin or other prostaglandin
inhibitors on the subsequent eggs in a sequence (ovulation
and oviposition of) has not been previously reported. In
this experiment, the ovulation of the second ovum in the
sequence was not blocked by the single bolus of indo—
methacin-—given approximately 4-5 hr before LH-release
and 11-12 hr before ovulation of this ovum would have
occurred. This observation is not in agreement with previous
work reported in mammals, that inhibitors of prostaglandins

(specifically indomethacin) will block the process of

77

78
ovulation and the belief by several researchers that pros-
taglandins may have an essential role in gonadotropin
release and ovulation (see Review of Literature).

The time of oviposition of the second and third egg
were not Significantly delayed. Five of 7 of the second
eggs were prematurely expelled by 6.9 to 15.0 hr. The
fact that the first egg was laid, in most cases, during
the dark portion of the photoperiod, and the time of the
first egg and the second soft-shelled egg, were reported
as being the same time (0215 hr) may cause these recorded
expulsion times to be in error by several hours. However,
the fact that 4 of 5 of these soft—shelled eggs had a
”truncated" appearance indicates that these eggs spent
some period of time in the shell gland with the first egg
in the sequence. It is possible that these soft-shelled
eggs were expelled from the shell gland at about the same
time as the first eggs in the sequence.

The 2 birds which delayed the second egg in the
sequence, as well as the first egg, also did not receive
a third ovum in the shell gland for the production of a
third egg-~as could be predicted by their past laying
records of 4 and 5 eggs per sequence. It was not deter-
mined whether ovulation had not occurred or that the fim-
briae of the oviduct had failed to pick up the ovum after

ovulation. The bird with the greatest delay of expulsion

79
(No. 4 - Table l) of both the first and second egg (21.8
and 20.1 hr, respectively), also had a lower production
rate (4 egg sequences) as compared to Bird No. 6 (5 egg
sequences).

0f the 5 birds which a third egg in the treatment
sequence, 3 of these eggs were delayed slightly from their
normal time of expulsion-~as compared to the average time
of expulsion of the third egg in four previous control
sequences. The 2 other birds expelled the third egg 1.2
and 10.2 hr prematurely.

The data for oviposition time from the second and
third egg (Table 1) offer no real pattern or trend, in
that time of expulsion of the eggs, were both premature
and delayed. The fact that Birds No. 4 and 6 delayed the
expulsion of the second egg, may have some relationship
to the fact that no third ovum was ovulated or present in
the oviduct. Ovulation of these ovum would have normally
occurred about 30 min after the normal eXpulsion of the
second egg. In the case of Bird No. 4, this ovulation
would have occurred about 19.6 hr before the time the
delayed second egg was oviposited. If we assumed a 4.4 hr
time for transport of this ovum to the shell gland, this
would place the egg in the shell gland 15.2 hr before the
delayed second egg's expulsion. In the case of Bird No. 6,

ovulation for the third egg would have been 7.0 hr before

80
the delayed expulsion of the second egg, and this ovum would
have arrived at the shell gland 3.6 hr before the expulsion.
The other 5 birds prematurely expelled the second egg by
9.6 I 1.4 hr (Mean I SE) or about 9 hr prior to the ovulation
of the third ovum. At the time of the expulsion of the
second egg, no soft-shelled eggs were found in the shell
gland as determined by palpation. The exact time of arrival
of the third egg to the shell gland was not determined.

If one assumes that no ovulation occurred for the third
ovum in the sequence for Birds No. 4 and 6, there are
several plausible explanations for this occurrence. The
indomethacin, via inhibition of prostaglandin synthesis,
may have inhibited the release of LHRH (luteinizing hormone
releasing hormone) and LH for this ovulation. By this same
mechanism, FSH (follicle stimulating hormone) release may
be depressed, causing the lack in the maturation of devel-
oping follicles, preventing them from responding to LH.
Another possibility is that indomethacin is interfering
with normal levels of other hormones or factors (such as
ion levels), that are necessary for the completion of the
ovulation process. If ovulation did occur, then the egg
was not received by the fimbriae of the oviduct, for trans-
port to the shell gland.

