Reproductive patterns of female house shrew (Suncus murinus) inhabiting central Taiwan were studied from September 1990 to February 1992 with animals collected monthly. The annual average body weight was 37.4 g for adults (N=198), and 23.2 g for juveniles (N=18), respectively. The reproductive females (pregnant and lactating) were found all the year round with higher percentages (more than 80%) in spring-summer. The average litter size was 2.9 (range: 1–6). The levels of plasma estradiol-17 β in adult females were higher in March-June (140 pg/ml), and decreased gradually to 98 pg/ml in January-February. Plasma levels of estradiol-17 β in pregnant and lactating females were significantly greater than those of non-pregnant adults and of juveniles. Ovarian weights, plasma levels of estradiol-17 β, and pregnancy rate were correlated with day length. The pre-implantation mortality and post-implantation mortality of the embryos were 6.9% and 1.6%, respectively. The present study has demonstrated that the female S. murinus inhabiting central Taiwan breeds all year round with higher reproductive activity in spring and summer.
INTRODUCTION
The house shrew or musk shrew (Suncus murinus) is a primitive eutherian mammal of the order Insectivora, which inhabits tropical and subtropical regions throughout Asia (Churchfield 1990; Ruedi et al., 1996). This species breeds opportunistically, and pregnant females and fertile males can be found all the year in most of the regions studied (Louch et al., 1966; Hasler et al., 1977; Brooks et al., 1980; Yang and Zhuge, 1989).
In Taiwan, S. murinus is commonly found in gardens, villages, and backyards of urban houses, but seldom found in the wild environs. The altitude of their habitation is seldom over 500 meters (Chang, 1992). Previous studies on S. murinus inhabiting Taiwan were concerned about serological survey, cytogenetical study and a preliminary investigation with respect to the shrews' reproductive ecology (McNeill et al., 1968; Yosida, 1982; Lin and Chang, 1996). However, no work has been attempted to investigate the seasonal reproductive patterns of S. murinus, and their relations to environmental factors.
We have investigated the annual reproductive patterns of S. murinus of males and females inhabiting central Taiwan. In previous paper, we reported the male reproductive patterns (Chang et al., 1999). The present paper reports on monthly patterns of reproduction in female S. murinus with respect to: 1) changes in weights of body and ovary, 2) changes in reproductive status (pregnancy, lactation, and non-pregnancy), 3) levels of plasma estradiol-17 β and their relations to reproductive status, 4) litter size and embryonic mortality, and 5) correlations among changes of various reproductive parameters and environmental factors.
MATERIALS AND METHODS
Trapping of animals, handling techniques and histological method
This study was carried out in the dormitory area of Tunghai University, located on the central Taiwan (24°10.68′N, 120°35.90′E; 184 m in altitude). From September 1990 to February 1992, house shrews were captured in the middle of each month with metal live-traps baited with sausages by setting different traplines with 100 traps (about 4 hectares) for 4 consecutive days. The traps on the study sites were examined every morning (0800–0830 h). The captured animals were immediately transported to the laboratory for examination. Animals were sacrificed within 2 hr after they were collected from the study sites. They were anesthetized with ether, and body weights and lengths (body and tail) were measured before they were bled by puncture of the vena cava with a heparinized syringe. The blood was centrifuged at 103×g for 10 min at 5°C, and plasma was collected and then stored ar −20°C until the assay of estradiol-17 β. Ovaries were checked for the Graafian follicles and the number of corpora lutea by the routine histological methods (Ann, 1978). The teats in females were also examined for any sign of lactation. The conditions of the uterus and the number, size and weight of embryos were recorded.
Classifications of reproductive status
The trapped females were assigned a reproductive status as follows: 1) Pregnant females had visible embryos in their uteri; 2) Lactating females were those in which milk could be expressed from one or more teats by hand pressing. During lactation the nipples became enlarged, bare, dark, and roughened; 3) Non-pregnant females were those having no visible embryos, and their nipples were minute and light-colored, but their ovaries were developed with Graafian follicles; 4) Juveniles whose ovaries were not much developed.
