BEHAVIORALBIOLOGY,8, 399-405 (1973), Abstract No. 1-155R
Cyclical Wheel-Running Behavior in Pregnant Rats 1 DAVID CHISZAR and BETTY J. PANNABECKER
University of Colorado, Boulder, Colorado Wheel-running behavior was monitored in five rats prior to and during gestation. All subjects showed peaks of activity during pregnancy and statistical analysis conf'trmed the cyclical occurrence of these peaks. Possible implications of these data for conceptualization of pituitary-ovarian gametogentic relationships are discussed,
It is well known that non-pregnant rats housed in activity wheels run in a manner which correlates with their estrus cycle. That is, the animals run at a higher rate during the stage of heat, as indicated by vaginal smears and copulatory behavior, than during the other stages of the cycle (Richter, 1927; Slonaker, 1924; Wang, 1923). These activity cycles are readily interpreted in terms of gonadotropic-induced cyclical release of ovarian estrogen (Richter, 1933; Wang and Guttmacher, 1927; Wang, Richter and Guttmacher, 1925) and progesterone of either ovarian or adrenal origin (Uphouse, Wilson and Schlesinger, 1970; Young, 1961). Although it is difficult to find data concerning wheel-running behavior of pregnant rats, it is commonly believed that "this vivid cyclicity is destroyed and the level of activity markedly reduced during pregnancy, lactation, or in pseudopregnancy" (BoUes, 1967, p. 116). Wang (1923) and Richter (1927) are usually cited to support assertions like the previous one. Wang presented activity records for two pregnant rats, both of which showed dramatic decreases in activity level after insemination. However, one of these animals showed three distinct peaks of activity during gestation. Although these peaks did not reach the level of prepregnant peaks, they were, nonetheless, clearly cyclical ( P < .01 by an adaptation of Noether's 1956 test of cyclical trend). Wang's second pregnant rat showed less clear evidence of activity cycles during gestation. Yet, even this subject's record contained a peak of activity, suggesting that pregnancy may not completely eliminate activity rhythms. The 1Contribution No. 57 from the Laboratory of Animal Physiology and Behavior. This research was supported by a Nurse-Scientist pre-doctoral fellowship awarded to the second author and supervised by the first. 399 Copyright © 1973 by Academic Press, Inc. All rights for reproduction in any form reserved.
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first of Wang's records was reprinted in Richter's (1927) discussion of activity during gestation. It should be mentioned that Wang, but not Richter, concluded that hormonal conditions of gestation eliminate activity cycles in female rats. However, Wang's data provides evidence for the opposite conclusion. The present study was conducted to generate additional data concerning the presence or absence of cyclical wheel-running in pregnant rats. METHOD
Subjects and apparatus. Five female albino rats of the Sprague-Dawley strain (Redwood Game Farms, Salt Lake City, Utah) were maintained in activity wheels (Wahmann Company) with ad lib. food and water. The animals were approximately 80 days old at the start of the study and hadnever been mated previously. The study was conducted in a windowless, air-conditioned room in which a reversed light-dark cycle was maintained with overhead fluorescent lights on between 1900 and 0700 hr. Whenever the experimenter entered this room from 0700 to 1900 hr, three red 25-w incandescent bulbs provided illumination. Procedure. During the prepregnant phase of the study (approximately 2 mo), daffy counts of wheel turns were recorded at 0900 hr (i.e., 2 hr after lights out) to provide, for each subject, a baseline record of wheel-running cycles. Small saline-soaked cotton swabs were gently inserted (approximately .25 in.) into each female's vagina daily at 0900 hr. Swabs were smeared on slides and examined microscopically immediately afterward. Smears were judged to indicate proestrus-estrus if: (a)leucocytes were rare or nonexistent and (b) nucleated epithelial cells were frequent. A slide containing disintegrating epithelial cells was judged to indicate proestrus-estrus only if non-disintegrated cells were also present in at least equal amounts. No attempt was made to distinguish between the stages of anestrus (i.e., metestrus, diestrus). This dichotomous scoring was quite reliable in that independent raters show more than 95% agreement in their classification of smears. After this prepregnant period, the animals were mated on a day when wheel running and smear indicated estrus. Subjects were placed back into their activity wheels and remained there until day 18 of gestation. Daily records of wheel running were kept. However, vaginal smears were no longer taken and the pregnant females were never handled since several reports have indicated that handling during gestation influences maternal physiology and foetal development and may even alter the length of gestation (Ader and Conklin, 1963; Ader and Plaut, 1968; Chiszar and Kappel, 1970; Werboff, Anderson and Haggett, 1968). RESULTS
Prepregnant phase. _A total of 47 activity cycles were observed. The modal length of these cycles (trough-to-peak) was 4 days while the mean
