Age at Puberty and First Litter Size in Early and Late Paired Rats 2

BIOLOGY OF REPRODUCTION 34, 322-3 26 (1986) Age at Puberty and First Litter Size in Early and Late Paired Rats 2 ALIDA M. EVANS Cancer Prevention Program 1300 University Avenue-7C University of Wisconsin Madison, Wisconsin 53706 ABSTRACT The rate of sexual maturation among female Sprague-Dawley rats was measured in a variety of intraspecific social environments. It was found that females of this strain differ from at least one other strain of laboratory rat in that neither age at vaginal perforation nor age at first estrus was affected in Sprague-Dawley females by the presence or absence of male, regardless of his age or breeding history. Sizes of first litter among females who mated at their first estrus were compared with those among females who were first inseminated at older ages. On average, females bred at first estrus produced litters that contained more than 3 fewer pups than females mated at older ages. This observation suggests that female Sprague-Dawley rats do not attain full reproductive competence until sometime after the onset of puberty. INTRODUCTION Age at puberty and first reproduction among females of several species of rodents is affected by the social environment in which the female matures. The presence of an adult male mouse, Mus musculus, results in acceleration of sexual maturation among females of that species, followed rapidly by first mating (Vandenbergh, 1967). Female voles of several species are exceedingly sensitive to the proximity of males with regard to the females apparent rate of sexual maturation, and indicators of puberty such as vaginal perforation and cornification (first estrus) are frequently not displayed in the absence of an inducing stimulus such as a male (Richmond and Stehn, 1976). Sexual maturation in laboratory rats (Rattus norvegicus) typically occurs between 30 and 50 days of age among singly housed nonstressed females when the criteria of puberty are vaginal opening and first estrus (Kennedy and Mitra, 1963; Vandenbergh, 1976; Ojeda and Jameson, 1977). Male rats typically attain sexual maturity between 30 and 40 days of age (Nalbandov, 1976). The rate of female sexual maturation Accepted September 3, 1985. Received May 10, 1985. This research was supported by NIH training grant #82 and National Institute of General Medical Sciences grant #7757. 2This paper is based on a portion of the author s dissertation submisted to Indiana University in 1981 in partial fulfillment of the requirements for the Ph.D. degree. among laboratory rats is less affected by social environment than in voles or house mice. Age at vaginal perforation does not seem to be affected by the presence or absence of a male in any strain of laboratory rats examined to date (Rutledge et al., 1974; Vandenbergh, 1976; Slob et al., 1985). However, age at first estrus has been found to be susceptible to influences of the social environment in at least one strain of lab rat; Vandenbergh (1976) found that females of the Holtzman strain respond to the presence of an adult male by displaying earlier first estrus than females housed in the absence ofamale. Rutledge et al. (1974) did not score for ages at first estrus in the unspecified strain of inbred rats utilized as subjects in their experiment. First ovulation or estrus in 2 substrains of Wistar rats was not accelerated by male stimuli (Slob et al., 1985). Therefore, it is unknown whether the effect noted by Vandenbergh (1976) occurs in strains of lab rat other than the Holtzman strain. In the current study, Sprague-Dawley females were tested to determine if either ages at vaginal opening or first estrus would be affected by the presence versus absence of a male during female sexual maturation. Previous investigations of the role of social environment in determining age at female sexual maturation have focused primarily on the influences engendered by adult males (Miss musculus, reviewed by Vandenbergh, 1973; Rattus norvegicus, Holtzman strain, Vandenbergh, 1976). In Mus, the major stmmulus in the acceleration of maturation effect seems to be an 322

