Crusio, W. E. & Schmitt, A. (1996) Prenatal Effects of Parity on Behavioral Ontogeny in Mice. Physiology and Behavior 59 1171-1174

Running head: EARLY BEHAVIORAL DEVELOPMENT AND PARITY

Send correspondence and proofs to Dr. W.E. Crusio, Génétique, Neurogénétique et Comportement, URA 1294 CNRS, UFR Biomédicale, Université Paris V René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France.

Tel.: 33 1 4286 2206; Fax.: 33 1 4286 2250; E-mail: crusio@citi2.fr

CRUSIO, W.E. AND A. SCHMITT. Prenatal Effects of Parity on Early Development in Mice. PHYSIOL BEHAV 00(0) 000-000. 0000.-In mice, parity and previous experience with pups may influence a mother's behavior towards her pups, thus possibly causing postnatal maternal effects on the subsequent development of the pups. The present experiment addressed the question whether parity also might have prenatal effects. We studied 622 pups from second or third litters that originated from 25 genetically different populations and had been fostered to random-bred lactating females. Development of responses was significantly delayed in mice from third litters, when compared to pups from second litters in 3 out of 5 sensorial and 4 out of 8 motor tests. In addition, pups from second litters initially were slightly heavier than those from third litters. This difference in body weight disappeared after the 10th day postnatally. However, it should be noted that effect sizes were quite small.

Prenatal effects - Early development - Sensorial and motor responses - Parity - Inbred strains - Mice

EARLY development of sensorial and motor responses in mice have been extensively studied employing the battery of tests developed thirty years ago by Fox [9] for this purpose. Since then, Fox's tests repeatedly have proven their value for revealing, sometimes very subtle, individual differences in ontogeny. Perhaps unsurprisingly, already quite early it became evident that an individual's genotype plays an important role in the regulation of early development as Van Abeelen and co-workers demonstrated abnormal development in some neurological mutants of the mouse 21, 22]. This work was followed by numerous reports of strain differences [e.g., 10, 11, 12, 23].

Although strain differences are often taken as an indication of genetic effects, this is not necessarily true [4]. By means of a series of carefully designed experiments, Roubertoux, Carlier and co-workers showed that both pre- and postnatal maternal effects might influence early behavioral ontogeny [2, 13, 14, 16,]. Postnatal maternal effects may occur because of, e.g., differential composition of the milk [15] or different treatment of pups by mothers of different genotypes [3] or having different previous experiences [5]. Prenatal maternal effects comprise cytoplasmic effects (differential compositions of either the cytoplasm of the egg or of the mtDNA) or effects of the uterine environment [18].

The experiments cited above mostly tried to explain differences between inbred strains in terms of genetic influences and either pre- or postnatal maternal effects. A notable exception is the study of Cohen-Salmon and co-workers, who showed differences in maternal behavior between primiparous and biparous mice in eight inbred strains [6]. Thus, differences in parity might cause postnatal effects on pups' behavior. The present experiment addresses the question whether differences in parity might also exert effects prenatally on pups' subsequent behavior. To this end, new-born pups from 25 genetically different groups were fostered to lactating females from a random-bred strain in order to provide all pups with a similar postnatal maternal environment. Subsequently, we compared pups derived from second litters with those derived from third litters.

Animals and Treatment

We bred 25 genetically different groups by systematically intercrossing 5 inbred strains in all possible combinations, including reciprocal crosses (diallel cross). As parents for the diallel cross we used mice from strains BA//C, C57BL/6J, C57BR/cdJ, BALB/cJ, and DBA/2J [20], bred for at least 10 generations in our own laboratory.

From all 25 crosses, 30 litters were raised simultaneously: one for each of the 20 hybrids and two for each of the 5 parentals. The 30 litters obtained in this way constituted one replication. Four such replications were bred consecutively. For the second and fourth replications we used only the second litter of a particular dam. Third litters were taken for the first and third replications. Breeding pairs were housed in plastic breeding cages (165 x 220 x 140 mm) with a metal cover and a bedding of wood shavings. Cages were cleaned once a week. Food pellets (Altromin) and tap water were always available. The animals were maintained in an air-conditioned mouse room (22 ± 2 oC; humidity 55 ± 5 %) with a 12h light/dark schedule (lights on 0600h; 225 Lux). Males were removed when females became visibly pregnant.

Cages were inspected twice a day for births (8.00 a.m. and 4.00 p.m.) and the day a litter was first seen was considered to be day one. Litters smaller than 5 were discarded, as were litters without male pups. Litters were culled to a maximum of 8 pups. To standardize the received quantity of milk and maternal pup care, litters smaller than 8 were brought up to 8 with pups with another coat color. This happened in about 50% of cases but in about the same proportions over crosses and parity groups. To randomize possible postnatal effects, all newborn pups were routinely fostered to lactating mothers from a random-bred NMRI stock.

