An animal’s behavior is a product of both external and internal stimuli. In the series of experiments in this exercise you will observe some effects of certain internal stimuli, i.e. sex hormones, upon the behavior of the rat and guinea pig. You will observe the effect first upon a non-sexual behavior pattern (running) and later upon various aspects of sexual behavior.
PART I. RUNNING ACTIVITY IN THE RAT
Since the study of behavior invariably involves activity measures of some sort, an appreciation of how normal, as well as unusual, factors may influence a fundamental behavior pattern is crucial. In this exercise you will measure the influences of three factors upon running activity: (1) time of day: (2) food deprivation: (3) sex hormones.
A. SUBJECTS AND EQUIPMENT
Each group of students should be supplied with the following:
- Adult rats: two males and two females.
- Four activity wheels, with counters.
- Surgical instruments (for castration): scalpel and blades; forceps; wound clips and applicator; tissue scissors; hemostats.
- Two 1-cc tuberculin syringes: two 1-inch 26-gauge hypodermic needles.
- 10 μg of estradiol benzoate: 10 μg of testosterone propionate.
B. DAILY VARIATION
Place each of two male and two female rats in an individual cage equipped with an activity wheel linked to a revolution counter. Allow the animals to feed and drink ad lib. Locate the cages so that they are disturbed as little as possible and are subject only to the normal daily variations in light: i.e. do not leave them in a laboratory or room where the lights remain on all night or where there is much activity. Record the number of revolutions of the wheel at precise 12-hour intervals, preferably at 8:00 a.m. and at 8:00 p.m. Make additional readings if you wish to determine hourly variation more precisely. Keep records on 12-hour activity of each animal for at least 10 days. Graph the results in Figure 33-1 (a) for all animals, (b) for males and females, (c) for each individual.
1. What do the graphs tell you about normal behavior?
2. During which half-day periods is activity greater?
3. Are the animals more active on some days than on others? Why?
4. Would you intuitively expect the activity levels to be fairly consistent from day to day? We will look further into this point in Section D.
C. FOOD DEPRIVATION
Remove the food (leave the water) from the cages and record the activity for the next two days. Would you expect food-deprived animals to be more active or less active than when they have food available? This is not a simple question to answer. Hunger is generally considered as arousing and activating for most animals, but is this always so? If the animals are left without food for more than 48 hours what do you think would happen to the activity? How are hunger and activity related? See Richter and Treichler and Hall for additional information.
D. SEX AND HORMONES
How did the activity of the males differ from that of the females? Did the males show a constant activity level? With what could the cyclic activity of the females be correlated (see Wang)?
Using a simple castration technique (see Zarrow, Yochim, and McCarthy). remove the testes from one of the two males and the ovaries from one female. Do sham operations on the other two rats. (In a sham operation one does all the operative procedure except actual removal of the testes. In this way one has a control over the effects of removal of the testes over and above any effects of the operation itself.) Record the number of wheel revolutions for all animals for the next 10 days. What has happened to the activity? Why? Is it the gonad itself that is crucial for normal activity? Instead of castrating the animals you might replace them with prepubertal ones (30-40 days of age) and make a similar comparison.
For the next 10 days give each female rat daily injections of 1.0 μg estradiol and give each male 1.0 mg of testosterone. These may be either subcutaneous into the axilla or intramuscular into the hamstring and gluteal muscles.
1. What happens to the running activity?
2. Does it seem strange to you that running activity is related to sex hormones and reproductive processes?
3. What relationships might be involved?
4. A study using animals from prepuberty to post-puberty (approximately 40 days of age to 95 days of age) should give you similar results. Why?
5. Why is it generally good scientific technique to do sham operations and to inject both males and both females?
E. ADDITIONAL PROBLEMS
What other factors might influence running? As a special project, work out with your instructor simple methods for testing the factors you propose. You will be surprised at the range of influencing factors you can find.
Do you think we would have gotten similar results if we had used guinea pigs instead of rats? Discuss.
Although we have demonstrated that running behavior in a wheel can be affected by certain manipulations of the environment, we must not be deluded into believing that all activity is necessarily affected similarly. Other measures of activity may yield different results. For example, Treichler and Hall compared the effect of food deprivation on activity as measured by activity wheel, stabilimeter, and maze investigation. Richards reviews this point too, as well as discussing activity during various reproductive (endocrine) situations.