PUBERTY AND FIRST LITTER SIZE IN RATS 323 androgen-dependent urinary pheromone normally produced only by adult males (Colby and Vandenbergh, 1974; Drickamer, 1974; Vandenbergh, 1976). Yet, the specificity of the social cues involved in early maturation of females housed with males may not be relegated to adult males in all species. For example, Hasler and Nalbandov (1974) found that Microtus ochrogaster females housed with unrelated immature males matured at approximately the same rate as females housed with adult males. Thus, if Sprague- Dawley female rats are found to mature earlier when housed with a male than without, it would be relevant to identify the relative impact of immature versus adult males in promoting this effect. Rapid postpubertal reproduction among female rats housed with an adult male is a frequent result of the pairing regime (Rutledge et al., 1974; Vandenbergh, 1976). Potential variations in litter sizes as a function of age at first reproduction may reflect differences in physiological maturation not observable by scoring of vaginal opening and first estrus. Rutledge et al. (1974) noted that the reproductive performances in terms of litter size (number of young born per litter) among early mated female rats are nearly comparable to those of late mated females. The general applicability of these results to other strains of rats was tested in the current study by examining the relative reproductive competence of Sprague-Dawley rats mated at various ages. In summary, the 3 objectives of the current study were 1) to determine if females of the Sprague- Dawley strain of laboratory rats are comparable to Holtzman in their response to the presence or absence of a male during maturation, 2) to test the possibility that a male s maturational status when paired with an immature female rat affects the extent of his impact on the female s age at maturation, and 3) to determine whether female rats mated at first estrus are as reproductively competent as females inseminated at older ages. MATERIALS AND METHODS The Sprague-Dawley female and juvenile male rats used in this study were third generation progeny of rats purchased from Laboratory Supply, Inc., Indianapolis, IN. Adult males were second generation progeny from the same breeding stock. All animals were kept in a room in which the light: dark cycle was 15L:9D and the ambient temperature ranged from 21#{176}Cto 25#{176}C.Housing for all animals consisted of plastic cages with wire mesh lids that served as water bottle holders and food hoppers. Animals were fed Purina rat chow (Ralston-Purina Co., St. Louis, MO) ad libitum. All experimental animals were from first litters of females that had been less than 100 days of age when bred. At 20 days of age, each experimental female was removed from her maternal home cage and placed with a nonsibling female in a clean plastic cage with pine shaving bedding; both females had the same birth date. Each 20-day-old female pair was assigned randomly to one of the following treatment groups: 1) early paired females with an immature male (EPF/ IM)-all animals were 20 days of age when placed in the cage together; 2) early paired females with a mature virgin male (EPF/VM)-the males had been housed singly until 60 days of age, at which time they were placed in a cage with two 20-day-old females; 3) early paired females with a known fertile male (EPF/FM)- a fertile male (age 110 to 140 days) was placed in the cage with a 20-day-old female pair; 4) late paired females with an immature male (LPF/IM)-a 20-day-old male was placed in a cage with two 60-day-old females; and 5) late paired females with a mature virgin male (LPF/VM)-a 60-day-old male, previously housed alone, was placed in a cage with two 60 day-oldfemales. Five female pairs were assigned to each treatment group. The late paired females (LPF/IM and LPF/VM groups) were expected to mature sexually prior to 60 days of age and, thus, prior to exposure to a male. As a result, they constituted an absence of male group during the phase of this study in which ages at sexual maturation were scored. Each of the EPF groups contained 10 females whereas the absence of male (LPF) contained 20 females. In order to maintain equal sample sizes with respect to the maturational data described below, 5 female pairs from the LPF groups were selected randomly for scoring of maturational events. Each female in the EPF groups and of the 5 randomly selected LPF pairs was checked daily for vaginal opening. Once a female s vagina was found to be open, a vaginal smear was taken by rinsing the vagina with about 0.2 ml of water. Vaginal smears were taken daily until the female attained first estrus as scored either on the basis of cornification of the vaginal epithelium, or the presence of spermatozoa or a seminal plug in the vagina. The 2 dependent measures in this phase of the study were age at vaginal opening and age at first estrus.

