Reproduction in a laboratory colony of the pouched mouse, campestris L. M. Westlin-van Aarde Research Institute, University of Pretoria, Pretoria 0002, South Africa

Summary. Pouched mice ovulate spontaneously and have a 4-day cycle (3\p=n-\5days). The variation was caused by prolonged oestrus. The vagina opened at about 34 days of age and the first oestrus was experienced at 44 days of age. Females experienced several sterile cycles before their first conception, which occurred at an age of about 56 days. The gestation period in most cases was 21 days and implantation occurred about 4\p=n-\5 days after mating. The females were not receptive post partum. Litter size varied from 3 to 13 with a mean of 7\m=.\1in young primiparous females and 7\m=.\9in adult multiparous females, indicating that fecundity did not increase with age or parity in this . Mortality of young was highest during the first 2 days post partum. The young were not attached to the nipples and were weaned at 25 days of age. Females did not cycle during lactation. After lactation most females exhibited one or two oestrous cycles without mating or became oestrous and mated without conceiving, resulting in a litter interval of about 53 days. Keywords: pouched mice; reproduction; puberty; pregnancy

Introduction

The pouched mouse, Saccostomus campestris, is included in the subfamily Cricetomyinae (Cricetidae; Smithers, 1983). The subfamily comprises only 3 species, all of them occurring exclusively in Africa. The pouched mice are only found south of the Sahara, where they are widely distributed (Smithers, 1983). They are nocturnal and terrestrial and will excavate their own burrows in sandy soils, but in hard ground use holes established by other species. The pouches are used for transporting food to the burrows (Smithers, 1983). Little is known about reproduction in the pouched mouse, most of the information being on seasonality and litter sizes originating from field work with small sample sizes (Hanney, 1965; Smithers, 1971, 1983; Swanepoel, 1976; Smithers & Wilson, 1979; see also Delany, 1975, for references to studies in which ^ 2). Limited data on reproduction have been published on laboratory , mostly under non-constant laboratory conditions and/or no information on material and methods or results and without giving sample sizes (Davis, 1963; Wrangham, 1969; Pitchford & Visser, 1970; Earl, 1978, 1980). The present study describes different aspects of reproduction of female pouched mice in the laboratory under assumed optimal breeding conditions in a constant environment.

Materials and Methods

A study colony of pouched mice (chromosome number 2n = 46) was established using animals from a mother colony, which originated from free-ranging animals caught in the Nelspruit district in 1958. The animals were kept in monogamous pairs when possible; females were often very aggressive towards the males after completed mating or just after parturition, and killing of males occasionally occurred. When a female was aggressive, the male was removed. Housing consisted of standard plastic laboratory mouse cages (Labotec, Johannesburg, South Africa) measuring 40 cm 24 cm 12 cm, with wire-mesh covers. Wood shavings were used

Downloaded from Bioscientifica.com at 09/29/2021 12:21:52PM via free access Table 1. Mean length of oestrous cycles in young, nulliparous and adult multiparous females, kept singly for 3 weeks or singly for 3 weeks and then caged with a male separated by a wire mesh

Length of oestrous cycle (days) No. of No. of Group females -Mean s.d. cycles Young, single $ 19 4-1 0-75 71 Young $ + adult ¿ separated 5 3-9 0-56 29 Adult, single $ 8 41 1-35 38 Adult $ + adult S separated 8 4-1 1-35 35

for bedding and sand was provided in a corner for sandbathing. A commercial rat food (Rat pellets, Epol (Pty) Ltd, Pretoria, South Africa) and water were given ad libitum. Cages were cleaned twice a week and supplementary food such as carrots, apples, sunflower seeds and rabbit pellets (Rabbit pellets, Epol (Pty) Ltd) were then provided. The animals were kept in a photoperiod of 14 h light and 10 h dark (lights on at 07:00 h) and at a temperature of 22 + 2°C. Relative humidity was maintained at 52%. Vaginal smears (by using a spatula) were taken daily in the early morning and late afternoon and a copulatory plug or spermatozoa in the smear was taken as evidence for mating. Cages with pregnant females were checked daily for litters. Checking included vaginal smears and weighing of females, to rule out the possibility of litters being born and eaten without discovery. The day of discovery of young was designated Day 0 of age and of the succeeding interbirth interval. Records were made of the number of young born and the litter weight at birth. The number of surviving young from Day 0 to Day 25 was recorded. The number of young found on Day 0 was used as litter size at birth, since it appeared that no young were completely eaten before discovery. Young were sexed, weighed and individually marked by toe clipping on Day 10 and weaned and weighed on Day 25. Young pouched mice were kept in groups of 3-5 animals of the same sex until used for experiments. Young females (from the day of weaning) were checked daily for vaginal opening and first oestrus. Fecundity was recorded in young primiparous females (first conception) as well as in multiparous females (1-1-5 years old). To study ovulation and ovarian cyclicity adult multiparous females (> 1-5 years old) as well as young nulliparous females (40 days of age) were used. The females were kept singly for 3 weeks or singly for 3 weeks followed by another 3 weeks being paired with a male but separated from him by wire mesh, allowing contact but not mating. Vaginal smears were taken twice daily. After the experimental conditions, the females were killed 2 days after the last oestrus, and the ovaries were examined for corpora lutea. Time of implantation was studied in 15 females; the animals were killed 3-5 (N = 5), 4-5 (N = 5) and 5-5 (N = 5) days after mating. From the day of parturition females which had been kept singly during pregnancy were daily paired with a male to study post-partum receptivity. Vaginal smears were taken daily from these females as well as from those which at parturition were still with a male. An oestrous cycle was defined as the time from when the vaginal smear of a female contained only cornified cells and mucus ( = oestrus) up to the next time such smear occurred. denotes number of animals and number of observations. All males used for mating were of proven fertility.