Radioimmunoassay of estradiol-17 β
Blood plasma samples were extracted with diethyl ether and allowed to freeze in a dry ice-ethanol medium. The ether was decanted into another tube and dried in a water bath at 38°C under continuous flow of nitrogen gas in a ventilator. The dried residue was dissolved in 0.01 M phosphate buffer saline (pH 7.40) containing 0.1% gelatin (PBSG) and incubated at room temperature (25°C) for one hour. Aliquots of the PBSG-dissolved ether-extracted plasma sample were incubated with 3H-estradiol-17 β [2,4,6,7,16,17-3H (N), New England Nuclear, Boston, MA, USA] and antiserum of estradiol-17 β; the antibody bound estradiol-17 β was separated with dextran-coated charcol and then counted in a liquid scintillation counter. The antiserum was produced in rabbits by immunization with estradiol-6-CMO:BSA (Steraloids Co.), and was highly specific for estradiol-17 β (Yu et al., 1988). The coefficients of variation (CV) of intra- and inter-assays were 3.0% and 12.1% respectively (N=5).
Climatic data and statistical analysis
Data on rainfall, temperature, photoperiod and relative humidity were obtained from the Taichung Weather Station, and are summarized in Fig. 1.
Fig. 1
Monthly changes in climatic factors (temperature, day length, rainfall, and relative humidity) in Taichung, Taiwan. (1990–1992)
Analysis of variance (ANOVA) was used to examine differences among the numerous variances. The correlations between reproductive parameters and climatic factors were examined by the methods of Pearson's correlation coefficients. Duncan's multiple range test was used to analyze the differences among reproductive status. Probability levels of 0.05, 0.01, and 0.001 were taken to indicate significance in comparison of means and correlations. Mean values were given with standard errors.
RESULTS
Seasonal changes in body weight and body length
A total of 216 female shrews were examined with 198 adults and 18 juveniles (Table 1). The mean body weights of adults was 37.4±6.1g (SEM), and changed monthly with the lowest in May (31.7±7.0 g) and the highest in April (40.8±6.4 g), but there was no significant monthly difference (F17,180 =1.52, P>0.05). The lowest and highest body weights in collected 198 adult females were 22.3 g found in February 1991 and 59.2 g found in November 1991, respectively. As for juveniles, the mean body weight was 23.2±2.3 g (SEM), with the highest being 27.9 g found in February 1991, and the lowest 20.1 g in May 1991.
Table 1
Mean monthly body weight and body length in adult and juvenile female Suncus murinus inhabiting central Taiwan
The mean monthly body length was 205.4±10.2 mm with a range from 165 mm to 225 mm (Table 1). The average body length of the juveniles was 179.7±9.3 with a range from 163 mm to 196 mm.
Seasonal changes in reproductive status and characters
Both pregnant and lactating females were found throughout the year and the prevalence ranged from 20% to 100%. There were higher proportions (over 70%) of both pregnant and lactating females in the population from March to September (Fig. 2). Thus, the intensity of reproduction were higher in spring-summer and lower in autumn-winter.
Fig. 2
Seasonal changes of the percentages of reproductive status of adult females of Suncus murinus. Sample sizes are shown at the top of respective columns.
The average plasma estradiol-17 β concentration in adult females was 109.8±18.9 pg/ml, and there was no significantly monthly change (F8,145=0.86, P>0.05). There appeared to be an elevation in plasma levels of estradiol-17 β in March-May (140 pg/ml) in contrast to other periods (Fig. 3).
Fig. 3
Monthly changes in plasma estradiol-17 β levels of adult female Suncus murinus. The data are expressed as mean±SEM. Arabic numerals above the bars are the numbers of animals.
Comparisons and differences in various reproductive parameters among different reproductive stages are indicated in Table 2. There were significant differences in body weights, body lengths, ovary weights and plasma estradiol-17 β levels between adult and juveniles. There were also differences between non-pregnant and pregnant adults in body weight, ovary weight and plasma estradiol-17 β levels. There were no differences between pregnant and lactating females in body weight, body length, ovary weight, and plasma estradiol-17 β levels.
Table 2
Reproductive parameters among different reproductive status of female Suncus murinus (Mean±SEM). Different letters indicate a significant difference (p<0.05)
Embryo numbers were obtained by counting of the number of visible uterine swelling. The number of embryos per female ranged from 1 to 6, with an average of 2.94±1.00 (N=107)(Table 3). Out of a total of 315 embryos, only 5 embryos were in the processes of re-absorption, i.e., the postimplantation mortality was 1.6%.
