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Delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx
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ZOOLOGY Zoology 108 (2005) 131–140 www.elsevier.de/zool
Delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx Karukayil J. Meenakumari, Amitabh Krishna Department of Zoology, Banaras Hindu University, Varanasi 221 005, India Received 14 August 2004; received in revised form 28 January 2005; accepted 10 February 2005
Abstract The unusual feature of the breeding cycle of Cynopterus sphinx at Varanasi is the significant variation in gestation length of the two successive pregnancies of the year. The aim of this study was to investigate whether the prolongation of the first pregnancy in C. sphinx is due to delayed embryonic development. The first (winter) pregnancy commences in late October and lasts until late March and has a gestation period of about 150 days. The second (summer) pregnancy commences in April and lasts until the end of July or early August with a gestation period of about 125 days. Changes in the size and weight of uterine cornua during the two successive pregnancies suggest retarded embryonic growth during November and December. Histological analysis during the period of retarded embryonic development in November and December showed a slow gastrulation process. The process of amniogenesis was particularly slow. When the embryos attained the early primitive streak stage, their developmental rate suddenly increased considerably. During the summer pregnancy, on the other hand, the process of gastrulation was much faster and proceeded quickly. A comparison of the pattern of embryonic development for 4 consecutive years consistently showed retarded or delayed embryonic development during November and December. The time of parturition and post-partum oestrus showed only a limited variation from 1 year to another. This suggests that delayed embryonic development in C. sphinx may function to synchronize parturition among females. The period of delayed embryonic development in this species clearly coincides with the period of fat deposition. The significance of this correlation warrants further investigation. r 2005 Elsevier GmbH. All rights reserved. Keywords: Cynopterus sphinx; Short-nosed fruit bat; Embryonic development; Pteropodidae; Bats
Introduction The order Chiroptera includes 916 species, about one quarter of all known mammalian species and ranked second only to rodents (Koopman, 1993). Two suborders of bats have long been recognized: MegachirCorresponding author.
E-mail address: [email protected] (A. Krishna). 0944-2006/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2005.02.002
optera (Megabats, old world fruit bats) and Microchiroptera (Microbats, echolocating bats). The suborder Megachiroptera includes a single family, Pteropodidae with 42 genera and 166 species (Koopman, 1993). The members of the family Pteropodidae are largely distributed throughout the tropics and subtropics of the old world from Africa to South-East Asia, Australia, Samoa and the Carolines (Koopman, 1993). Despite this widespread distribution, patterns of
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reproductive timing and embryonic development of old world fruit bats (Megachiroptera) are poorly known as compared to those of other mammals. Reproductive biology of certain Megachiropterans is particularly interesting because of an unusual adaptation that extends the period between implantation and parturition. One of the longest gestation periods, including a period of about 8 months of delayed development is reported in Haplonycteris fischeri, a member of the family Pteropodidae (Heideman, 1989). Mutere (1967) noted the occurrence of delayed implantation in the tropical African fruit bat, Eidolon helvum. Our knowledge of the reproductive pattern in members of the family Pteropodidae is restricted to records of breeding in a few species of the genera Pteropus, Epomops, Rousettus, Eidolon, Epomophorus, Cynopterus, Haplonycteris, Otopterus, Micropteropus, Macroglossus etc. (Marshall, 1947; Gopalakrishna, 1964; Mutere, 1967; Carter, 1970; Okia, 1974a, b; Krishna and Dominic, 1983; Heideman, 1989). Marshall (1947) reported that Pteropus giganteus from Sri Lanka has a sharply defined annual breeding season despite the climatic uniformity. However, the report on the breeding cycle of P. giganteus from central India differed from Marshall’s report in gestation length and also regarding the timing of embryo development and parturition (Moghe, 1951). Studies of the reproductive cycles of pteropodid bats indicate that breeding in most of the species is restricted to one or two sharply defined seasons. The larger pteropodid bats, e.g. Pteropus breed once in a sharply defined season and smaller pteropodid bats, e.g. Rousettus leschenaulti breed twice a year (Gopalakrishna and Choudhari, 1977). The short-nosed fruit bat, Cynopterus sphinx is found throughout India. C. sphinx breeds twice a year in quick succession at Varanasi (Krishna and Dominic, 1983). Mating in October is followed by pregnancy, which terminates in March. Within a short period, females mate again and deliver young in late July. The unusual feature of the breeding cycle of C. sphinx at Varanasi is the variation of gestation length in those two successive pregnancies (Krishna and Dominic, 1983). The aim of the present study was to investigate whether the prolonged gestation length of the winter pregnancy in C. sphinx is due to delayed embryonic development.