Results Ovulation and oestrous cycle Ovulation occurred spontaneously, whether the females were young nulliparous or adult multi¬ parous, kept singly or with a male separated by wire mesh. In all females the ovaries contained several corpora lutea at dissection 2 days after the last oestrus. The length of the oestrous cycle was 4 days with some inter- as well as intra-individual variation (mostly between 3 and 5 days) (Table 1). The variation in the length of the cycle was caused by variation in the length of oestrus (1-3 days, mean = 1-3, s.d. = 0-48; = 193). There was no differ¬ ence in the length of the oestrous cycle between single young nulliparous females (mean = 41, = s.d. 0-75; = 71) and single adult multiparous females (mean = 41, s.d. = 1-35; = 38). Females first caged singly and then caged together with a male but separated from him by wire

Downloaded from Bioscientifica.com at 09/29/2021 12:21:52PM via free access Table 2. Vaginal smear patterns during an oestrous cycle, pregnancy and lactation in pouched mice

Reproductive status Vaginal smear

Oestrus (1-3 days) Cornified cells, mucus. The epithelium is shed at the end of the period Metoestrus (0-5-1 day) Round nucleated cells, few or no leucocytes Dioestrus (1-1-5 days) Cells scarce. Leucocytes, some epithelial cells without nucleus and some cornified cells Pro-oestrus (1 day) Mucus, cornified cells and nucleated epithelial cells; very few or no leucocytes Pregnancy 3-5 days after Leucocytes, mucus, rounded cells without nucleus, soon declining in numbers mating Pregnancy > 5 days after Mucus, leucocytes mating Pregnancy about Days 13-16 Mucus, erythrocytes, leucocytes Lactation 0-4 days after Mucus, erythrocytes, leucocytes parturition > 4— > 28 days after Mucus, leucocytes parturition mesh did not change their cycle length, and there was no difference in this respect between adult multiparous females (mean = 41, s.d. = 1-35;« = 35) and young nulliparous females (mean = 3-9, s.d. = 0-56; = 29). The vaginal smears of an oestrous cycle are described in Table 2. On the day of oestrus (or on the last day of a prolonged oestrus) the females formed a 'vaginal plug'; the whole vaginal epithelium was shed with masses of cornified cells inside the 'plug'. When a smear was taken the plug often easily came out, and behind it the smear consisted of rounded nucleated cells, also in large numbers. Females shed the epithelium whether they had mated or not. Males formed a copulatory plug at mating which filled the vagina. Such plugs were only found in the mornings. Sometimes no plug but only spermatozoa or fragments thereof were found in the vaginal smear. After conception the vaginal smear changed into a very mucous smear containing only leucocytes. It stayed like that also after parturition until the time when the female started cycling again, which was after the weaning of the young.

Pregnancy Mean gestation length was 21 -2 days (s.d. = 0-45; = 36). The exact time of implantation was not established in the present study. No sign of implantation was found 3-5 days after mating (N = 5). Implantations were apparent 5-5 days after mating (N = 5) and at that time the tropho- blasts were about 3 mm long (n = 35). At 4-5 days after mating 4 out of 5 females showed clear implantations. Sample sizes are small, but suggest that implantation occurred 4-5 days after mating.

Lactation and weaning Young were not attached to the nipples. They started eating solid food at a mean age of 17 days (s.d. = 1-22; = 71) and were fully weaned at 25 days of age (N = 181). Females did not experi¬ ence a post-partum oestrus and they did not cycle during lactation, as shown by vaginal smears. The time from one parturition to the next one varied from 46 to 74 days (mean = 51-8, s.d. = 906; = 14). One female gave birth 33 days after the previous parturition. The litter was lost 2 days after delivery and oestrus occurred 7 and 11 days later, with mating and conception at the latter time. The first oestrus after parturition occurred about 28 days post partum (mean = 28, s.d. = 1-4; = 12), a few days after weaning. Five females mated and became pregnant on this occasion, 4

Downloaded from Bioscientifica.com at 09/29/2021 12:21:52PM via free access females mated without conceiving and 3 females cycled without mating (i.e. 1-3 sterile cycles/ female; mean = 1-6, s.d. = 0-79; = 7).

