Download Can Pavlovian conditioning overcome the negative effects of stress

Stress, Fertility, & Sexual Conditioning 1
Can Pavlovian Conditioning Overcome the Negative Effects of Stress?: Fertility Effects in
Female Japanese Quail (Coturnix japonica)
Leah Davis and Brian Cusato
Sweet Briar College
In Pavlovian conditioning, an arbitrary stimulus that does not initially elicit a particular
response is referred to as the conditioned stimulus or CS. The unconditioned stimulus, or US,
elicits a biologically relevant response called the unconditioned response. After pairing the CS
with the US, the CS acquires the ability to elicit the unconditioned response without the US
being present. For example, Pavlov presented dogs with a metronome (CS) paired with food
(US). After several CS–US pairings the metronome elicited salivation in the dog without the
food being present. Thus, Pavlovian conditioning is the process by which organisms come to
associate two stimuli, a conditioned stimulus with an unconditioned stimulus.
Pavlovian Conditioning of Sexual Behavior
In sexual conditioning, a specific form of Pavlovian conditioning, the US is biologically
relevant to reproduction. For example, after pairing a tone (CS) with the presentation of a male
(US), a female will show sexually receptive behaviors toward the tone. Most research in sexual
conditioning has been conducted in the field of male sexual behavior. Relatively little research
has investigated female behavior in terms of sexual conditioning. As an exception, Gutierrez and
Domjan (1997) conducted sexual conditioning trials with female quail as subjects. They found
that female sexual behavior is much less overt than that of the male and they identified squatting
as a sexually receptive behavior in female quail. Locomotion was identified as a sexually nonreceptive behavior. Using this criterion the researchers found that sexual conditioning does not
Stress, Fertility, & Sexual Conditioning 2
increase the number of squats but does increase the duration of squatting in females. This study
is important because it showed that female sexual behavior can be conditioned.
Emily Harris, a recent graduate of Sweet Briar College, recently completed a series of
sexual conditioning experiments using female quail. She paired a tone (CS) with the release of a
male quail (US) and found that, relative to the unpaired control conditions, the duration and the
frequency of female squatting increased as a result of sexual conditioning. In a separate
experiment, Kelly Gibbons used physical cues of the animals surrounding (i.e. contextual cues)
as the CS paired or unpaired with a male US. Gibbons found not just a significant increase in
squatting as a result of sexual conditioning, but also a significant increase in the production of
fertile eggs. Based on these findings, it seems as though sexual conditioning facilitates female
sexual behavior as well as female reproductive success.
The Adverse Effects of Stress on Fertility
What the scientific community knows about stress and its effect on reproductive
behaviors is largely due to research in the field of behavioral endocrinology (e.g. Greenberg,
Carr, & Summers, 2001). For example, hormones such as corticotropin (ACTH), prolactin,
oxytocin, and rennin have also been found to be “stress sensitive.” Another system that mediates
stress is the hypothalamic-pituitary-ovarian axis, which has been associated with environmental
stress related infertility (Wasser, 1994). This is important because this same neural architecture
also is involved in the regulation of reproductive behaviors.
Lane and Hyde (1973) combined the fields of stress and fertility research by measuring
fertility after environmental stress was administered. The researchers restricted the movement of
female rats to induce stress four hours a day for seven days. The movement of the non-stress
control females was not restricted. Both groups were then permitted to mate with non-stressed
Stress, Fertility, & Sexual Conditioning 3
male rats. (The use of non-stress male stimulus rats ensured that they were measuring the effects
of stress only on the female.) The researchers found that the females that were stressed produced
significantly less offspring than the non-stressed females (Lane & Hyde, 1973).
In summary, sexual conditioning has been shown to significantly increase female
fertility, while environmental stress has been shown to significantly decrease female fertility.
How might these two phenomena interact? The current research project attempted to address this
question by investigating the negative effects of environmental stress on fertility in female quail
and whether or not sexual conditioning could overcome these negative effects. A two by two
between-subjects design was used. During conditioning, all birds were exposed to a 30 second
tone acting as the CS. For the paired females, the CS was presented immediately before they
were given access to a male for copulation (the US). Unpaired females were allowed access to a
male 45 minutes after the CS tone was presented. Within the paired and unpaired conditions half
of the birds were food deprived to induce stress. Thus, four groups were created: stress paired,
stress unpaired, no stress paired, and no stress unpaired. All of the animals were video taped
during the experiment to determine the effects of conditioning and environmental stress on
behavioral measures such as squatting (a sexually receptive behavior in females) and locomotion
(a sexually non receptive behavior in females). Eggs were collected, incubated, and candled to
determine each female’s fertility.
Method
Subjects
Eighteen sexually naïve female Japanese quail (Coturnix japonica) and 18 sexually naïve
male Japanese quail served as experimental subjects. The males were pre-tested to ensure their
ability to copulate, and female egg production was monitored to ensure their reproductive
Stress, Fertility, & Sexual Conditioning 4
potential. The birds were 6 months old at the start of experimentation and each bird weighed
between 200 and 300 grams. Weights were monitored daily and the birds in the stress conditions
were maintained at 80% of their free feeding body weight.
