Reproduction in a laboratory colony of the pouched mouse, Saccostomus campestris L. M. Westlin-van Aarde Mammal 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 species. 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 animals, 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.