Litter size and weight at birth

Mean litter size of young primiparous females was 7-1 (s.d. = 3-99; = 18, range 3-13) and of adult multiparous females 7-9 (s.d. = 2-34; = 27, range 3-11). The number of nipples per female varied between 9 and 12, 10 being the most usual. Five females had 9, 11 or 12 nipples. Six preg¬ nant females had 1-2 more fetuses than nipples. The mean weight at birth was 2-4 g (s.d. = 0-47; = 173). Sex ratio at 10 days of age was 1 male: 1-24 females which did not differ significantly from the expected 1:1 ratio ( 2 = 1-84; = 157).

Mortality ofyoung Preimplantation loss (fertilization failure, loss of embryos), as indicated by a larger number of corpora lutea than embryos, was 7-91% (11 out of 139 embryos; 15 females). Post-implantation loss (résorptions) was only suspected in one female, which had 11 corpora lutea (6 and 5 respect¬ ively in each ovary) but only 3 embryos, all implanted in one uterine horn. However, the empty horn showed no clear signs of résorptions. Overall, prenatal loss was 13-38% (19 out of 142 embryos; 16 females). Total post-natal loss of young up to an age of 25 days (after which age no young died) was 35 out of 199 (17-6%). Mortality was highest during the first 2 days after delivery when 60% of the total loss occurred (21 out of 35).

Sexual maturity

The vagina became perforate at an average age of 34-6 days (s.d. = 2-27; = 62) and first oestrus was experienced at an age of 44-6 days (s.d. = 317; = 19). The females experienced a number of sterile cycles (mean = 1-3, s.d. = 1-42, range 1-5; = 20) before their first mating and age at first conception varied between 44 and 71 days (mean = 56-4, s.d. = 812; = 21). Only one female in the present study did not conceive at first mating.

Discussion

Free-ranging female pouched mice are seasonal breeders (see Smithers, 1983, for references), with a peak in births during the warm wet summer months. Earlier attempts to breed this species in captivity have varied in success (Davis, 1963; Pitchford & Visser, 1970; Earl, 1980). The animals in the present study were kept under apparent optimal conditions for breeding, and although the study was performed during the austral autumn and winter, no decline in reproductive success was noted. When the colony was established in March, all females bred immediately, and during the first 3 months almost 200 young were born. The oestrous cycle length was not affected by the presence of a male in adult multiparous or young nulliparous females, as it is in mice (Whitten, 1957, 1958; Aron, 1979; Sánchez-Criado, 1982). Vaginal smears (see Table 2) confirmed that pouched mice are spontaneous ovulators. This was borne out by the presence of corpora lutea in the ovaries of females kept singly. To our knowledge shedding of the outer part of the vaginal epithelium, like a sac filled with cornified cells has not been described earlier. It occurred in cycling as well as in newly mated females on the day of oestrus. The occurrence of a copulatory plug in the vagina or spermatozoa in the vaginal smear exclusively in the morning indicates that mating occurred only during the dark period.

Downloaded from Bioscientifica.com at 09/29/2021 12:21:52PM via free access The differences between vaginal smears of conceiving and non-conceiving females 3 days after mating (see Table 2) were consistent in all females. This suggests that vaginal smears for 3 consecu¬ tive days are sufficient to estimate whether a female has conceived when at inspection no copulatory plug, or only fragmented spermatozoa, is found in the smear on the day of oestrus. Suckling in the pouched mouse has been reported to occur up to an age of about 55 days in some cases (Earl, 1978). In the present study the young were all weaned at an age of 25 days (n = 181). Shortly after weaning the females started cycling again. The reason for some females (7 out of 12) experiencing sterile cycles before the next conception, and 4 of these to mate without conceiv¬ ing, is not clear, but together with gestation and lactation periods it results in a litter interval of about 53 days. Mean litter size of young primiparous and adult multiparous females did not differ from each other, indicating that fecundity did not increase with age or parity in the present study. In several microtine (also cricetids) litter size increases with parity (Cowan & Arsenault, 1954; Frank, 1956; Kalela, 1957; Negus & Pinter, 1965; Richmond & Conaway, 1969; Hasler & Banks, 1975; Gustafsson et al, 1980). The mean litter size of all females in the present study (8-2, s.d. = 2-37; = 45) was slightly larger than reported earlier for this species (in Malawi, mean = 51, = 10: Hanney, 1965; in Botswana, mean = 7-4, = 8: Smithers, 1971; in Zimbabwe, mean = 6-7, = 7: Smithers & Wilson, 1979). The difference may be due to small sample sizes in the earlier studies or to litter size increasing with the number of generations in captivity. The latter has been shown to occur in bank voles (Gustafsson et al, 1980). In field studies a perforate vagina is often considered as evidence of sexual maturity. In the present study vaginal opening occurred at the age of about 34 days. First oestrus was experienced about 10 days later and first conception after a further 12 days, at an average age of 56-4 days. This gives an average age at the first parturition of 76 days, which is 20 days younger than has been reported earlier for pouched mice (Earl, 1978).

This study was financially supported by the University of Pretoria and the Council for Scientific and Industrial Research. Stock animals for the colony were donated by the South African Medical Research Institute, Nelspruit.

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