Apparatus
Nine identical testing cages were used. The cages were constructed of sealed plywood,
and each of the 9 cages was separated into 2 compartments so that interactions between the male
and female birds could be regulated. The female side of the cage measured 49.53 cm (high) x
64.14 cm (wide) x 105.41 cm (deep). The male side of the cage was the same heights and depth,
but was only 45.72 cm (wide). An 18.42 cm (wide) x 20.32 cm (high) opening was centered
along the dividing wall to connect the male and female sides of the cage, and a guillotine style
door could be opened or closed from outside the cages to regulate the timing of the male-female
copulations. An additional one-way, wire-mesh door was also located at the opening. It ensured
that all of the male-female interactions occurred on the female side of the apparatus. The front of
the cages was hinged to allow for service, and the hinged service door was made of chicken wire
so that behavioral observations could be made. Hardware cloth was used as flooring, and the
floor on the female side was divided into 15 equal zones using brightly colored string (each zone
20.3 cm2). The number of times a female crossed in and out of each zone during the conditioning
trials was used as a means of assessing conditioned receptivity. Water was available to the birds
at all times.
A Coulbourn Instruments tone/noise generator was connected to a 5.08 cm (wide) x 6.35
cm (high) speaker in each testing chamber. The speaker was used to present a 30 second, 1000
Hz tone (-21 attenuation) as the CS. The CS speaker was located on the female side of each
testing chamber but the tone could be easily heard by the male on the other side of the dividing
Stress, Fertility, & Sexual Conditioning 5
wall. The speaker was located 16 cm from the cage floor, centered on the wall opposite the
opening that separated the 2 compartments. Three sound attenuation booths were used to enclose
the testing chambers (each booth contained 3 chambers). The CS tone was simultaneously
presented to the 3 testing chambers in each booth, so the 3 pairs of birds housed in each booth
experienced their conditioning trials at the same time.
Procedure
The birds were separated into 2 squads, and each squad was housed in the testing cages
on an alternate 24-hour rotation for the duration of the experiment. A 13-day habituation period
preceded the beginning of conditioning to ensure that all birds were used to the rigors of the
everyday testing procedure. During habituation, free food was removed from the birds in the
stress conditions until they reached 80% of their free feeding body weight. Thereafter, they were
weighed every day to ensure they did not drop below this level, and access to food was restricted
to only certain periods of the day so that their 80% target weights could be maintained.
Following habituation, 1 conditioning trial was conducted each day for a period of 19
days. The squad that was housed in the experimental chamber the previous night experienced
their conditioning trials first. Twenty minutes elapsed after the first squad’s trials ended before
the second squad was placed in the testing chambers, and the second squad was given 20
additional minutes to habituate to the chambers before their conditioning trials began.
In the paired conditions, the 30-second CS tone was presented to the male and female
bird. The guillotine door between the 2 compartments was then raised and the male was
permitted to enter the female compartment. The 2 birds were allowed to interact for 5 minutes
(the US period). The male and female birds in the unpaired conditions also experienced the 30second CS tone and 5 minute US period, but the CS and the US were separated by at least 45
Stress, Fertility, & Sexual Conditioning 6
minutes. After the 5-minute US period, the males were returned to their side of the testing
chamber. Females experienced their conditioning trials in the same testing chamber each day.
The male birds, however, were assigned to 1 of the 4 testing conditions and rotated daily within
their condition so that they did not copulate with the same female on consecutive days.
Response Measures
The conditioning trials conducted on days 1,4,7,10,13,16, and 19 were video taped so that the
behavior of the females during the CS presentations could be analyzed. These behaviors included
the number and duration of squats, and the extent to which the females moved around the cage.
(Receptive females remain relatively immobile, squat more often, and for longer periods of
time.) The mobility of the females was determined by measuring the number times the female
crossed in and out of the zones marked on the cage floor (line crosses). A squat consisted of the
female remaining immobile and lowering her body until it touched the cage floor. Eggs were
collected from the females each day, incubated for at least 10 days, and candled to determine
their fertility.
Results
Preliminary analyses indicated that food deprivation was an effective means of inducing
stress, and that such an environmental stressor has a negative effect on female reproductive
functioning in Japanese quail. As can be seen in Figure 1, females in the stress conditions laid
Figure 1
18
16
Mean Eggs Laid
14
12
10
8
6
4
2
0
no stress
stress
Stress, Fertility, & Sexual Conditioning 7
significantly less eggs than those in the no stress conditions, F (1, 14) = 18.17, p < .05.
Difference scores were used to determine the behavioral effects of the conditioning trials
on female mobility and squatting (The response level of the birds on the first day of conditioning
was used as a baseline, and this baseline level was subtracted from the response levels during
each of the subsequent conditioning days captured on video taped). Using the differences scores,
it was determined that paired birds squatted more frequently (F (1, 14) = 3.816, p < .05), and for
longer durations (F (3,14) = 30.077, p < .05) than the unpaired birds (see Figures 2 and 3,
respectively). Statistical analysis of the mobility data did not show significant differences
between the paired and unpaired conditions. The stress manipulation also did not affect any of
the behavioral measures.