CYCLICALACTIVITY IN PREGNANTRATS
length was 3.6 days (SD = 1.09). Although most peaks (66%) of wheel-running activity were co-incident with the day of vaginal estrus, there were a number of exceptions. Nineteen percent of the activity peaks occurred during the day before vaginal estrus; the remaining 15% occurred during the day after squamous cells appeared in the vagina. Such discrepancies have been reported previously (Cooley and Slonaker, 1925). Pregnant stage. Figure 1 presents the prepregnant and pregnant records for each subject. All animals show a decline in activity during gestation. 20[ s J
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Fig. 1. Daily number of wheel turns prior to and during pregnancy for five rats. Although the overall amount of running decreases during pregnancy, oscillations are present in the records. During the 18 days prior to conception, the mean daily number of wheel turns was 5990 while the daily mean during the first 18 days of gestation was
CHISZAR AND PANNABECKER
2409 (t(4) = 3.74, P < .05). However, it is clear that activity peaks occur during pregnancy. This is true in all subjects, although some animals show less regular periods than others. For example, subjects 2 and 3 show the most irregular patterns during gestation; y e t it is possible to identify three peaks of activity in each case. However, the gestation records for the remaining three subjects are more regular. Indeed, it is possible to superimpose these records upon segments o f the respective prepregnant baselines and obtain regular co-incidence between successive peaks and troughs (although the heights of the pregnancy peaks will be lower than the prepregnant counterparts). Table 1 presents the analysis o f each subjects' prepregnant and pregnant records. 2 Clearly, the prepregnant records contain cyclical activity fluctuTABLE 1 Significance of Cyclical Trends Shown by Each Subject Prior to and During Pregnancy
1 2 3 4 5
P = .109 P =.049 P = .051 P=.001 P=.091
P = .093 P =.093 P = .234 P = .093 P=.234
P represents the probability that the observed number of monotonic groups could have occurred by chance. Rejecting Ho (and inspecting the records) implies that a cyclical trend exists. ations. If the three-score groups are pooled across subjects, 46 o f 6 4 ( 7 1 . 8 % ) prepregnant groups are monotonic (X2(1)= 12.24, P < . 0 1 ) and 23 of 30 (76.6%) gestation groups are monotonic (X2(1) = 8.52, P < . 0 1 ) . Lewis and Burke (1946) argue that the generality o f these straightforward tests should be restricted only to the present sample of organisms. To extend generality, tests should be performed on individual subjects, after which the X2s can be 2A modified version of Noether's (1956) test was employed to evaluate the significance of the apparent cyclical trends during pregnancy. The test partitions a continuous record into successive and nonoverlapping groups of three scores. Such groups are considered to be independent and the test evaluates the frequency of monotonic groups against the binomial distribution with P= 1/3. Tied (adjacent) scores within a group present a problem which is often resolved by eliminating the tie and regrouping the scores. The present modification considers adjacent scores to be tied if their values are within 10% of each other. However, instead of dropping the tied scores, they are counted as being part of a monotonic group if the remaining score is greater or less than the tied scores by at least 10%. In all other respects, the present version is exactly like the original except that the binomial probability of a monotonic group becomes 1/2. These modifications tend to increase the power of the test (especially for data as variable a, wheel running) and, thereby, provide a convenient tool for the analysis of eyelieity.
CYCLICAL ACTIVITY IN PREGNANT RATS
pooled. Employing Cochran's (1954) pooling procedure, the average normal deviates (AND) for prepregnant and pregnant records are 3.54 ( P < . 0 1 ) and 2.46 (P<.01), respectively. Hence, it is reasonable to conclude that both kinds of records contain cyclic trends. Finally, it is meaningful to ask if the relative frequency of monotonic sequences differs between the prepregnant and pregnant stages. Chi-squares were calculated when three-score groups were pooled over subjects (X2(1) =.002, P > > .05)and for individual subjects AND = 0.53, P > > .05). Thus, during the first 18 days of gestation, the percentage of monotonic groups is comparable to the percentage of such groups prior to pregnancy. This implies that the frequency composition of prepregnant and pregnant records are similar. Figure 2 presents relative frequency distributions of cycle lengths (trough-to-peak) for both phases of the study. Clearly, there is O0
PREGNANT (5 Ss ,18 CYCLES)
ILl 5 0
PREPREGNANT (5 Ss, 47 CYCLES)
Fig. 2. Relative frequency distributions of cycle lengths during the prepregnant and pregnant phases of the study. considerable overlap. But, the modal length of activity cycles during gestation is 3 days (mean = 3.1, SD = 0.83). However, this apparent modal shift may not be important since the prepregnant and pregnant frequencies of 4-day or 3-day cycles are not significantly different (X2s-- 1.94 and 1.67, respectively, df's = 1, P's > .05).
