Strain-typical patterns of pregnancy-induced nestbuilding in mice: Maternal and experiential influences

Physiology & Behavior, Vol. 25, pp. 153-157. Pergamon Press, 1982. Printed in the U.S.A.

Strain-Typical Patterns of Pregnancy-Induced Nestbuilding in Mice: Maternal and Experiential Influences JOHN BROIDA AND BRUCE SVARE

Department of Psychology, State University of New York at Albany, Albany, NY 12222 R e c e i v e d 28 N o v e m b e r 1981 BROIDA, J. AND B. SVARE. Strain-typical patterns of pregnancy-induced nestbuilding in mice: Maternal and experiential influences. PHYSIOL. BEHAV. 29(1) 153-157, 1982.--Pregnant C57BL/6J mice incorporate less material into maternal nests and build fewer fully enclosed nests than do pregnant DBA/2J mice. These strain differences are not ameliorated

by additional reproductive experience since multiparous animals also exhibit a similar pattern. Reciprocally-crossedhybrid females exhibit DBA-like levels of pregnancy-induced nestbuilding and cross-fostered C57BL and DBA females retain the phenotype of their strain. Experiential and maternal environmental factors apparently are not responsible for strain differences in pregnancy-induced nestbuilding. Differences in ovarian function and/or central neural tissue sensitivity to ovarian hormones may modulate strain differences in pregnancy-induced nestbuilding. Inbred mice Pregnancy Cross-fostering

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Maternal environment

IN female rodents, nestbuilding during pregnancy is instrumental for the successful rearing of young following parturition. Pregnant mice build nests which are larger and more fully enclosed then the "sleeping" nests built by nonpregnant females [5, 6, 19]. Construction of these "maternal" nests during pregnancy is dependent upon the release of ovarian hormones. F o r outbred mice, nestbuilding behavior and circulating progesterone (P) exhibit a steady increase from Gestation Days 1 to 11 followed by a decline in the behavior and plasma steroid levels beginning around Gestation Day 15 (e.g., [5, 6, 12, 15]). Virgin female mice can be induced to exhibit maternal type nests if they are first exposed to a priming injection of estrogen (E) followed by continuous exposure to P [6, 10, 11]. Genotype is an important modulator of nestbuilding activity. F o r example, Lee [7, 8, 9] reported significant strain differences in the nestbuilding activity of virgin C57BL/6J and DBA/2J female mice in that the former incorporated more material into the nest than the latter. In the present series of experiments, we sought to further explore genotypic influences on nestbuilding activity by examining this behavior in pregnant C57BL/6J and DBA/2J female mice. These strains were utilized because it is well known that they dramatically differ with respect to many different aspects of endocrine function and hormone-dependent reproductive behavior (e.g., [3,4]). In the first experiment, nestbuild~ng behavior was monitored in female C57BL/6J and DBA/2J mice throughout pregnancy. EXPERIMENT 1 METHOD

C57BL/6J and DBA/2J mice, born and reared in our lab-

Experience

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oratory at SUNY-Albany, were used in these experiments. The animals were descendents of stock originally purchased from the Jackson Laboratory, Bar Harbor, ME. The animals were maintained in a temperature controlled environment (24___I°C) on a 12/12 hr light/dark cycle (lights on at 6:00 a.m.). The animals had free access to food (Charles River Mouse Chow) and water, and were housed in 11 1/2×71/2x5 in. polypropylene cages with pine shavings on the floor. The mice were reared from birth in same sex litters of 4--6 animals and were weaned at 21 days of age. At 60-70 days of age, ten C57BL virgin females and the same number of DBA animals were timed-mated with similarly aged males of the same strain. When a vaginal plug was found (Day 0 of pregnancy), the animals were individually housed and provided with preweighed absorbent cotton placed into the food well of the wire grid cage cover (cf., [2,18]). Nestbuilding assessments were made the following morning and each morning thereafter until parturition. Qualitative assessments were m a d e ~ c c o r d i n g to the following scale (modified from [2]): Type 1: no nest; Type 2: saucer shaped; Type 3: raised sides; Type 4: fully enclosed. All cotton pulled into the cage was then removed and the cotton remaining on the cage top was weighed and replenished. This method provides an accurate assessment of nestbuilding behavior since most, if not all, of the cotton pulled into the cage is incorporated into the nest [2,18]. Analysis of Variance (ANOVA) was used in this experiment as well as the others reported here in order to determine main effects on the amount of cotton pulled. Where significant effects were obtained post-hoc comparisons using Fisher's Least Significant Difference (LSD) Test (a=0.05) with appropriate error terms were performed [17]. The median nest-quality score over the 19 days of pregnancy was