To avoid cannibalism in the first few hours after birth, breeders were allowed to keep their first litters (discarded after weaning) and received 4 pups from the random-bred foster mothers after the second litters were fostered away. This procedure ensured exposure to pups and provided the breeders with nursing experience [5].

From each litter, one (litters with 5 pups) or two male pups (litters with 6 to 8 pups) were not tested but raised to adulthood for other experiments, so that between 4 and 6 pups were finally tested per litter. In total, 622 animals (233 males and 389 females) from 120 litters were tested, resulting in 17-22 (parentals, 4 litters) and 8-12 subjects (hybrids, 2 litters) per parity group per cross. In total, 317 animals from second litters and 305 animals from third litters were studied.

Procedure

Thirteen sensorial and motor responses and body weight were recorded in each pup on the mornings of days 3, 5, and 8 (neonatal period; see refs. 9 and 23), days 10 and 13 (postnatal transitional), and days 17 and 22 (postnatal infantile; day of birth = day 1). The same individuals were tested at all ages. For identification purposes, pups were daily marked with a non-toxic purple dye. Tests took place between 8.30 a.m. and 16.00 p.m. in a sequence that varied randomly over groups.

Although it should be noted that all tests include both motor and sensorial components, we classified tests as either primarily motor (M) or primarily sensorial (S) following Wahlsten [24]. Tests in the order administered and a brief description of each were as follows. 1) Rooting (M): bilateral stimulation of the face induces the pup to crawl forwards, pushing the head in a rooting fashion. 2) Righting (M): when the pup is placed on its back, it turns over and rests with all four feet on the ground. 3) Postural flexion and extension (M): when suspended by the scruff of the neck, the pup either flexes or extends its limbs. 4) Vibrissae placing (S): the pup is suspended by its tail. When its vibrissae are stimulated with a pencil it raises its head and performs a placing response with the extended forelimb. 5) Visual placing (S): the pup is suspended by its tail and lowered toward a solid object (e.g., a pencil or table top), without its vibrissae being stimulated. It will raise its head and perform a placing response. 6) Forelimb grasping reflex (M): occurs when the inside of the forepaw is stimulated with an object. 7) Hindlimb grasping reflex (M): as previous, with the hindlimb. 8) Vertical screen test (M): the pup is placed on a vertical wire-mesh screen and the time until the animal falls down is registered. 9) Bar holding (M): the pup must be able to hold onto a bar (1.5 cm diameter) for more than 5 sec. 10) Negative geotaxis (S): when the animal is placed on a 45o incline with its head pointing down it will turn around and crawl up the slope. 11) Cliff-drop aversion (S): When the pup is placed on the edge of a cliff or table top, it will turn around and crawl away from the cliff drop. 12) Popcorn behavior (M): exaggerated jumping and running behavior in response to a gentle puff of the experimenter's breath. 13) Auditory startle response (S): a loud snap of the fingers close to the ears (but without tactile stimulation) causes an immediate startle response.

Following Nosten [13], animals were scored for the first day on which an adult response was displayed for the first time for all behavioral variables. If an animal did not display the adult response on day 22, it was arbitrarily assigned a score of 25.

Statistical analysis

For the behavioral variables, uni- and multivariate analyses of variance (ANOVA and MANOVA) with group (25 levels), parity (2 levels), and sex (2 levels) as main factors were carried out. Body weights were analyzed using repeated-measures ANOVA. In both cases Type III sums of squares were used for significance testing, given the unbalanced nature of our experimental design. Similarly, we calculated least-squares estimates of population marginal means, which are the values of class means as would be expected for a balanced design. The GLM procedure of the SAS package for PC [19] was employed in both cases. The effect sizes of the differences between second and third litters were calculated using Cohen's d statistic: d = M₂ - M₃ / SD, where M₂ is the mean obtained for second litters and M₃ the mean obtained for third litters. SDs were approximated as the product between the S.E.M. and the square root of the corresponding number of subjects. As a genetic analysis of the differences between crosses will be the subject of another paper, they will not be elaborated upon here.

A repeated measures ANOVA for body weight (with days, cross, parity, and sex as factors) indicated no significant effect of sex (F_1,523 = 3.13, P > 0.075). However, sex interacted with days (F_6,3138 = 6.41, P < 0.0013), because a sex difference developed from day 22 onwards. As no significant effects of sex were obtained on any other day, only results of analyses obtained from data pooled over sexes are presented for simplicity. Least squares means can be found in Table 1. Pups from third litters generally weigh less than pups from second litters, although the difference declines progressively and becomes insignificant from the 13th day onward (day 3: F_1,572 = 7.08, P < 0.01; day 5: F_1,572 = 7.57, P < 0.01; day 8: F_1,572 = 13.42, P < 0.001; day 10: F_1,572 = 10.43, P < 0.01; day 13: F_1,572 = 1.92, P > 0.16; day 17: F_1,572 = 0.16, P > 0.68; day 22: F_1,572 = 0.42, P > 0.51). Accordingly, a repeated measures ANOVA indicated an almost significant effect of parity (F_1,572 = 3.17, P = 0.076), which interacted with the factor days (F_6,3432 = 4.33, P < 0.001). Cohen's d statistic indicated that effect sizes were generally very low (mean d = 0.173).