324 EVANS A female s age at first successful conception was determined either by observation of spermatozoa or a seminal plug in the vagina 21 to 23 days prior to parturition, or by subtracting 22 days from her age at parturition. All young in a litter were counted and the number of dead neonates was noted. The 3 dependent measures in this portion of the study were percentage of females per group producing litters, total number of young born per litter, and percentage of young born dead per litter. Nested analyses of variance (Sokal and Rohlf, 1969) were used to test differences between groups, with a critical probability level of 0.05. Planned comparisons were used to test differences between particular groups. RESULTS The presence of a male from weaning (20 days of age) through sexual maturation failed to have any significant impact on a female s age at puberty, regardless of his maturational status (Table 1). Neither mean age at vaginal opening (F(3,16)1.08) nor mean age at first estrus (F(3,16)=1.22) differed between groups, and most females displayed first estrus when they were first found to have open vaginas. When females were housed in the presence of an adult male during maturation (EPF/VM, EPF/FM groups), conception occurred at first estrus in all cases; females paired early with immature males (EPF/IM) or paired late with mature or immature males (LPF/VM, LPF/IM groups) conceived their first litters at ages of 59.0 ± 1.02 days, 74.5 ± 1.82 days, and 112.3 ± 1.78 days (mean ± SEM), respectively. The mean litter sizes of the groups of females were significantly different (F(4,20)=6.70, p<0.005, Figure 1). Planned comparisons indicate that the mean litter size of the early conceiving groups (EPF/VM and EPF/FM) were significantly smaller than those of the later conceiving groups (EPF/IM, LPF/VM, LPF/IM) (F( 1,20)=20. 17, p<0.001). The EPF/IM mean litter size was not significantly smaller than the litter sizes of the late paired females (F(1,20)=1.89), nor did the litter sizes of the LPF/IM and LPF/VM groups differ significantly (F(1,20)=0.96). One female in the LPF/IM group had a litter size of 9, all of which were born dead. Aside from this one stillborn litter, the greatest number of young born dead per litter in any group was 1. No significant differences were found in the mean percentage of young born alive per litter between groups (F(4,20)=0.47; range of mean percentage born alive: 90%-99.2% including stillborn LPF/IM litter; 97.3%-100% excluding stillborn LPF/IM litter). Every female in the experiment produced a litter. DISCUSSION The results with respect to age at maturation, whether measured as age at vaginal opening or first estrus, suggest that Sprague-Dawley rats differ from Holtzman rats in their ability to respond to the social environment. Since first estrus in Holtzman rats does not occur simultaneously with vaginal opening, the presence of a male seems to decrease the time interval between these two maturational events (Vandenbergh, 1976). Bowever, first estrus in about 85% of the Sprague-Dawley females in the present study was observed at the same time as each female s vagina was first found to be open. Ramaley and Bunn (1972) noted that Sprague-Dawley females housed in the absence of a male typically ovulated on the same day vaginal opening occurred, indicating that vaginal opening and first estrus are associated with functional maturity in these rats. The lack of latency between vaginal opening and first estrus or ovulation in Sprague- Dawley rats precludes the possibility that the presence of an adult male hastens first estrus, relative to vaginal opening. These results parallel those of Slob et al. (1985) among Wistar rats. In no case has age at vaginal opening in female rats been observed to be influenced by presence or absence of a male (current results; Rutledge et al., 1974; Vandenbergh, 1976; Slob etal., 1985). The major finding of the current study is that females bred at an early age produce about 3 to 4 fewer young per litter than females bred at an older age. This difference cannot be explained by the length of a female s exposure to a male prior to conception. Females housed with maturing males from female age of 20 (EPF/IM) or 60 days (LPF/IM) had successful TABLE 1. Mean ages in days at vaginal opening and first estrus of female Sprague-Dawley rats housed in the presence of males of various maturational statuses or in the absence of a male. Group n Vag inal Opening Age ± SEM Age First Estrus ± SEM EPF/lM 10 36.5 0.54 37.3 0.67 EPF/VM 10 35.3 0.50 35.7 0.62 EPF/FM 10 35.4 0.72 36.0 0.70 LPF 10 35.8 0.59 36.4 0.76