Previous research has indicated that prostaglandins
may be involved in calcium regulation in mammals (see

Review of Literature). In this experiment, the eggs after

81
indomethacin appeared to have thinner egg shells (even
though a considerable amount of time was spent in the Shell
gland), suggesting that this drug may have an effect on
calcium.levels, and by this mechanism, dictate the amount
of Shell that is deposited, and directly or indirectly
dictate the time and amount of gonadotropin release. Some
of these possibilities were looked into in other experi-

ments within this research project.

Experiment 2

 

The results from Experiment 2 (Table 5 and Figure 1)
indicated that indomethacin was found in the plasma within
1 hr was at maximum levels at 2 hr was at very low levels
at 18 hr, and was essentially cleared in 32 hr after oral
dosing at levels of 50 and 100 mg/kg BW.

The statistical analysis of these two curves suggests
that they are parallel and essentially different at all
sampling times up to 18 hr.

The reliability of these levels, due to the large
amount of blood removal from repeat sampling (40 ml), was
partially substantiated by the results in Part 2 of Experi-
ment 2. The results indicate that there was no significant
difference in the plasma indomethacin levels between two
groups of birds which were bled twice when compared to the
serially-bled birds at two different time periods, for the

100 mg/kg BW level.

82

The 100 mg/kg BW oral dose of indomethacin was not
administered in any of the subsequent experiments, because
15 of 21 birds given this dose died within 50 to 80 hr
after dosing. The exact cause of death was not completely
substantiated, however, the fact that the total plasma
calcium was extremely low just prior to death and the
presence of an off-white precipitate in the internal organs,
indicate that the low blood calcium and kidney dysfunction
may have been contributing factors.

This toxic dose also caused follicular atresia in all
15 of these birds. This may indicate a reduction in the
levels of the ovarian supporting hormones. However, the
mechanisms of this level change via reduction in the appro-
priate levels of essential prostaglandins, would have to

be verified by measuring these substances.

Experiments 3 and 4

 

Experiments 3 and 4 were different only in the fact
that the birds in Experiment 4 were continuously checked
at 30 min intervals during the dark portion of the photo-
period in the treatment phase of this experiment. For this
reason, the results of these two experiments will be dis-
cussed simultaneously.

The last egg of the laying sequence in both eXperiments
was quite dramatically delayed in its expulsion from the

shell gland, after an oral dose of 50 mg/kg BW of

83
indomethacin--combined, the mean I SE = 14.7 I 2.28 hr
(n = 14).

The average combined time of the difference from admin-
istration time to the time of delayed oviposition of this
egg was 21.4 I 3.10 hr (Mean I SE; n = 14). This would
indicate that the delayed expulsion occurred a few hours
after the indomethacin in the plasma had reached a very
low level (Figure 1). Assuming that this level of prosta-
glandin inhibitor is only expressing its delaying effect
by way of prostaglandin reduction, then this recovery and
subsequent expulsion of the delayed egg seems to offer some
evidence that prostaglandins play an essential role in
oviposition.

The expulsion time of the delayed last egg of the
sequence and the soft-shelled LH-induced egg were estimated
in Experiment 3; whereas, in Experiment 4, the hens were
checked every 30 min during the dark portion of the photo-
period in the treatment period. This more precise timing
of these expulsions of eggs during this period, may in part,
have contributed to the differences in these mean recorded
values. The mean I SE delay for Experiment 3 was 12.4 I
1.70 hr and in Experiment 4 this value was 17.0 I 4.23 hr.
It appears that the delay of the last egg's expulsion may
have been poorly estimated in Experiment 3. However, one

bird in Experiment 4 delayed the expulsion of this egg by

84
42.2 hr. If this value was not included, then the mean
I SE delay for Experiment 3 was 12.9 I 0.70 hr.