captured alive between 1998 and 2004 on and around Banaras Hindu university premises and in Ramnagar, Varanasi, India. They were then transported to the laboratory immediately. Body weight of bats was recorded as soon as they were brought to the laboratory (within 2 h of capture). Females weighing 43 g or more with a wing-span exceeding 46 cm were sexually mature (Krishna and Dominic, 1983).
Study site Cynopterus sphinx is available in Varanasi and adjacent sites in a reasonable number throughout the year. Varanasi is located in Northeast India (251N, 831E), where the cold and dry season lasts from November to February and the warm and dry season from March to June. Rainfall is seasonal. The rainy season normally begins in July and lasts until September. Temperature in Varanasi ranges from extremely hot in May and June (mean high 4074 1C and mean low 2674 1C) to cold during mid-December and late January (mean high 1874 1C to a mean low of 874 1C).
Histological procedures Usually twice a month (about 1st and 3rd week) between 15.00 and 16.00 h four female bats were killed by decapitation, blood was collected and their reproductive tracts were immediately dissected out. Serum was separated out within 1 h and stored at 20 1C until assayed. Reproductive tracts were fixed in Bouins fluid for 24 h, dehydrated through a graded series of ethyl alcohols, cleared in xylene and embedded in paraffin wax. After fixation of the reproductive tract, the greatest diameter of the uterine horn, which normally carried the conceptus, was measured. The uteri with diameters o5.0 mm were sectioned and examined histologically to determine the status of early pregnancy. Bats with uterine diameters 45.0 mm were in mid-pregnancy. Some of the reproductive tracts were sectioned in a longitudinal plane whereas some others were serially sectioned transversely at a thickness of 6–7 mm. The histological sections were stained with hematoxylin and eosin or by the periodic acid-Schiff (PAS) technique.
Determination of gestation length
Materials and methods Source of animals All experiments were conducted in accordance with principles and procedures approved by Banaras Hindu University, Departmental Research Committee. About 276 female bats (C. sphinx) utilized in this study were
Because all females collected at any time from the same locality had embryos at nearly the same stage of development, it appears that ovulation and fertilization are nearly synchronous in the colony. Thus the gestation period can be calculated as the interval between the first recorded ovulation (presence of corpus luteum in the ovarian sections) and the first recorded parturition (appearance of lactating young one). For calculating
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abdominal region by mid-November prior to the severe winter season. Adults show a decrease in the deposited fat as winter passes and the accumulated fat is completely utilized by late December.
80 Body weight (g)
133
75 70 65 60
Reproductive seasonality
55 50 45
O
N D
J
F M-1 M-2 A
M
J
J
A
S
Months M-1 = March Pre-partum M-2 = March Post-partum
Fig. 1. Seasonal changes in the body weight of Cynopterus sphinx.
the age of the conceptus, day 1 of pregnancy was taken as the mid-point between first recorded ovulation and last recorded non-pregnant female.
Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA). Where appropriate, student t-tests were applied. Differences were considered significant if po0:05. Data were expressed as mean7SEM.