A MANOVA of the behavioral variables (with cross, parity, and sex as factors) showed no significant effects for sex, neither alone, nor in interaction with cross or parity (p>0.05). Therefore, for simplicity, results were pooled over sexes for the subsequent analyses in this case, too. Least-squares means for the different behavioral variables are presented in Table 2. All, or almost all, animals reached adult scores for rooting, righting, postural flexion/extension, visual placing, forelimb grasping, vertical screen test, bar holding, and negative geotaxis. In addition, at least 90% of all animals reached adult scores for hindlimb grasping, cliff-drop aversion, and popcorn behavior. For the auditory startle response only 53.5% of all pups reached adult scores, whereas this proportion dropped to 30.2% for vibrissae placing. Significant developmental retardations were found in third litters for 6 out of 13 tests: righting (F_1,572 = 8.33, P < 0.01), vibrissae placing (F_1,572 = 57.76, P < 0.0001), forelimb grasping (F_1,572 = 4.32, P < 0.05), hindlimb grasping (F_1,572 = 10.12, P < 0.001), bar holding (F_1,572 = 20.35, P < 0.0001), and the auditory startle response (F_1,572 = 8.46, P < 0.01). A MANOVA on all 13 variables combined yielded a highly significant effect of parity (F_13,560 = 6.77, P < 0.0001). Here, too, effect sizes were generally very low as indicated by Cohen's d statistic, which varied from 0.005 to around 0.25, vibrissae placing being an exception with a somewhat larger effect size of 0.61. Over all variables, the mean d equaled 0.171.

The present data show that mouse pups stemming from third litters are developmentally retarded when compared to pups from second litters. We would like to stress, however, that the effects are rather small and subtle. They could only be detected in the present experiment because of the large numbers of animals employed.

Furthermore, the data on body weight suggest that compensatory mechanisms may be at work, as the mild retardation becomes relatively even less important over time. Nevertheless, some behavioral responses (e.g., auditory startle response) are affected, despite the fact that they develop rather late (for the auditory startle response: around 22 days). An analysis of both behavioral and neuroanatomical data obtained at an age of 3 months from one male animal per litter (animals that remained untested here; see refs. 7, 8) did not show any effects of parity, indicating complete recovery (data not shown).

At this moment, we can only speculate on the physiological basis of the effect of parity here reported. First, maternal age may be a factor. Evidently, females were older at the time of birth of the third litter. However, as the ages of our breeders at the start of breeding varied between 10 and 14 weeks, this explanation appears unlikely. Second, when the third litters were born, breeders had already been through two other periods of gestation and nursing. Although high-quality food was available ad libitum, this may have led to some physiological strain. Further experiments are clearly necessary to elucidate this question.

In humans, it has been reported that the IQ of subsequent children in a family diminishes progressively (with the exception of the first child; see ref. 1). However, we would like to stress that the present results obtained with inbred and hybrid mice need not necessarily be extrapolatable to another species. Although such may appear attractive, more information regarding underlying physiological mechanisms is needed before early behavioral development in the mouse can be accepted as an animal model of the decreasing IQ phenomenon.

In conclusion, the present results show that in studies of early behavioral and physical development, the parity of the mother is a variable of some importance that ideally should be incorporated in the experimental design.

The experimental parts of the work described here were carried out while the first author was working as a guest at the Institute of Human Genetics and Anthropology, University of Heidelberg, Germany, supported by a NATO Science Fellowship, awarded by the Netherlands Organization of Pure Research (ZWO; Den Haag, The Netherlands), and an Alexander-von-Humboldt stipend, awarded by the Alexander-von-Humboldt Foundation (Bonn, F.R. Germany). The analysis of the data and the writing-up profited from support of the Centre National de la Recherche Scientifique (CNRS URA 1294, Paris, France). We gratefully acknowledge the generous hospitality of Profs. F. Vogel and W. Buselmaier (Heidelberg, F.R. Germany), who provided all materials necessary to carry out this study. Profs. Pierre L. Roubertoux and Michèle Carlier (Paris, France) critically read the manuscript.

We dedicate this article to Prof. Friedrich Vogel (Heidelberg, F.R. Germany), on the occasion of his 70th birthday.

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+Least-squares means ± SEM

++Effect size: d = M₂ - M₃ / SD.

*p<0.05; **p<0.01; ***p<0.001.

+Least-squares means ± S.E.M.

++Effect size: d = M₂ - M₃ / SD.

*p<0.05; **p<0.01; ***p<0.001.