Bird No. 6 in Experiment 4 did not respond to the
LH injection after indomethacin, as it had in the previous
control period. This response could be contributed to an
interference by indomethacin in the maturation and rupture
of the largest follicle, an anti-LH response of the bird
due to antibody development after the LH injection in the
control period, or a poor i.v. injection of the LH. The
latter is highly unlikely due to caution taken to make
consistently clean injections, but due to the positive
response of the other 13 birds in these 2 experiments,
under the same conditions, it is probably the best explan-
ation.

The data in Tables 7 and 10 indicate that the egg
shells are thicker than the controls for 9 of the 14 birds
in these experiments (p < 0.05 for Experiment 3; p < 0.01
for Experiment 4). In the remaining eggs, there was no
difference (0.000) to - .0004 inches difference in thinner
egg shells. This indicates that the calcium deposition
process was not completely stopped during this extended
period of time in the Shell gland (about 50-60% longer
than normal). In contrast, all of the LH-induced eggs
were thinner (p < 0.01) in the treatment period than they

were in the LH—induced control period. The thinner Shell

85
condition was partially confounded with premature expulsion
of some of these eggs. However, 9 of 12 of these thinner
treatment eggs were only slightly premature or slightly
delayed when compared to their control's expulsion time.
This would indicate that indomethacin was causing some dis-
ruption of the calcium deposition process.

The results from the total plasma calcium sample taken
during the control and treatment period offer some evidence
that there may be a reduced calcium deposition rate in the
shell gland due to the lower amount of calcium available
for this process.

In both experiments, the post-indomethacin total plasma
calcium levels were significantly reduced as compared to
pre-drug control levels, for all 14 birds.

The time periods for blood sampling (1 m1 aliquots)
were selected as 11, 24, and 32 hr after indomethacin, in
accordance with the time of shell formation. Luteinizing
hormone was injected 1 hr after the indomethacin bolus was
given. Under these conditions, ovulation would occur at
about 9 hr after the drug was given and the soft-Shelled
egg would arrive in the shell gland 4-5 hr afterwards, thus
the egg would spend more than 20 hr undergoing shell forma-
tion. This would place the time of the first blood sampling
at about 2-3 hr before the soft-shelled egg arrived in the
shell gland, the second at about midway in the Shell depo-

sition process, and the third at a period after oviposition

86
and at a time when indomethacin was almost completely
cleared in the blood.
For discussion and comparison purposes, the mean I
SE of all of the plasma control samples at each time period
for both experiments was 23.51 I 0.616 mg% calcium (n = 42)
--See Table 8 and 11. The mean I SE for all the treatment
samples was 15.90 it 0.664 mg% calcium (n = 42). This
represents about a 30% reduction in the total plasma cal-
cium after the oral indomethacin treatment.

The actual physiological events, which lead to this
reduction in the total plasma calcium, were not determined
in this study. However, several researchers have sug-
gested that prostaglandins may play a role in the regula-
tion of calcium in several mammals (Dietrich and Reiz,
1975). Indomethacin may cause lowered calcium by directly
or indirectly lowering dietary calcium absorption in the
gastrointestinal tract or lowering calcium.mobilization
from the medullary bone.

During the dark portion of the photoperiod, in the
treatment phase of Experiment 4, it was noted that Bird
No. 2 had expelled the LH—induced soft-shelled egg about
15 min prior to the expulsion of the delayed last egg in
the sequence. This soft—Shelled egg was not plumped out
with water and salts from the Shell gland and was there-
fore, quite small in size compared to a normal egg,thus

allowing this egg to leave the shell gland through a

87
smaller opening. Digital palpation of this bird, immedi—
ately after finding this soft-shelled egg, revealed that
the uterovaginal region was very taut, partially dilated,
and unelastic. This condition would inhibit the expulsion
of a normal hard-shelled egg. Verma et a1. (1976) has
hypothesized that the prostaglandin PGEl relaxes the smooth
muscle in the uterovaginal region, while at the same time,
causing the shell gland smooth muscles to contract and
thereby expel the egg.