Figure 3
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
Paired- Stress
Paired-No Stress
Unpaired- Stress
Unpaired-No
Stress
Mean Duration of Squats
Mean Frequency of Squats
Figure 2
6
4
2
0
-2
-4
-6
The stress manipulation did, however, affect the fertility of the female’s eggs. Statistical
analyses indicated that females in the stress conditions laid significantly fewer fertilized eggs
than the females in the no stress conditions (F (1, 14) = 5.689, p < .05). The CS–US pairing
manipulation also affected the fertility measure. Females that had the tone paired with copulation
laid significantly more eggs than the females that experienced unpaired presentations of the tone
and copulation (F (1, 14) = 9.498, p < .05).
Stress, Fertility, & Sexual Conditioning 8
Figur e 4
16
Mean Fertile Eggs
14
Paired No Stress
12
Paired St ress
10
8
Unpaired No Stress
6
Unpaired Stress
4
2
0
The stress x pairing interaction also was significant (F (1, 14) = 5.189, p < .05). As can be seen
in Figure 4, the fertility of the females in the stress–paired condition did not significantly differ
from the fertility of the females in the no stress conditions. This latter finding suggests that
sexual conditioning can compensate for the stressful effects that food deprivation has on
reproduction.
Discussion
Previous research in the Pavlovian conditioning of Japanese quail has shown that sexual
conditioning can significantly increase female fertility. Research in behavioral endocrinology
has shown that environmental stress can significantly decrease female fertility. The present
experiment investigated the negative effects of environmental stress on fertility in female quail
and whether or not sexual conditioning could overcome these negative effects. Half of the birds
were food deprived to induce stress, and during conditioning, all birds were exposed to a 30
second tone acting as the CS. For the female subjects in the paired condition, the tone CS was
presented immediately before they were given access to a male for copulation (the US). The
behavior of the females during the conditioning trials was video taped and analyzed to determine
the effects of conditioning and environmental stress on the squatting response and locomotion.
Stress, Fertility, & Sexual Conditioning 9
Eggs were collected from the females, incubated, and candled to determine each female’s
fertility.
The experiment revealed a number of interesting findings. First, food deprivation
significantly reduced the number of eggs the females produced. This suggests that food
deprivation is an effective means of creating stressful reproductive conditions in the laboratory.
Second, sexual conditioning occurred in the paired conditions. The females that experienced
paired presentations of the CS tone and copulation squatted significantly more, and for
significantly longer durations, than the females in the unpaired control conditions. This finding
illustrates that auditory cues are effective in the sexual conditioning of females, and that sexual
conditioning can significantly improve the sexual receptivity of female Japanese quail. Finally,
the fact that the females in the paired conditions laid significantly more fertile eggs revealed that
sexual conditioning can also improve a female’s reproductive fitness. Moreover, the fertility of
the females in the paired–stress condition did not differ from the no-stress condition. This
suggests that sexual conditioning can overcome the negative effects of environmental stress.
Collective these results indicate a clear connection between a female’s reproductive
behavior and her reproductive success. They also illustrate a functional role for sexual
conditioning in compensating for the harsh living conditions so often experienced by animals in
their natural environment. But how might these findings be applied to animals living under
stressful conditions in artificial environments?
The stresses of captivity and living in artificial environments often reduce an animal’s
reproductive success. Furthermore, captive breeding programs are often costly and in their
current form, not extremely successful. This makes each sexual encounter extremely vital – vital
to the breeding program’s success, and in the case of endangered species, vital to the survival
Stress, Fertility, & Sexual Conditioning 10
and welfare of the species as a whole. The present research findings suggest that sexual
conditioning can increase the effectiveness of these programs by compensating for the stressful
artificial environments. The ease with which such sexual conditioning procedures may be
implemented in situations outside the laboratory awaits further experimentation.
References
Cusato, B., & Domjan, M. (2000). Facilitation of appetitive conditioning with naturalistic
conditioned stimuli: CS and US factors. Animal Learning and Behavior. 28, 247-256.
Domjan, M., Cusato, B., & Villarreal, R. (2000). Pavlovian feed-forward mechanisms in the
control of social behavior. Behavioral and Brain Sciences. 23, 235-282.
Gutierrez, G., & Domjan, M. (1997). Differences in the sexual conditioned behavior of male and
female Japanese quail (corturnix Japonica). Journal of Comparative Psychology. 111,
135-142.
Greenberg, N., Carr, J., & Summers, C. (2001). Ethological causes and consequences of the
stress response. Integrative & Comparative Biology. 42, 508-516.
Lane, E., & Hyde, B. (1973), Effects of prenatal stress on fertility and sex ratio: Pilot study with
rats. Journal of Abnormal Psychology. 82, 78-80.
Wasser, S.K. (1994). Psychosocial stress and infertility – Cause or effect? Human Nature. 5,
293-306.