Results Body weight Changes in the body weight of female bats (Fig. 1) were primarily related to the seasonal cycle of fat deposition and during pregnancy to increase in fetal mass. The bat accumulates fat in the posterior Table 1.
Ovulation and fertilization Ovulation in C. sphinx was observed between late October to mid-November and then again in April (Table 1). In exceptional cases ovulation may be observed in late November or early January also. A single corpus luteum was always found in the ipsilateral ovary suggesting that polyovulation or unsuccessful pregnancy is rarely found in the species. Spermatozoa were found in uteri of females captured during late October to early November and April suggesting that mating occurred only during the periovulatory period. Females with sperm in their uteri were not found during other times of the year. The presence of spermatozoa in uteri of females having mature preovulatory follicles suggests that mating is closely tied to ovulation in C. sphinx. Pregnant females had only a single corpus luteum and ovulation was found to have occurred from the ovary ipsilateral to the side of the pregnant uterine cornu. The number of ovulations from right and left ovaries did not differ significantly. Postpartum ovulation occurred in the ovary contralateral to the ovary containing the corpus albicans. Post-partum oestrus In late March, the majority of bats captured were in advanced stages of pregnancy and/or with suckling young indicating recent parturition. By mid-April, most of the females had delivered and were with suckling young. By late April, several females were found with
Reproductive seasonality of Cynopterus sphinx at Varanasi, India
Month
August September October November December January February March April May June July
Number of animals collected
14 14 20 35 20 25 24 24 28 26 18 28
Pregnant females EP
MP
LP
— — 12 26 17 4 3 3 20 3 — —
— — — — 3 21 4 — 2 21 1 —
2 4 — — — — 15 13 4 2 14 5
Non-pregnant
Lactating
— — 8 9 — — — — 2 — 2 1
12 10 — — — — — 8 20 17 4 22
Early Pregnancy (EP) ¼ Uterine diameter p4 mm. Mid Pregnancy (MP) ¼ Uterine diameter ranges from 5–10 mm. Late Pregnancy (LP) ¼ Uterine diameter X10 mm.
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recently ovulated follicles in the ovary and spermatozoa in the genital tracts. Blastocysts were also seen in the uteri of some females and in a few, implantation was already completed. The ovary ipsilateral to the parturated uterine horn contained a single corpus luteum in an advanced stage of regression. Almost all the lactating females examined in the last week of April had an implanted embryo and well-developed corpus luteum in the ovary ipsilateral to the pregnant uterine horn. The occurrence of simultaneously pregnant and lactating females indicates the incidence of a post-partum oestrus. The pregnancies follow one another in quick succession.
Early embryonic development and implantation Morphology of early embryonic development in C. sphinx was similar to that of other cynopterine fruit bats. The embryo’s stay in the oviduct was relatively short in C. sphinx. Females collected during early November and/or early April generally had embryos in different stages of cleavage in the oviduct. The embryos in the oviduct were enclosed in a zona pellucida. The latest embryonic stage observed in the oviduct was a morula or early blastocyst. The earliest stage of embryo found in the uterus was a blastocyst (Fig. 2a). Blastocysts shed their zona pellucida soon after reaching the uterus. All preimplantation and implanting blastocysts were centered within the progestational (swollen) region of the uterus, where hypertrophy of the uterus and proliferation of uterine glands were most extreme. The preimplantational early blastocyst consists of an undifferentiated inner cell mass surrounded by a single layer of columnar trophoblast. Initial contact of the blastocyst was superficial and at one side of the uterus. Soon it expanded and attached around its entire circumference. The uterine endometrium was either missing