In addition, observation of birds in the dark and
early lights-on portion of the photoperiod, after indo-
methacin treatment, revealed that all of the birds 1 to
2 hr prior to ovipositing the delayed last egg of the
sequence, experienced quite rhythmic and deep abdominal
contractions. It appeared that they were trying (unsuc-
cessfully) to rid their shell gland of the delayed egg by
use of their skeletal (voluntary) muscles. When observed
closely, this phenomenon was also noticed in other birds

given this level of indomethacin.

Experiment 5

 

Indomethacin (50 mg/kg BW), administered 1 hr after
an LH-injection, did not interfere with the physical process
of ovulation of the LH stimulated follicle. Figure l indi-

cates that the plasma levels are more than twice as high

88
at the time of ovulation (about l9.ug/ml) as the plasma
levels when indomethacin was given 1 hr before the LH
injection (about 7,0g/ml), as in the previous experiments.

The last egg in the sequence, which was in the Shell
gland at the time indomethacin was given, was delayed an
average of 15.8 hr from their normal time of expulsion.

As was Shown in the previous experiments, this period of
delay can be correlated with a time when indomethacin was
at very low levels in the blood. Results from Experi-
ment 3 and 4 indicate that this expulsion time occurred

at a time when total plasma calcium levels were about 30%
lower than their levels in the control period (11 to 32 hr
after indomethacin). There is a possibility that the
calcium levels in the blood may be dictating, in part, the
period of time that an egg Spends in the Shell gland. This
hypothesis can be supported, in part, by the fact that in
this eXperiment, as well as in the previous, all or a large
number of the LH-induced eggs were prematurely expelled
after indomethacin treatment.

In this experiment, as well as the previous ones in
which egg shell thickness was measured, premature expulsion
of the LH-induced egg resulted in thinner egg shells. How-
ever, when one looks at individual birds which did not pre-
maturely expel this egg (or only expelled this egg by a
Short period before the control time), these eggs were

still significantly thinner than controls.

89

Experiment 6

 

The results of this experiment, indicate that the
presence of a hard—shelled egg in the shell gland was not
the reason that premature expulsion of the additional
LH-induced egg was being prematurely eXpelled from the
Shell gland, as seen in the previous experiments. These
data Show that even with no indomethacin-delayed hard-
Shelled egg in the shell gland that 5 of 7 eggs were pre-
maturely expelled (ranging from 0.4 to 17.5 hr) when com-
pared to their own control oviposition time for this
LH-induced egg. The thinner egg shell of the treatment
LH-induced egg reflects this shorter period of time in
the Shell gland.

There appears to be some difference between the con-
trol and treatment periods (though not significant) as to
the time from LH injection to the time that the soft-shelled
egg (which was the result of this injection) arrived at
the shell gland. Their was a trend toward an acceleration
of these events in the treatment period (after PGF2oz and
indomethacin) in that 5 of 7 of these birds reduced this
period by 0.1 to 2.2 hr.

The significance of this trend toward acceleration
could implicate prostaglandins in the regulation of these
two events. However, when considering the total time

period that we are dealing with (12-13 hr from LH injection

90
to the time of soft-Shelled egg's arrival to the Shell
gland), one might be forced to conclude there, in fact,

may be no difference at all in these time periods.

Experiment 7

 

The results of this eXperiment suggest that there
was no blocking effect of indomethacin on the LH stimu-
lated follicle, as verified in the previous experiments,
even when given orally (50 mg/kg BW) 3 hr after an
LH injection. This time of administration of indometha-
cin, subjected the animal to levels of 30 to 60,ug/m1 of
indomethacin in the plasma for 3 to 4 hr before the time
of ovulation.