PUBERTY AND FIRST LITTER SIZE IN RATS 325 I.- -J 5-4 z o 13 0 >- II * NJ U, a: I- I- -J I0 2 EPF/VM EPF/FM EPF/lM LPF/IM LPF/VM FIG. 1. Mean litter size (number of young born per litter) and 95% confidence limits for female Sprague-Dawley rats paired at 20 (EPF) or 60 (LPF) days of age with males that were either adult virgins (VM), known fertile (FM), or juvenile (IM) at time of pairing. n=1o females in each group. conceptions after 3 0-65 days of cohabitation whereas LPF/VM females conceived within about 2 wk of pairing; even so, no differences were found in litter sizes between these groups. Any differences between early and late mated females in parameters of reproductive performance may be construed to reflect differences in reproductive competence, a measure of physiological maturation independent of vaginal opening and first estrus. The smaller litter sizes in the EPF/VM and EPF/FM groups as compared to the other three, later conceiving groups suggests that the reproductive competence of these early mating females is not equivalent to that of the later mating females. Puberty, as measured by vaginal opening and first estrus, reflects the onset of the ability to reproduce per se since all females inseminated at first estrus successfully conceived and carried their litters to term. Yet, full reproductive competence may require a longer period of time to develop. Eisen (1973) points out that fewer young per litter or a higher percentage of stillbirths among early mating females probably indicates that the uteri of the young females are less physiologically mature than those of females that conceive at older ages. It is also possible that ovulation rates may be lower in young females, thus indicating that ovarian output may not reach an asymptotic level until several weeks past puberty. Since the early conceiving females in the current study did not have a higher percentage of young stillborn, the deficit in reproductive competence among the early mated Sprague-Dawley females is more likely attributable to lower ovulation rates. Further investigations of postpubertal development of reproductive physiology in these rats seem to be warranted. Given that the EPF/IM rats conceived at approximately 60 days of age and their mean litter sizes were not significantly different from the females conceiving at considerably older ages, it is possible to infer that full physiological maturation has occurred by 60 days of age in the Sprague-Dawley females, and, from this age on, reproductive competence changes very little. The current litter size data contrast with the findings of Rutledge et al. (1974) in that the latter observed only about a 1 pup differential between litter sizes of early mated and late mated females. The ages at which female rats conceived in the Rutledge et al. study were essentially the same as those in the EPF/ VM, EPF/FM (early mated) and LPF/VM (late mated) groups in the current experiment; overall, their mean litter sizes were lower than those in the current study, with early mated females having an average of 8.2 young and the late mated females an average of 9.2 young per litter. Rutledge et al. (1974) suggest that early breeding procedures would satisfactorily shorten generation intervals without serious impact on the reproductive performance of the breeding females. The current results indicate that caution should be used in applying this suggestion as various strains of rats may differ in the severity of reproductive performance or competence deficits caused by early breeding practices. In summary, Sprague-Dawley females do not demonstrate variation in age at puberty as a function of the types of social environments examined in this study, and early breeding females have smaller litter sizes than females bred at 60 days of age or older. REFERENCES Colby DR, Vandenbergh JG, 1974. Regulatory effects of urinary pheromones on puberty in the mouse. Biol Reprod 11:268-79 Drickamer LC, 1974. Contact stimulation, androgenized females and accelerated maturation in female mice. Behav Biol 12:101-11 Eisen, EJ, 1973. Genetic and phenotypic factors influencing sexual maturation of female mice. J Anim Sci 37:1104-11 Hasler MJ, Nalbandov AV, 1974. The effect of weanling and adult males on sexual maturation in female voles (Microcus ocbrogaster). Gen Comp Endocrinol 23:237-38

326 EVANS Kennedy GC, Mitra J, 1963. Body weight and food intake as initiating factors for puberty in the rat. J Physiol 166:408-18 Lombardi JR, Vandenbergh JG, Whitsett JM, 1976. Androgen control of the sexual maturation pheromone in house mouse urine. Biol Reprod 15:179-86 Nalbandov AV, 1976. Reproductive Physiology of Mammals and Birds. San Francisco: W. H. Freeman Ojeda SR, Jameson HE, 1977. Developmental patterns of plasma growth hormone in the female rat. Endocrinology 100:881-89 Ramaley JA, Bunn EL, 1972. Seasonal variations in the onset of puberty in rats. Endocrinology 91:611-13 Richmond M, Stehn R, 1976. Olfaction and reproductive behavior in mictrotine rodents. In: Doty R. L. (ed.), Mammalian Olfaction, Reproductive Processes and Behavior. New York: Academic Press, pp. 197-217 Rutledge JJ, Kalscheur JA, Chapman AB, 1974. Effect of age at mating on the prenatal and postnatal performance of the female rat. J Anim Sci 39:846-48 Slob AK, van Es G, van der Werff ten Bousch JJ, 1985. Social Factors and Puberty in Female Rats. J Endocrinol 104:309-13 Sokal RR, Rohlf FJ, 1969. Biometrics. San Francisco: W. H. Freeman Vandenbergh JG, 1967. Effect of the presence of a male on the sexual maturation of female mice. Endocrinology 81:345-49 Vandenbergh JG, 1973. Acceleration and inhibition of puberty in female mice by pheromones. J Reprod Fertil 19:411-19 Vandenbergh JG, 1976. Acceleration of sexual maturation in female rats by male stimulation. J Reprod Fertil 46:451-53 Vandenbergh JG, Whitsett JM, Lombardi JR, 1975. Partial isolation of a pheromone accelerating puberty in female mice. J Reprod Fertil 43 :515-23