Table 3
Monthly distribution of litter sizes in Suncus murinus inhabiting central Taiwan
The pre-implantation mortality was assessed by comparing the differences between the numbers of corpus lutea through histological sections of ovaries (Fig. 4) and the implanted embryos. In 78 pregnant females examined, there were 288 corpora lutea in ovaries, but only 268 embryos present in uterus; the pre-implantation mortality was thus 6.9%.
Fig. 4
Histological photographs showing different ovarian follicles and/or corpora lutea at various reproductive status of Suncus murinus. Panel A: A juvenile with immature follicles (F) in ovary. Panel B: A mature but non-pregnant female Suncus murinus with a Graafian follicle (G) in ovary. Panel C: A pregnant female Suncus murinus with corpus luteum (C) in ovary. Panel D: A lactating female with Graafian follicles and regressed corpus luteum in ovary. Scale = 0.1 mm.
Climatic factors and reproductive patterns
The correlations between reproductive parameters of adult female S. murinus and the climatic factors (mean temperature, rainfall, relative humidity, and day length) were compared. As shown in Table 4, ovary weight, pregnancy rate, and levels of plasma estradiol-17 β were all correlated with temperature or day length. In addition, the pregnancy rate was also correlated with rainfall. Although their correlations were statistically significant at p <0.01 or p <0.05, their correlation coefficients were not high except those between pregnancy rate and length of day or temperature.
Table 4
Pearson's correlation coefficients between reproductive characters of adult Suncus females and the climatic factors (* p<0.05; ** p<0.01)
DISCUSSION
The annual breeding patterns of S. murinus have been investigated in various tropical and subtropical regions. Except in certain desert areas (Rajasthan, India and Punjab, Pakistan), the pregnant female Suncus were found to be present all, or nearly all year round (Table 5). In the present study, we observed that the pregnant female Suncus were present every month, with higher prevalency in March-September and lower prevalency in November-February, exhibiting large difference between seasons. The seasonal variation of reproductive activity of the female Suncus were essentially synchronized with those of the male Suncus that exhibiting maximal spermatogenic activity in March-September (Chang et al., 1999). As indicated in Table 6, the female Suncus in Rangoon had more stable prevalency of pregnancy on seasonal basis; while those in Calcutta, Guam, Malaya, Chekiang and Taiwan showed more variable prevalency of pregnancy. It is interesting to note that female Suncus in Chekiang, China exhibited a bimodal reproductive activity occurring in March-April and July-August (Yang and Zhuge, 1989). The observations of previous investigators from different laboratories revealed that female Suncus is either a continuous or non-continuous breeder depending on the localities of habitation (Table 5). For those Suncus being continuous breeders, however, they all exhibited, more or less, seasonal variation in reproductive activity. Thus, the variability of prevalency of pregnancy and other reproductive status of S. murinus among various localities studied are correlated to and reflected by, different climatic factors and other ecological conditions.
Table 5
Reproductive characters of Suncus murinus at 9 localities
Table 6
Comparison of prevalence by seasons in Suncus murinus of different localities
The litter size in S. murinus shows a vast geographical variation (Table 5). The two largest litter sizes are from India and Pakistan with 4.7 and 4.2, receptively, from Rajasthan (Rana and Prakash, 1979) and Punjab (Khokhar, 1990). Rana and Prakash (1979) suggested that it is a compensated way for the shorter breeding season of shrews in the desert environment to maintain a higher turnover rate of the population in the yearly cycle. At other localities with continuous breeding all the year round, the litter size appears to be a function of body weight in female shrews. The Calcutta shrews were heaviest in body weight (67.7 g) and possessed the relatively large litter size (3.8)(Louch et al., 1966); while the Guam shrews were the lightest (21 g) and had smallest litter size of 2.1 (Hasler et al., 1977). The litter size of shrews in central Taiwan as observed in this study was 2.9, which is similar to those in Rangoon (Brook et al., 1980), Okinawa (Oda and Shigehara, 1978) and Chekiang (Yang and Zhuge, 1989). It can be inferred that the shrews inhabit in central Taiwan exhibit higher reproductive efficiency with a lower pre-natal mortality (both pre- and post-implantation mortality) due likely to a more stable ecological environment.