or degraded at the site where the developing trophoblast and endometrium were in contact. Following implantation, the embryo and its trophoblast were completely embedded within the uterine wall and a decidua capsularis had developed suggesting that implantation in C. sphinx is secondarily interstitial. During early implantation, the embryo was a roughly spherical mass of cells (Fig. 2a). The embryo at this stage was composed of an outer layer of hypoblast along Reichert’s membrane and an inner layer of columnar epiblast surrounding a central region of undifferentiated cells. The hypoblast cells subsequently formed the yolk sac membrane lining the yolk sac cavity. Prior to gastrulation, two primary schizoamniotic cavities developed in the inner core by a process of cavitation (Fig. 2b). The primary cavity in the center of the inner core of cells expanded as the cells at the cavity borders degenerated. The secondary cavity expanded as the
Fig. 2. Early embryonic stages of Cynopterus sphinx during the winter pregnancy (a–d) and the summer pregnancy (e–h). (a) Bilaminar blastocyst stage prior to gastrulation (11 November), 75. (b) Mid-gastrula stage showing the primary (1) and secondary (2) schizamniotic cavities (28 November), 75. (c) Late gastrula stage (11 December), 75. (d) Post-gastrula presomite stage embryo (28 December); three embryonic germ layers are seen, 34. (e) Oviductal embryos; note the presence of zona pellucida (arrow) (2 April), 300. (f) Implanting blastocyst in uterine lumen, trophoblast cells (arrow) are closely apposed to the uterine endometrial cell lining (8 April); ICM ¼ inner cell mass, 250. (g) Late gastrula stage (22 April), 75. (h) Post-gastrula pre-somite stage embryo (5 May), 34.
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presumptive ectoderm at its boundaries elongated, enclosing an increasingly larger volume. Finally, prior to gastrulation the two schizoamniotic cavities fused to form a single proamniotic cavity filled with cell debris. Following fusion of the two schizoamniotic cavities, presumptive mesoderm appeared between the presumptive hypoblast and epiblast (Fig. 2c). In the advanced gastrula stage, the mesoderm completely lined the yolk sac, and a small head fold and notochord were found, but the neural groove and somites had not yet appeared (Fig. 2d).
Delayed development At Varanasi, C. sphinx breeds twice a year in rapid succession. The winter pregnancy in C. sphinx commences in late October and terminates in March, with a duration of about 150 days. The summer pregnancy commences in early April and terminates in late July and
Table 2. The range of days post ovulation during which different developmental stages are found during the winter and summer pregnancies of Cynopterus sphinx Stage of embryonic development Implanting blastocyst Gastrula Somite stage Limb bud stage
Winter pregnancy (days post ovulation)
Summer pregnancy (days post ovulation)
9–13
9–13
13–50 48–64 64–71
13–25 26–40 40–47
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has a duration of about 125 days. A difference of about 25 days in gestation length between the two successive pregnancies of the year suggests delayed embryonic development during the winter pregnancy in C. sphinx (Table 2). Differences in embryonic development during winter and summer pregnancies are also reflected in highly significant differences in fetal weight, size and placental weight. Interestingly, late embryos from winter pregnancies were significantly heavier than late embryos from summer pregnancies, while crown-rump length was the same (Table 3). The changes in the size and weight of uterine cornua of bats killed at regular intervals throughout the two pregnancies are shown in Figs. 3a and b. The data clearly suggest a slow increase in the size (diameter) of the uterus at the site of implantation during November and December. The uterine swelling ranged between 2 and 4 mm for about 6–8 weeks during this period. By the end of December a marked increase in the size of uterine swelling was noted. Subsequently