C o p y r i g h t © 1982 P e r g a m o n Press---0031-9384/82/070153-05503.00/0

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TABLE 1 QUALITATIVE ASSESSMENTS OF PREGNANCY-INDUCED NESTBUILDING

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FIG. 1. Results of Experiment 1 showing mean amount of cotton pulled as a function of strain and day of pregnancy (left panel), and mean total amount of cotton pulled during pregnancy as a function of strain (right panel).

computed for each subject in this experiment as well as the others reported here and analyzed by the Mann-Whitney U Test. RESULTS Because pregnant C57BL females tended to deliver their young on the 19th day of gestation while DBA females delivered their young on the 20th day, observations of nestbuilding were restricted to the first 19 days of pregnancy. Figure 1 portrays the mean amount of cotton pulled as a function of strain and day of pregnancy and the mean total amount of cotton pulled during pregnancy as a function of strain. The figure shows that animals of both strains exhibited the characteristic rise and subsequent decline in the amount of cotton pulled with advancing gestation. However, the mean amount of cotton pulled by DBA mice peaked at a higher level and did not decline as rapidly as that of their C57BL counterparts. Thus, the figure shows that pregnant DBA mice pulled more cotton than C57BL animals during the second half of pregnancy. An ANOVA performed on the mean amount of cotton pulled supported the above observations in that significant effects of strain, F(1,18)=7.4, p<0.01, and day, F(18,324)=7.9, p<0.001, were detected. Importantly, the interaction of these variables was also significant, F(18,324)=13.7, p<0.001. Fisher's LSD Test showed that, with the exception of Gestation Day 17, pregnant DBA females pulled significantly more cotton than C57BL females between" Days 9 and 19 of pregnancy. Table 1 summarizes the median nest quality scores for the two strains. The table shows that pregnant DBA mice exhibited higher median values than their C57BL counterparts. A Mann-Whitney U Test showed that this difference was significant, U(10,10)= 13,p<0.05. Thus, pregnant DBA females tended to build more enclosed nests and to incorporate more cotton into their nests than did pregnant C57BL females. Additional reproductive experience might ameliorate the apparent strain differences observed in the above experiment. To explore this possibility, animals in Experiment 2 were

Experiment 1 C57BL DBA Experiment 2 1st pregnancy C57BL DBA 2nd pregnancy C57BL DBA Experiment 3 C57BL B6D2 D2B6 DBA Experiment 4 C57 Cross-fostered Within-fostered Control DBA Cross-fostered Within-fostered Control

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*Median (and range) of median nest quality scores. The quality of the nest was rated each day during pregnancy according to the following scale: 1=no nest, 2=saucer shaped, 3=raised sides, 4=fully enclosed. A median score was then computed for each animal.

examined for nestbuilding behavior on two successive pregnancies.

EXPERIMENT 2 METHOD Nulliparous 60-70 day old C57BL (N =9) and DBA (N = 11) females were timed mated with same strain stud males and tested for pregnancy-induced nestbuilding as described earlier. Testing was temporarily discontinued on the 19th day of pregnancy and the dams were left with their litters following parturition until Postpartum Day 21. At this time, the litters were weaned and the mother was remated with a same strain stud male. When a vaginal plug was found, testing was reinstituted in the manner described earlier. RESULTS Figure 2 portrays the mean amount of cotton pulled as a function of strain, parous state, and day of pregnancy, and the mean total amount of cotton pulled during each pregnancy as a function of parous state and strain. It is evident that during each pregnancy animals of each strain exhibited an initial increase in the amount of cotton pulled followed by a decline towards the end of gestation. The figure also shows

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and DBA females, respectively). The above findings clearly show that additional reproductive experience does not ameliorate strain differences in nestbuilding behavior. Another factor which is known to mediate some strain differences in behavior is the prenatal and postnatal maternal environment (e.g., [13, 14, 16]). One way to explore the potential influence of maternal environmental factors is to examine nestbuilding behavior in reciprocally-crossed animals. Thus, C57BL males are crossed with DBA females and their offspring are compared to the offspring of C57BL females crossed with DBA males. The resulting hybrid offspring have the same genotype but their mother should make a difference in the pups' level of the behavior if prenatal and/or postnatal maternal factors are important for nestbuilding. Reciprocal crosses were used in the following experiment in order to examine maternal environmental influences on nestbuilding behavior. EXPERIMENT 3