Studies indicated that musk shrews are induced ovulators, but exhibit no cyclic changes in reproductive structure or in sexual behavior (Dryden, 1969; Fortune et al., 1992). Sexual maturity in female Suncus is generally reached by 30 days of age and mating behavior is induced by contact with a male, and its gestation period lasts about 30 days (Dryden, 1969; Fortune et al., 1992). Dryden and Anderson (1977) reported that uterine and vaginal weights and histologies were not altered by ovariectomy or estrogen treatment in female shrews. Hasler and Nalbandov (1978) also revealed that the levels of plasma estradiol-17 β were not detectable in adult female shrews. Thus, the role of ovarian estradiol in the control of reproductive status of this species was once considered not conformable to the accepted mammalian pattern. However, Rissman and Bronson (1987) demonstrated that ovariectomy eliminated sexual behavior, and treatment with estradiol restored it in almost all shrews. Such observations indicated that estradiol plays a role in regulation of sexual behavior in house shrews. Fortune et al. (1992) further demonstrated that matings stimulated ovulation in musk shrews also triggered specific changes in ovarian steroidogenesis; increased estradiol-17 β production by the ovary and elevated level of estradiol-17 β in the blood during periovulatory period suggest a role of estradiol during the final stage of follicle development and/or ovulation. They also demonstrated that ovarian secretion of estradiol was increased during the early luteal phase, thus suggesting a role of estradiol in luteal function in shrews. The present study also investigated the levels of plasma estradiol-17 β in female shrews during an annual reproductive cycle. We have demonstrated that the ovarian weights of both pregnant and lactating female Suncus were larger than those of non-pregnant adults and juveniles (Table 2), and that the levels of plasma estradiol-17 β were correlated to different reproductive status of the Suncus with higher levels of plasma estradiol-17 β present in both pregnant and lactating females in comparison to the non-pregnant and juvenile female (Table 2). In addition, the present study revealed that the levels of plasma estradiol-17 β tended to be elevated during March-September when the percentage of pregnancy and lactation of Suncus being greatest. Our findings thus support the proposal that ovarian estradiol-17 β plays a role at various stages of reproduction of female Suncus. Furthermore, we found that the elevated levels of plasma estradiol-17 β of female Suncus in spring-summer are very much synchronized with the elevated levels of plasma androgen of the male in the same period (Chang et al., 1999).
Correlations between environmental cues and reproduction of female Suncus inhabiting in different localities have been investigated by various laboratories (Table 5). In general, the peak breeding activity of female Suncus occurs in spring-summer when the day lengths are longer. It was shown that the female Suncus housed in short day lengths had significantly lighter uterine cervices, and were less likely to demonstrate sex behavior than animals kept in under long day lengths, although ovarian and uterine weights did not differ (Rissman et al., 1987; Wayne and Rissman, 1990). In the present study, we have observed that ovarian weight, pregnancy rate, and levels of plasma estradiol-17 β of the female Suncus were all correlated with day length and temperature, although their correlation coefficients were not consistently high (Table 4). The weights of testis, epididymis, seminal vesicle-prostate, and plasma levels of androgen of the male Suncus, investigated simultaneously, were all correlated to the day lengths and temperature (Chang et al., 1999). Consequently, the long photoperiod and higher temperature are likely stimulatory to reproductive activity in house shrews. The effects of rainfall and relative humidity on reproductive activity of house shrews are variable and complex. Factors such as amount and length of rainfall, different latitude and elevation, desert or swamp area, and the length of rainy season may have different influences on reproductive activity. Rainfall was correlated with the reproductive peak of S. murinus (Rana and Prakash, 1979). We have observed that rainfall, but not relative humidity, was correlated with pregnancy rate of female Suncus. Our present study thus demonstrated that day length and temperature are likely the most important factors in relation to reproductive activity of S. murinus inhabiting central Taiwan.
Acknowledgments
We thank Mr. Y. H Kao, Mr. C. Y. Lin, and Ms. M. R. Yang for assistance in laboratory work. Thanks are also due to Ms L. G. Chen, Mr. C. P. Lin and Mr. W. Y. Ker for their helpful work in field.