PART II. NORMAL SEXUAL BEHAVIOR OF THE GUINEA PIG
Although sexual behavior has been scientifically studied for less time than other areas of behavior. for few behavior patterns is the associated biology so well understood. There is a wealth of information on the anatomy, physiology, and biochemistry of sexual behavior. The credit for this is due to those early investigators who demonstrated the strong relationships between sexual behavior and the endocrine and nervous systems. An introduction to this extensive field may be gained from the following experiments in this part of the exercise.
A. ANIMALS AND ADDITIONAL EQUIPMENT
Each group of students should be supplied with the following.
- Guinea pigs: six adult females and three adult males.
- Six cages.
- Observation enclosure with at least 4 square feet of floor space.
- Estradiol benzoate (in oil); progesterone (in oil).
- Stop watch with sweep-second hand.
- Colored airplane dope.
B. FEMALE BEHAVIOR
1. Behavior of normal females
Obtain six female guinea pigs and cage each separately. Introduce them one at a time, at approximately 5-minute intervals, into a single observation cage. Observe the interactions. You will notice the animals will, usually after a period of hesitation, investigate their new environment and each other. Mutual investigation may be categorized into several forms of observable behaviors. These are sniffing and nibbling, which refer to a general exploratory behavior as one animal brings its head into body contact with another animal, and nuzzling, which designates sniffing and licking confined to the anogenital region. For the first two females record the number of times each sniffs and nuzzles during the 5-minute period of initial encounter (Table 33-1). Record the behavior of females 1, 2, and 6 when female 6 is introduced.
Table 33-1. Investigatory behavior of normal female guinea pigs
|Female 1||Female 2||Female 6|
Time interval (minutes after introduction of first female)
Number of sniffs/5 minutes
Number of nuzzles/5 minutes
1. How does the behavior of a female guinea pig that is introduced to the cage when only one other female is present compare with that of one introduced to the cage when five others are present?
2. Who initiates the investigation?
Some fighting may occur but this will usually cease within a few minutes. Return the animals to their individual cages.
Most of the behavioral interactions you note are social and due to curiosity, although intuitively you may prefer to consider them sexual or homosexual. (How do you think the terms used in this area of behavior may bias your observations, discussions, arid conclusions? In what respect is the loose use of terms a general problem for the study of behavior?) Mutual genital investigation occurs in many species, notably dogs.
2. Behavior of castrated females
Castrate the animals (see Zarrow, Yochim, and McCarthy) and after a week repeat the above procedures and observations (Table 33-2). What differences, if any, do you note in the animals’ behavior?
Table 33-2. investigatory behavior of castrated female guinea pigs
|Female 1||Female 2||Female 6|
Time interval (minutes after introduction of first female)
Number of sniffs/5 minutes
Number of nuzzles/5 minutes
3. Production of estrus in castrated females
To each of three of the six castrated guinea pigs give a subcutaneous injection of 6.0 μg estradiol benzoate followed 26 ± 1 hours later by a 0.4-mg injection of progesterone. Mark the three animals that have received the hormones so they can be readily identified (placing red airplane dope on their ears the day before testing is a convenient method). Place all six test animals in the observation cage and for 15 minutes after the injection of progesterone unobtrusively observe and record the frequency of sniffing and nuzzling demonstrated by each female. In addition, record the frequency and type of mounting seen (as in Table 33-3). Record the behavior of the treated animals above the dotted line, and that of the nontreated females below the line. There are three types of mounting: abortive, posterior, and ectopic. In Abortive Mounting the animal places one or both forepaws on another without direct body contact. In both Posterior and Ectopic Mounting body contact occurs with or without accompanying pelvic thrusts: the two differ only in the location of body contact. An ectopic mount may occur anywhere but posteriorly; i.e. one animal may mount the head or flank of another.
|Behavior||Time units (in hours or minutes for females; in minutes for males)|
After observing for 15 minutes, test each female for lordosis (the upraising and posterior presentation of the vagina) by repeatedly stroking her anogenital and lumbar region with your fingers in one slow deliberate and continuous cephalad movement of the hand. Try this on each female several times.
1. Which females respond to your stroking?
2. Are the females which lordose the same ones which mount?
3. How might you account for this?
Repeat these 15-minute observations and lordosis tests hourly for 10 hours. Record the occurrence of each behavior category on the record sheet (Table 33-3). Then return the females to their individual cages. Indicate the behavior of the injected females above the dashed line and that of the noninjected ones below it.