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Nulliparous 60-70 day old C57BL and DBA females were mated with similarly aged males of the same (C57BL female × C57BL male=C57BL offspring (N=21); DBA female × DBA m a l e = D B A offspring (N=21)) or opposite strain (C57BL female × DBA male=D2B6 offspring (N=21); DBA female × C57BL male=B6D2 offspring (N=20)). The animals were isolated when visibly pregnant and, following parturition, the resulting litters were adjusted to 4--6 female offspring. The animals were weaned at 21 days of age. At 55 days of age, the females were timed-mated as described earlier and individually housed. Nestbuilding was assessed as described in Experiment 1. RESULTS

that pregnant DBA females pulled more cotton than pregnant C57BL females on the first and second pregnancies with these differences most pronounced during the latter half o f gestation. An A N O V A test on the mean amount of cotton pulled during pregnancy revealed significant effects due to strain, F(1,18)=16.4, p<0.001, and day of pregnancy, F(18,324)=15.6, p<0.001, but not for parous state, F(1,18)=1.99, p>0.05. The day by strain interaction was significant, F(18,324)=7.1, p<0.001, as was the pregnancy by day interaction, F(18,324)=4.0, p<0.001. Importantly, the three way interaction (strain by day by parous state) was not significant, F(18,324)= 1.1, p>0.05, thus further indicating that parity was without effect on the amount of cotton pulled by animals of the two strains. Fisher's LSD test confirmed the fact that pregnant DBA mice pulled significantly more cotton than pregnant C57BL females on each pregnancy. On days 6, 7, and 9-19 of the first pregnancy and on days 2, 7-9, and 11-19 of the second pregnancy, DBA females pulled significantly more cotton than did C57BL females. The median qualitative scores for nestbuilding during the first and second pregnancies were similar to those of the quantitative scores (See Table 1). Mann-Whitney U Tests showed that pregnant DBA females exhibited higher nest quality scores than pregnant C57BL females on each pregnancy, U(9,11)< = 15, p<0.01. Wilcoxin Signed Ranks Tests showed that neither strain exhibited a change in median nest quality from the first pregnancy to the second pregnancy (T(9 and 11)=30.5 and 102.5, p > 0 . 0 5 for C57BL

The mean amount of cotton pulled as a function of strain and day of pregnancy, and the mean total amount of cotton pulled during pregnancy as a function of strain is illustrated in Fig. 3. Once again, as pregnancy advanced, animals of each line exhibited a rise followed by a decline in the amount of cotton pulled. Most importantly, however, it is evident from this figure that C57BL pregnant females pulled much less cotton than pregnant females of the DBA strain and pregnant animals of both hybrid lines. From midgestation onward, pregnant C57BL mice appeared to pull less cotton than pregnant DBA and hybrid females. A N O V A tests on the mean amount of cotton pulled during pregnancy revealed significant effects due to strain, F(3,79)= 32.5, p<0.001, and day of pregnancy, F(18,1422)=60.9, p<0.001. Post-hoc tests using the LSD test showed that pregnant DBA, D2B6, and B6D2 females pulled significantly more cotton than did pregnant C57BL females and that pregnant D2B6 animals pulled significantly more cotton than did pregnant DBAs. Although pregnant B6D2 animals appeared to pull significantly more cotton than did pregnant DBAs, this difference just missed statistical significance. Importantly, however, the pregnant hybrid lines did not significantly differ from each other with respect to the amount o f cotton pulled. Pregnant DBA and hybrid females pulled significantly more cotton than pregnant C57BL females on Gestation Days 9 through 19. Also, Mann-Whitney U Tests on the median nest quality score showed that C57BL female exhibited lower nest quality scores than pregnant B6D2, D2B6, and DBA females, U(20,21)<=5, p<0.001. Once again, pregnant D2B6 females

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did not significantly differ from pregnant B6D2 females in terms of the median nest quality score. Because both hybrid lines exhibited high quantitative and qualitative nestbuilding scores, the findings show that there is dominance (indeed, probably overdominance) for the DBA phenotype of nestbuilding behavior. Moreover, the results also suggest that the strain of the mother has little influence on levels of nestbuilding activity. The extent to which postnatal maternal environmental factors modulate strain-typical patterns of behavior should also be assessed by use of the cross-fostering technique (see Broadhurst [1] for a discussion). Briefly, neonates are transferred at birth to mothers of the opposite strain (crossfostered). Postnatal maternal influences on nestbuilding behavior would be implicated if pups reared by a foster mother behaved like pups of the foster mother's strain; Postnatal maternal influences would not be implicated if cross-fostered pups behaved true to their own genotype. The above tactic was used in the following experiment. EXPERIMENT 4 METHOD

Nulliparous 60-70 day old C57BL and DBA female mice were timed-mated as described earlier. At parturition, litters were adjusted to 4-6 pups and either cross-fostered to recently parturient dams of the opposite strain (Group CROSS, N = 11 and 10 respectively for C57BL and DBA mice), infostered to recently parturient dams of the same strain (Group WITHIN, N = 10 and 15 respectively for C57BL and DBA mice), or handled and returned to their natural mother (Group CONTROL, N = 9 and 12 respectively for C57BL and DBA mice). The animals were weaned at 21 days of age. At 55 days of age, the animals were timed-mated and tested for nestbuilding as previously described.

