1) Various prostaglandin inhibitors were administered to female red-sided garter snakes to measure their effects on male attraction to females and estrogen levels. 2) The inhibitors had no significant effect on mating behavior or male courtship compared to the control. However, two females treated with the prostaglandin inhibitor indomethacin had lower estrogen levels than other treated females. 3) While most results were inconclusive, treatment with the inhibitor indomethacin may have suppressed estrogen levels in some females, warranting further study of its impact.

1. Effects of Inhibitors to Prostaglandin’s Effects on Female Garter Snakes Pg. 1
Portland State University
P.O. Box 751, Portland, Oregon 97207-0751
Effects of Inhibitors to Prostaglandin’s Impact on the Behavior and
Estrogen Release in Female Red-Sided Garter Snakes
Prepared by
Radford C. Bean
Student
For Dr. Joan Whittier
Associate Professor of Biology
Portland State University

2. Pg. 2 American Journal of Animal Physiology
PGF2 is found to be a uterine luteolytic
hormone in guinea pigs and sheep, thereby
terminating corpus luteum (reviewed by
Moore 1985). It is also a uterine vaso-
constrictor.
Prostaglandins stimulate FSH and LH
release from the pituitary gland. Prostaglandin
also may be responsible for the estradiol
feedback on GRH.
In sheep, systemic administration of PGF2
caused plasma LH to increase, while admin-
istration of indomethacin caused a decrease in
plasma LH. Aspirin given to cows caused a
decrease in LH. Prostaglandins have many
characteristics of inflammatory mediators.
Increased levels of prostaglandin in the
plasma of female red-sided garter snakes has
been found to increase the latency of females
to mate (PGF2 – 0.05ug/g BW). A higher
dosage of PGF2 (5.0ug/g BW) has rendered
females unattractive to males within 24 hours
of treatment (Whittier and Crews 1985). This
experiment attempts to establish the effects
inhibiting prostaglandin will have on male and
female courtship behavior, and plasma levels
of estrogen in the females.
When a sexually active female is
introduced into an aquarium of adult males,
the males form a mating ball around the
female. Male courtship in garter snakes, as
with other species of snakes, involves the
stroking of the female’s back by the male’s
chin. This is a result of a pheromone released
by the female (Whittier and Crews 1986;
Crews and Garstka 1982). This chin stroking
behavior provides a means of counting the
number of males courting, determining if the
certain prostaglandin inhibitors have an effect
on male and female behavior.
Methods
Animals and animal maintenance
Adult, sexually mature male and female
red-sided garter snakes were collected in
Manitoba, Canada. All snakes were marked by
clippings to various ventral scales. The
females were kept in hibernation for a period
of 6 months at 60
C, 0L, and 24D. All the
females used for the experiments had snout to
vent lengths > 45cm and weights > 32.5g.
Six, 10-gallon aquariums housed seven
males each, except for one aquarium that
housed on six. A Vitalite provided illumination
for each aquarium. The substrate in each
aquarium consisted of hardwood chips.
All the males and females were fed smelt
and provided with water.
Abstract: Various prostaglandin inhibitors were administered to female red-sided garter snakes
(Thamnophis sirtalis parietalis) to measure the effects on male attractiveness to female garter
snakes and the impact on plasma levels of estrogen in females. A 1% solution of sodium
bicarbonate was used as the control. Inhibitors consisting of 8mg/ml indomethacin and 80mg/ml
acetaminophen were injected into the females. After a period of incubation, the females were
introduced to males and their receptivity monitored. If prostaglandin was inhibited by the various
treatments, it would be reasonable to expect that after the females mated they would still be
receptive and attractive to male garter snakes. Females observed during the mating and
immediately on completion showed that the mated females were unreceptive to other males, and
that the unmated males lost interest in the mated females.
Twenty-four hours after completion of the experiments, the females were bled. Inhibition of
prostaglandin should result in low levels of estrogen 24 hours later.

3. Effects of Inhibitors to Prostaglandin’s Effects on Female Garter Snakes Pg. 3
Treatments and administration
Three various treatments were made up
consisting of 1% NaHCO3, 8mg/ml
indomethacin (Sigma Chem. CO., Lot Date
129C-0052) and 80mg/ml acetaminophen
(Sigma Chem. Co., Lot Date 71F-0160).
Sodium bicarbonate was used as the control
was used as the control. These treatments were
kept refrigerated when not being used.
Three groups of twelve females received
one of the three treatments. Their body weight
x .001ml determined the dosage administered
to them. The dosage was injected into their
body cavity, and the females placed in a
holding aquarium for a period of 20 minutes
from the time of injection. The treatments
administered to each female were recorded
along with each female’s individual number
(determined by the scale clippings).
Behavioral measurements
The experiment was run six times during a
period of 3 weeks. One female each was
introduced into one of the five aquariums
housing seven males, or into the sixth
aquarium housing six males. Every 5 minutes
the number of males courting each female was
recorded. This recording continued over a
period of 1 hour. It also was recorded when a
female mated and the effect it had on the
unmated males.
Extraction, chromatography and RIA of the
blood samples
All mated and unmated females were bled
using the method of withdrawing blood from
the ventricle. The collected samples were sent
to the Oregon Regional Primate Research
Center for radio immunoassaying.
Statistical methods
The statistical significance of the results of
the inhibitors with regards to the control was
figured using the Chi square formula and the
2 X 2 contingency tables. P < 0.05 was
considered significant.
X2
=df=1
H0: Mating unrelated to treatment.
H1: Treatment influences mating.
If x2 value exceeds 3.84 (from table) reject
H0.
The Kruskal-Wallis test was used to
determine the ranking of the three treatments
to E2 levels in the female’s blood plasma. P <
0.05 was considered significant. The one-
tailed test was used to determine the
signifiacnace among 15 comparisons.
z = 2.713
x =
Results
One of three treatments was administered
to thirty-six females removed from
hibernation. Each run consisted of two females
from each treatment. A minimum of 10
minutes elapsed before observing the first
mating. The average number of courting males
for each treatment, whether mating occurred or
not, is listed in Table 1.
x
2 N AD BC∠ N 2)2⁄∠(
A B) C D) A C) B D)+(+(+(+(
--------------------------------------------------------------------------------=
)/1/1)(12/)131(31( vu nnz ++