4. Do the groups differ in all categories of behavior?
5. Do they differ all the time?
6. Is the distinction between different types of mounting informative?
7. Are all the mounts posterior mounts? Discuss.
8. Why do we have to observe the animals for an extended period of time?
9. Which behavior patterns would you now say constitute the best index of estrus?
Estrus (heat) is a behavioral term used to describe the behavior of a female that is sexually receptive to a male and will lordose when mounted by him. For females of most species this is a very limited period which seems to become less restrictive as we traverse the evolutionary path towards humans. (Often the term is used to refer to the vaginal condition seen during ovulation. In a broader sense, the term estrous cycle refers to the periodic changes in the ovaries and genitalia. This is confusing but common usage; the student should always establish whether the term is being used in its behavioral or somatic sense. For endocrinologists the latter usage is convenient and will probably persist.) Behavior during artificially induced estrus is indistinguishable from that which occurs spontaneously.
10. Do humans have any estrous period?
11. How long did estrus last for the female guinea pigs you observed? This span is usually considered the “duration of estrus.”
12. When did estrus begin and end in regard to the progesterone injection? The time interval from progesterone injection to first demonstration of lordosis is called the “latency to estrus.”
13. In terms of evolutionary adaptiveness, is a limited estrous period beneficial? Discuss.
14. With which physiological processes is estrus normally correlated?
15. Do these processes correlate with our use of estrogen and progesterone to induce heat?
16. Why was it more convenient to induce estrus in the females than to observe its spontaneous occurrence?
17. How frequently does a female guinea pig spontaneously come into heat?
18. Following estrus, the female is in a refractory period and cannot be immediately reinduced to come into heat. What may this infer in regard to the interactions of endocrines and the nervous system?
With the animals remaining in their home cages. induce estrous behavior in the three females previously uninjected. Select the two which demonstrate the best lordosis response to fingering; place them together in the observation cage, and record their mutual behavior in a test situation for 10 minutes.
19. What happened?
20. How did their behavior compare to that of the females tested as a group? We will refer to this later in Section C.
21. Do you think mutual mounting among animals of the same sex is unusual and seen only when the animals are segregated by sex or domesticated?
22. Will females lordose when mounted by other females? When stimulated by finger? Is a male necessary?
23. What does this mean in terms of the appropriateness of particular stimuli for a particular response?
24. How do you think the sexual behavior of a female would differ if she were tested with a male rather than a female? We will investigate this further in Section C.
25. What do you think might happen to the estrous patterns if the females were given larger doses of estrogen or progesterone?
We have dealt, in this experiment, with a female estrus that is cyclic, usually corresponding with the ovulating cycle of the animal. In contrast, many animals. e.g.. rabbit, ferret, and cat, are either always receptive or receptive continuously during certain seasons, and ovulate only after the stimulus of copulation. These are termed “induced ovulation” in contrast with “spontaneous ovulators.”
In observing and recording the sexual activity of males, use the categories of active behavior used previously for the females: lordosis. however, occurs rarely. In addition, look for and record intromissions and ejaculations. Intromission is often accompanied by slow pelvic thrusting in contrast to the rapid thrusting seen with mounting without intromission. Ejaculation is recognized by a convulsive drawing in of the flanks and pause in pelvic thrusting.
Place an adult male guinea pig in the observation cage and let him adapt to the new situation for a few minutes. Why? Quietly introduce a castrated female which has been brought into estrus (injected with 6.0 μg of estradiol benzoate for 3 days, the last injection 12 ± 1 hours prior to a 0.4 mg progesterone injection). To induce receptivity of a male by the female, several estrogen injections prior to progesterone injection are needed. These are to prepare properly (to open and cornify) the vagina for intromission. Because the animals are easily disturbed by loud noises and sudden motion, be as quiet and motionless as possible while you are observing. If startled, the animals will ‘freeze” and you will have to start over again after they relax.
On a score sheet similar to Table 33-3, record the behavior observed during a 10-minute period. Record the male’s behavior above the dashed line and the female’s behavior below the line. Consider the time units as minutes.
1. How does the behavior of the male compare with the behavior of the female?
2. Which is more aggressive and persistent in initiating contact?
3. Is there an equal amount of activity demonstrated during each minute of observation?
Repeat this 10-minute test with a new male and an anestrous female and record your data. What differences do you observe in the behavior of the male? In the behavior of the female?
Although the female may be in estrus, the male won’t always ejaculate during the 10-minute test session, and extending the session doesn’t necessarily help. Does this surprise you? Why? Do males have an estrous cycle? Repeat the test with another male and another estrous female. What might account for the failure of the male to ejaculate?