AS was the case in Experiment 6, there was a trend
in the data, suggesting that there was a reduction in the
time period from the LH injection to the time of the
arrival of this ovum in the shell gland. The difference
was significant at p < 0.05. Four of 6 birds demonstrated
an early arrival of the ovum to the shell gland when com-
pared to the controls. However, this researcher is not
convinced that this trend is the result of the treatment,
due to the very Short time periods involved and relatively
small number of animals used. The literature indicates
that prostaglandins possibly play a role in ovum.transport
in the S.C.W.L. hen and the quail. However, the effect

of an inhibitor of prostaglandins (indomethacin) on this

91
large time period (LH to soft-Shelled egg in the shell
gland), cannot be conclusively drawn from these experi-
mental data.

Four of the 6 birds responding to LH injection
started a slight premature expulsion of the LH-induced
egg after indomethacin. However, this premature expulsion
is not Significant (p > 0.5).

In Experiment 6, the birds demonstrated a more dra—
matic premature expulsion of the LH-induced egg during
the treatment period in that 5 of 7 birds expelled their
eggs prematurely (p < 0.01). There were two basic dif-
ferences in the procedures of these eXperimentS:

(l) The last egg of the sequence in both the con-

trol and treatment period were expelled by
PGF2a prior to LH and placebo/indomethacin
treatment in this experiment, whereas, in
Experiment 6, PGF20L was used to remove the
indomethacin blocked egg from the shell gland
just prior to the expected ovulation from

the LH-Stimulated ovary.

(2) The level of indomethacin in the plasma at the
time of this LH-induced soft—Shelled egg was
higher in Experiment 7 than Experiment 6 due
to the time of administration of indomethacin

in relationship to the LH injection.

92

The second difference (above) between the experiments,
may serve to partially explain this premature expulsion.
Indomethacin was given 1 hr before the LH injection,
which (according to Figure 1) would indicate that there
would be relatively low levels of indomethacin in the
blood at the time of the earliest expulsion of these soft—
shelled eggs.

In the case of Experiment 7, the indomethacin was
given 3 hr after the LH injection. This fact would place
the indomethacin levels at those of the 10 hr period on
Figure l ( 4,ug/m1) instead of the 14 hr period
( 2.5 ug/ml). The fact that the calcium levels have been
Shown to be reduced at 11 hr and to remain so until 32 hr
may also have some effect on the early expulsion of this
soft-shelled and hard-Shelled egg, as was previously

discussed.

SUMMARY

Seven experiments were conducted in which S.C.W.L.

laying hens were administered indomethacin (orally)

for the purpose of determining its effect on several

reproductive parameters and total plasma calcium.

The results were as follows:

1.

The ovulation of a mature follicle was

not blocked by indomethacin when given

orally:

a) One hr before or up to 3 hr after LH
injection

b) Eleven to 12 hr before time of normal
ovulation.

Ovum tramsport (measured indirectly from

time of LH injection to soft-shelled egg

in the shell gland) is not dramatically

effected by indomethacin when given 1 hr

before or 3 hr after LH injection--though

there is a trend toward a reduction in

this time period.

Total plasma calcium is lowered (approxi-

mately 30%) by at least 11 hr after indometh—

acin and remains low for at least 32 hr.

93

94
Normal time of oviposition is delayed and
the subsequent expulsion of this egg occurs
at or after a time when indomethacin is at
very low levels in the blood.
Egg shell thickness in eggs which are expelled
at about the normal time of oviposition
(I 2 hr), is significantly reduced after

indomethacin.

B. The conclusions drawn from these eXperiments with the

domestic laying hen, using the prostaglandin inhibitor

indomethacin, are as follows:

I.

Prostaglandins probably do not have an essen-
tial role in the processes of LH release or
ovulation, as previously Shown in mammals.
Prostaglandins may play a role in shell forma-
tion by regulating the absorption, mobiliza-
tion and excretion of calcium.

Prostaglandins appear to have a role in the
process of oviposition, as previously

reported.

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