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RESULTS

Figure 4 shows the mean amount of cotton pulled as a function of day of pregnancy, fostering condition, and strain, and the mean total amount of cotton pulled during pregnancy as a function of strain and fostering condition. Consistent with previous experiments, the figure shows that the animals in every group exhibited an increase followed by a decline in the amount of cotton pulled with advancing pregnancy. Also, it is evident from the figure that pregnant DBA female mice pulled more cotton than did pregnant C57BL females regardless of fostering condition. Once again, the figure also illustrates that strain differences in nestbuilding activity (i.e., DBA mice pulling more cotton than C57BL animals) emerge around midpregnancy and persist through late gestation. ANOVA tests on the amount of cotton pulled during pregnancy revealed significant effects due to strain, F(1,61)=46.1, p<0.001, and day of pregnancy, F(18,1098)=86.4, p<0.001, but not for fostering condition, F(2,61)= 1.0, p>0.05. Day of pregnancy was again found to interact with strain, F(18,1098)=22.2, p <0.001, and with fostering condition, F(36,1098)= 1.7, p<0.01, but the three way interaction of these factors was not significant,

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F(36,1098)=1.3, p>0.05. LSD tests showed that regardless of fostering condition pregnant DBA females pulled significantly more cotton than did pregnant C57BL mice. Also, regardless of fostering condition, pregnant DBA females pulled significantly more cotton than pregnant C57BL females on Gestation Days 9 through 19. Finally, regardless of fostering condition, pregnant C57BL females exhibited significantly lower median nest quality values than did C57BL mice, U(9,10)<=14, p<0.05. To summarize, crossfostering does not influence either the quality or quantity of nests constructed by DBA and C57BL mice. Therefore, postnatal maternal environmental factors do not influence strain-typical patterns of pregnancy-induced nestbuilding. G E N E R A L DISCUSSION The present findings show that female C57BL/6J and DBA/2J inbred mice exhibit significant qualitative and quantitative differences in their nestbuilding activity during pregnancy. From mid-gestation (Day 9) onward, C57BL females incorporate less material into maternal nests and build fewer fully inclosed nests than do DBA females. Additional reproductive experience does not ameliorate the above strain differences since animals of the two strains exhibit the same pattern of nestbuilding activity on their second pregnancy. Reciprocally-crossed hybrid females exhibit DBA-like levels of pregnancy-induced nestbuilding and cross-fostered C57BL and DBA females retain the phenotype of their strain. The similarity of the hybrid strains to the DBA strain would suggest that "high" levels of the behavior is a dominant trait and may represent an advantageous adaptation. More importantly, however, the findings show that experiential and maternal environmental factors are not responsible for strain differences in pregnancy-induced nestbuilding.

It is unclear at the present time as to how genes modulate strain-typical patterns of pregnancy-induced nestbuilding behavior. Because nestbuilding behavior is positively correlated with body surface area and negatively correlated with body temperature [9,19], genotypic variation in the behavior may be due to strain differences in body weight or temperature. This does not appear to be the case, however, since preliminary observations (Broida and Svare, unpublished observations) indicate that pregnant DBA/2J and C57BL/6J female mice do not differ with respect to core temperature or body weight. Alternatively, because the behavior is known to be progesterone (P) dependent [6, 10, 11], it is tempting to speculate that the genome mediates strain differences in pregnancy-induced nest construction by controlling circulating levels of P and/or the sensitivity of the brain to the steroid. Work currently in progress in our laboratory is designed to examine the above possibility. The study of nestbuilding behavior during pregnancy would seem to offer special advantages for those interested in behavior-genetic analysis. It is a species-typical behavior which is easily quantifiable and markedly influenced by genotype. Just how genes function to modulate physiological status and animal behavior is a problem central to our understanding of individual differences. The systematic study of the mechanisms underlying strain differences in nestbuilding may be a small step toward answering the above question. ACKNOWLEDGEMENTS This work was supported in part by a Research Grant from the Harry Frank Guggenheim Foundation, by Grant BNS80-08546 from NSF, and by Grant AG01319from NIA. We thank Dr. Bruce Dudek for statistical assistance. Send reprint requests to Dr. Bruce Svare, Department of Psychology, SUNY-Albany, 1400 Washington Avenue, Albany, NY 12222.

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