Describe the behavior of the male after ejaculation. It is usually very distinctive and can often be used to confirm the fact that ejaculation has occurred.
4. Was the behavior of the female placed with a male different from that of a female placed with another female (as in Section B)?
5. Observe two males placed together for 10 minutes. Can you now distinguish sexual behavior from social behavior and aggressive behavior?
6. What are the chief features of male sexual behavior?
7. Which of these differ from patterns seen in the female?
The beginning student may wonder why courtship and maternal behavior are apparently ignored in the above exercise. Sexual behavior in its broadest sense would indeed include these activities. In addition it could include any pattern in which sexually dimorphic actions occur, as in territoriality, aggressiveness, or dominance. For convenience of study these are considered separately. As separation of various activities is arbitrary. so may be the subpatterns we choose to record, such as lordosis, mounting, and ejaculation. The methods of recording, too, are arbitrary. You should consider other possible behavior categories or patterns to be observed and recommend any preferable method of recording and evaluating them. Discuss these possibilities with your instructor and consult the excellent chapter on the measurement and evaluation of behavior by Denenberg and Banks.
PART III. EFFECTS OF ANDROGEN ON SEXUAL BEHAVIOR
In the previous exercise the normal sexual behavior patterns of guinea pigs were observed. It was demonstrated that these were strongly dependent upon gonadal hormones, particularly in females. The present exercise will demonstrate sexual behavior in the rat and further explore the role of hormones, androgen in particular, on sexual behavior in both the rat and guinea pig.
Each group of students should be supplied with the following:
- Rats: nine experienced (old breeder) males: six adult castrated females.
- Guinea pigs: twelve females, four males.
- Observation cage: as in Part I.
- Surgical equipment for castration: as in Part I.
- 350 mg of testosterone propionate (50 mg/ml); 20 μg estradiol benzoate and 35 mg progesterone.
- Syringes and hypodermic needles: as in Part I.
B. RESPONSE TO CASTRATION IN MALES
Obtain nine sexually experienced (old breeder) male rats and castrate six of them (method of Zarrow, Yochim, and McCarthy). In the previous exercise guinea pigs were used since the speed of their various sexual activities is slow enough to permit observation while you are learning. The rat, in comparison, is much quicker and the various components of mounting are less readily distinguished. (See Stone for a description of the normal sexual behavior patterns of the male rat).
Two weeks or longer after the castration, test all the rats for sexual behavior. Present each male with an estrous female for 15 minutes and record the activity of each pair as in Table 33-3. Female rats may be brought into heat by injecting 1.0 μg of estradiol benzoate 30 ± 1 hours prior to a 2.0-mg injection of progesterone. The females will usually come into heat about 3-6 hours after the progesterone injection and will remain receptive for about 2-4 hours. Each female may be used with one or more males as long as she appears receptive (or lordoses) to fingering or mounting by the male.
1. How does the sexual behavior of intact males differ from that of castrate males?
2. Does castration affect components of sexual behavior equally?
3. What differences do you note between the castrate males themselves?
4. Do the females react differently to a castrate male than they do to an intact male?
5. Do you think it unusual for the behavior of one animal to influence the behavior of another?
6. The effects of castration are not always readily obvious. Why do you think an interval of at least two weeks between castration and testing of the males was allowed?
7. Is it necessary that we start off with sexually experienced males? Why?
8. How long do you think it would take before significant effects due to castration were seen in an experienced male cat or man?
9. There is a great deal of species and individual variation in the effect of castration. What might this suggest in regard to genetic, neural, and ontogenetic components of male sexual behavior?
10. How do the effects of orchidectomy compare with the effects of ovariectomy (the term castration may be used both for males and females) on the sexual behavior of the rat, cat, or human? (See Beach and Holz, Rosenblatt and Aronson, and Bremer.)
It takes more time to demonstrate the effect of castration on the guinea pigs than in rats.
C. RESPONSE TO HORMONE REPLACEMENT IN CASTRATED MALES
Every day for 1 week, inject intramuscularly (I.M.) into the hamstring and gluteal muscles of three of the six castrated males 1.0 mg of an androgen, testosterone propionate (T.P.), and similarly inject the three others with 10.0mg of T.P. Give the three intact males placebo (vegetable oil) injections of comparable volume (0.1 ml). After this series of injections test all nine rats again with estrous females. Compare the behavior of the three groups.
1. What do you observe now?
2. What can you infer about the need for androgen in eliciting male sexual behavior?
3. How might the hormone work in eliciting this behavior?
4. Does the behavior of the males receiving the higher dose of T.P. seem to differ markedly from that of those receiving the lower dose?