DISCUSSION The present data reveal estrus4ike cycles of wheel-running behavior during gestation in the Sprague-Dawley rat. Such cycles have also been observed in Long-Evans rats (Chiszar, unpublished data). Similar observations were reported by Slonaker (1925). In addition, Richards (1964, 1966) observed comparable fluctuation in frequency of home cage activities (e.g., sleeping, time spent in the n e s t ) i n pregnant golden hamsters. These phenomena suggest that underlying hormonal cycles may not be obliterated
CHISZAR AND PANNABECKER
during pregnancy. In fact, data reviewed by Nalbandov (1958) reveals that a number of domestic mammals (ewes, cows, sows, rats and mice) show considerable follicular development and, in many cases, ovulation during gestation. In many ewes and cows, as well as laboratory rodents, this ovarian activity has been reported to eventuate in behavioral heat during pregnancy (Nalbandov, 1958; M. Seer, personal communication3). This accumulating evidence forces certain changes in the conventional view of pregnancy. It seems unreasonable to believe that the ovaries are always gametogenetically dormant during gestation. In fact, the occurrence of ovulation, behavioral heat, and cyclical activity probably indicates that the gonadotrophic-ovarian axis remains at least partially functional throughout pregnancy. This may imply that the rhythmical release of pituitary gonadotropins (FSH and LH) in mammalian females is at least partially independent of feedback mechanisms reflecting circulating estrogen and/or progesterone levels which, during gestation, increase several fold through luteal and placental production. An additional possibility is that estrogen and/or progesterone of placental origin may not be as effective for pituitary feedback (because of chemical properties, circulatory effects, or inopportune titers for synergistic action) as their ovarian or adrenal counterparts. Since estrus-like activity cycles occur regularly during the rat's gestation and since superfetation is known to occur, it is probable that copulatory behaviors also occur during pregnancy. The fact that activity level is generally depressed during gestation may imply that supemormal stimulation is required to elicit lordorsis and other copulatory responses in the pregnant female. These considerations indicate that gestation does not eliminate behavioral and physiological reproductive processes, but probably interacts strongly with them, suggesting that current feedback concepts may be unable to handle effects of pregnancy without modification. REFERENCES Ader, R. and Conklin, P. M. (1963). Handling of pregnant rats: Effects on emotionality of their offspring. Science 142, 411-412. Ader, R. and Plaut, S. M. (1968). Effects of prenatal maternal handling and differential housing on offspring emotionality, plasma corticosterone levels, and susceptibility to gastric erosions. Psychosom. Med. 30, 277-286. BoUes, R. C. (1967). "Theory of Motivation," p. 116. New York: Harper and Row. Chiszar, D. and Kappel, H. (1970). Effects of handling pregnant rats on gestation and postnatal behavior of the young. Presented at meetings of Amer. Assoc. Lab. Anita. Sci., Upstate New York Branch, Utica, New York. Cochran, W. G. (1954). Some methods for strengthening the common tests. Biometrics 10, 417-451. Cooley, C. L. and Slonaker, J. R. (1925). The effects of early and late breeding on the mother and the sex ration in the albino rat. Amer. £ Physiol. 72, 597-613. 3Examination of breeding records at Mr. Seer's dairy indicates that nearly all cows ovulate and come into heat during gestation.
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Lewis, D. and Burke, C. J. (1949). The use and misuse of the chi-square test. Psychol. Bull. 46, 433-489. Nalbandov, A. V. (1958). "Reproductive Physiology," pp. 218-245. San Francisco: W. H. Freeman. Noether, G. E. (1956). Two sequential tests against trend. £ Amer. Stat. Assoc, 51, 440-450. Richards, M. P. M. (1964). Cyclical behavioral activity in the golden hamster (Mesocricetus auratus waterhouse). Nature, (London) 204, 1327-1328. Richards, M. P. M. (1966). Activity measured by running wheels and observation during the oestrus cycle, pregnancy and pseudopregnancy in the golden hamster. An#n. Behav. 14, 450-458. Richter, C. P. (I927). Animal behavior and internal drive. Quart. Rev. Biol. 2, 307-343. Richter, C. P. (1933). The effect of early gonadeetomy on the gross body activity of rats. Endocrinology 17 445-450. Slonaker, J. R. (1924). The effect of pubescence, oestruation, and menopause on the volitional activity in the albino rat. Amer. £ Physiol. 68, 294-315. Slonaker, J. R. (1925). The effect of copulation, pregnancy, pseudopregnancy, and lactation on the voluntary activity and food consumption of the albino rat. Amer. £ Physiol. 71, 362-394. Uphouse, L. L., Wilson, J2 R. and Schlesinger, K. (1970). Induction of estrus in mice: The possible role of adrenal progesterone. Horm. Behav. 1, 255-264. Wang, G. H. (1923). Relation between "spontaneous" activity and oestrus cycle in the white rat. Comp. Psychol. Monogr. 2, 1-27. Wang, G. H~ and Guttmacher, A. F. (1927). The effect of ovarian traumatization on the spontaneous activity and genital tract of the albino rat, correlated with a histological study of the ovaris. Amer. £ Physiol. 82, 335-349. Wang, G. H., Richter, C. P. and Guttmacher, A, F. (1925). Activity studies of male castrated rats with ovarian transplants, and correlation of file activity with histology of the grafts. Amer. J. Physiol. 73, 581-598. Werboff, J., Anderson, A., and Haggett, B. N. (1968). Handling of pregnant mice: Gestational and postnatal behavioral effects. PhysioL Behav. 3, 35-39. Young, W. C. (1961). The hormones and mating behavior. In W. C. Young (Ed.), "Sex and Internal Secretions," 2, Baltimore: Wi/liams and Wilkins.