4. Pg. 4 American Journal of Animal Physiology
The percentage of males courting females
in which mating occurred was similar between
the control and the two treatments (Figure 1).
The same case held true for the males courting
with no resulting copulation (Figure 2).
Courtship Bouts Where Mating Occurred
Figure 1. Percentage of males courting
females for each treatment in which copulation
occurred.
Upon intromission, all other males in the
aquarium lost their attraction for the female
and no further stroking of the female’s back by
the males was observed. This was the case for
all the females that mated, regardless of the
treatment. All the females that mated had
deposited in the cloaca a copulatory plug (Ross
and Crews 1977). It is not certain whether or
not a pheromone contained in the copulatory
plug interferes with further female
attractiveness to males (Whittier, Mason and
Crews 1985; Ross and Crews 1977). The
females administered the control substance
had a higher number of successful matings;
half of those injected with NaHCO2 mated.
Those females injected with the two inhibitors
had lower rates of mating success compared to
the control value (Figure 3). The difference in
success was not significant (x2 = 7.37 X 10-1
H0 was not rejected: x2 < 3.84).
Courtship Bouts Without Mating
Figure 2. Percentage of observed males
courting females for each treatment in which
no mating took place.
Treatments to Female Garter Snakes
Figure 3. Percentage of females that mated
based on the type of treatment each received.
Test results show that all the mated
females, regardless of treatment, had higher
levels of E2 in their blood plasma with the
Table 1: Average Number of Males
Courting
NaHCO3
Acetamino-
phen
Indometh-
acin
Mated 4 4 3
Unmated 2 2 2
0
1
2
3
4
5
6
7
N aHC O 2 A cetam inophen Indom ethacin
T reatm ent
0
2
4
6
8
10
12
14
16
18
N aHC O 2 A cetam inophen Indom ethacin
T reatm ent
0
5
10
15
20
25
30
35
N aHC O 2 A cetam inophen Indom ethacin
T reatm ent

5. Effects of Inhibitors to Prostaglandin’s Effects on Female Garter Snakes Pg. 5
exception of one case. Female #94 had an E2
level of 2.320ng/ml. Since this is not
consistent with the results for the other mated
females, I chose to exclude that E2 value from
the data. Two out of three mated females
treated with indomethacin had E2 levels
considerably lower than those mated females
that received the other two types of treatments.
The difference in E2 levels in females
administered indomethacin was significant,
compared to the E2 levels in those females
treated with the control or acetaminophen
(Table 2).
Discussion
From the results obtained during the
experiment, the inhibitors had no effect on
prostaglandin’s impact on male and female
mating behavior. Inhibiting prostaglandin
should have resulted in the females still being
receptive to further male courtship
immediately following copulation; this did not
occur. Furthermore, all unsuccessful males
should have shown continued interest in the
female, even though she had already mated.
Prior research showed that prostaglandins
increase latency and inhibit receptivity in the
females (Whittier and Crews 1986). Based on
Figures 1 and 2, acetaminophen and
indomethacin had no effect on male courtship.
In both cases, mated and unmated, the
percentages are similar to the control in each
graph.
Of the three females treated with
indomethacin that mated, the two females with
the lowest E2 levels are causing the
insignificance between those females and the
females administered with the other
treatments. Indomethacin may, therefore, have
impacted estrogen by suppressing the E2 levels
in those two females. A larger sampling of
females administered with indomethacin is
needed to see if the above results hold true in
most cases.
References
1) Moore P. K. Prostanoids: Pharmacological,
Physiological and Clinical Relevance.
England. Cambridge University Press,
1985.
2) Whittier J. M, Crews D. “Effects of
Prostaglandin F on Sexual Behavior and
Ovarian Function in Female Garter Snakes
(Thamnophis sirtalis parietalis).”
Endocrinology 119 (1986): 787-792.
3) Whittier J.M, Crews D. “Ovarian
Development in Red-sided Garter Snakes,
Thamnophis sirtalis parietalis,
Relationship to Mating.” General and
Comparative Endocrinology 61 (1982): 5-
12.
4) Crews D., Garstka W. R. “The Ecological
Physiology of a Garter Snake.” Scientific
American 247 (1986): 158-168.
5) Siegel S., Castellan N. J., Jr. “Table C.”
Non-parametric Statistics for the
Behavioral Sciences. McGraw-Hill, 1988.
6) Whittier J. M., Mason R. T., Crews D.
“Mating in the Red-sided Garter Snake,
Thamnophis sirtalis parietalis:
Differential Effects on Male and Female
Sexual Behavior.” Behavioral Ecological
Sociobiology 16 (1985): 257-261.
7) Ross P., Jr., Crews D. “Influence of the
Seminal Plug on Mating Behavior in the
Garter Snake.” Nature 267 (1977: 344-
345.
Table 2. Average Sum of Females
NaHCO3
Acetamino-
phen
Indometh-
acin
Mated 25.98a 26.63a 22.7b
Unmated 8.5b 10.7b 9.13b