5. Would you expect increased amounts of androgen to effect an increase in the incidence of display of sexual behavior components?
6. Would giving androgen to intact rats affect their behavior?
7. Do all behavior components seem to “suffer” equally following castration?
8. Do you think the age of the animals at castration is of any significance?
The conclusions you draw from working with the rat may not apply as thoroughly to other species. One must always study several species before making generalizations.
D. RESPONSE TO ANDROGEN IN THE FEMALE
We have previously examined the sexual behavior of normal and castrated animals. The following exercise will give some insight into behavior under the influence of “inappropriate” sex hormones.
Inject (I.M.) four of the twelve female guinea pigs (castrated or intact) with 2 mg of testosterone propionate daily for 2 weeks. Simultaneously. inject another four with a vegetable oil placebo. After this series of injections test each of these eight females as if they were males; i.e.. present the experimental female with a stimulus female for a 10- minute observation period (see Part II, Section C).
1. How did the real males differ from the androgen-treated females?
2. What might this infer about the relationship of hormones to behavior?
3. What might this infer about the underlying nervous system which is responding to the hormones? (As an interesting demonstration of the potency of the androgen compare the genitalia of both groups.)
We have demonstrated that we can induce females to display “male-like” patterns of behavior. Does this imply that all sexual behavior is determined by circulating hormones? Do females display “male-like” patterns without androgen? Do you understand why we have used quotation marks around the term male-like?
It would be a serious mistake to think that only endocrine factors are involved in the display of sexual behavior patterns. What else might be involved? See the works by Beach for a discussion of many such factors.
PART IV. EFFECTS ON ADULT BEHAVIOR OF HORMONE TREATMENT DURING DEVELOPMENT
In the development of the nervous system and dependent behavior patterns the concept of a critical period has real significance. A critical period may be defined as an interval during early development when certain physiological or environmental influences can have permanent effects on adult behavior. Usually, it is intuitively difficult to imagine that a single or relatively simple procedure early in development could irrevocably structure all future behavior. The following procedures will investigate how a physiological disturbance during early development can effect sexual behavior in the adult.
A. ANIMALS AND EQUIPMENT
Each group of students should have the following:
- Rats: Twelve females pretreated on the third day after birth; six sexually experienced males and six naive males.
- Activity wheels: as in Part I.
B. SEX BEHAVIOR
Your instructor will have prepared three groups of rats for your use. The first group will consist of six female rats injected (subcutaneously), 3 days after birth. with a single 2.0-mg dose of testosterone propionate. The second group of six female rats will have received a comparable volume of vegetable oil. The third group will consist of six normal males from the same litters.
After the rats are 100 days old, castrate all the females, and 2 weeks later test each for female behavior after administering estradiol benzoate and progesterone (see Part III, Section A). Note the behavior of each female rat when placed for 10 minutes with an experienced male (not the males of the third group). Record the number of times each female will lordose when mounted and note how receptive she is to the male’s advances.
1. How does the female behavior of the experimental group compare with that of the control group?
2. What mechanism might account for such a dramatic change in mating behavior due to a single neonatal injection?
3. Would the injection be more effective if given at a different stage in the rat’s development; e.g., during gestation, at puberty, at maturity? Why?
4. Do you think the effect would be different if a female hormone were injected neonatally?
Many papers discuss the above effects specifically. Most of these are reviewed by Whalen.
Histological studies of the ovaries removed from the neonatally treated females would reveal that ovarian functioning has been seriously affected as has genital structure. Would you expect non-reproductive functions to be affected also? This question will be investigated in the following section.
Place each animal from all three groups in an activity wheel for 10 days and record the total number of revolutions turned for each group as in Figure 33-1.
1. How do the three groups compare in their running activity?
2. Is the alteration in reproductive behavior accompanied by an alteration in sexually dichotomous running behavior?
3. How might you account for the findings?
That different endocrine-neural mechanisms may be involved in various sexually dichotomous behavior patterns is a significant finding. It demonstrates that an influence on certain behavior patterns does not necessarily affect other related patterns. See the papers by Kennedy and by Gray, Levine, and Broadhurst for discussions of this point. It must be realized, however, that more than one pattern may be affected by the same mechanism (see Goy).
The demonstration that simple and transient physiological and environmental influences can have permanent behavioral effects on an individual has far reaching implications, and the student will do well to consider what some of these might be (see Diamond).