a regular increase in uterine swelling (diameter) was found in all succeeding months until parturition. Similarly, the weight of uterine cornua also showed only a slow increase during November and December. No such retardation in uterine swelling during the early stage of embryonic development was noticed during the summer pregnancy. A gradual increase in the size of uterine swelling and weight of uterine cornua was observed throughout the summer pregnancy (Table 4). Histological analysis of embryos during the period of delayed embryonic development of the winter pregnancy (November and December) showed that the embryos were mainly undergoing the process of gastrulation (Figs. 2a–d). The majority of females collected during
Table 3. Changes in the fetal mass, crown-rump length and placental mass of Cynopterus sphinx during winter and summer pregnancies Month
Pattern of pregnancy
Fetal weight (g)
Crown-rump length (cm)
Placental weight (g)
Winter pregnancy November December January February March
EP EP MP LP LP
a
a
a
a
a
a
0.3470.05 6.6871.021,4,5 8.7770.781,4,5
2.7270.24 6.5270.431,4,5 7.8270.361,2,3,6
0.34970.05 0.88270.081,4,5 0.77870.081,4
Summer pregnancy April EP May MP June LP July LP ANOVA
a
a
a
0.9870.21 2.0970.59 7.6371.271,4,5 Po0:001
3.3270.353 4.4670.391,3 7.9670.601,2,4,5 Po0:001
0.38170.05 0.63870.031,4 1.05070.061,2,3,4,5 Po0:001
Values are mean7SEM of at least four animals. Values are statistically significant (Po0:05) by Duncan’s multiple range test as compared with January (1), February (2), March (3), May (4), June (5) and July (6). Early Pregnancy (EP) ¼ Uterine diameterp4 mm. Mid Pregnancy (MP) ¼ Uterine diameter ranges from 5–10 mm. Late Pregnancy (LP) ¼ Uterine diameter X10 mm. a Data not available.
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uterine diameter (mm)
25
Summer pregnancy
20 15 10 5 0
0-15
15-30
30-45
45-60
60-75
75-90
90-105 105-120 120-135 135-150
Days post coitum
(a) 12
Uterine weight (g)
10
Winter pregnancy Summer pregnancy
8 6 4 2 0 1-15
15-30
30-45
45-60
60-75
75-90
90-105 105-120 120-135 135-150
Days post coitum
(b)
Fig. 3. (a) Changes in the uterine diameter (mean of 4 years) of Cynopterus sphinx during winter and summer pregnancies. (b) Changes in uterine weight (mean of 4 years) of Cynopterus sphinx during winter and summer pregnancies.
Table 4. Changes in the uterine diameter (mm) and mean uterine weight of Cynopterus sphinx during the winter and summer pregnancies Days postcoitumb
Winter pregnancy
Summer pregnancy
t-test (P value)
Winter pregnancy
Uterine diameter (mm) 0–15 15–30 30–45 45–60 60–75 75–90 90–105 105–120 120–135 135–150
1.0370.05 2.0270.17 2.6770.12 3.0670.15 3.8670.35 9.7370.79 11.9370.66 19.7571.04 24.0070.58 28.5071.08
1.0170.05 2.1970.18 4.1170.29 8.5770.76 14.3270.55 18.2471.41
NS NS Po0:001 Po0:001 Po0:001 Po0:001
a
a
28.2271.38
Po0:001
a
a
a
a
Values are mean7SEM of at least four animals. NS ¼ Non-significant. a Data not available. b Determination of days post-coitum is described in Materials and methods.
Summer pregnancy
t-test (P value)
Uterine weight (g) 0.01370.001 0.01970.001 0.02470.002 0.02970.002 0.07270.016 0.91970.209 1.43070.172 5.04670.429 5.83071.302 9.61070.574
0.0270.001 0.0270.002 0.1370.028 0.6970.039 1.8470.188 3.5170.488
NS NS Po0:004 Po0:003 Po0:001 Po0:001
a
8.1970.764 a a
Po0:001
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mid-November had an embryo in early gastrulation showing the beginning of amniogenesis. The embryos were composed of a layer of endoderm surrounded by ectoderm and surrounding an inner sphere of cells. Embryos attained the primitive streak stage at a later phase of delay. Since relatively few specimens were found with embryos in either preimplantation or implantation stages between early to mid-November, the development up to this stage may be rapid and possibly took less than 13 days. The evidence suggests that the developmental rate suddenly slowed down considerably in the embryos that completed the implantation process. During the period of slowed embryonic development the trophoblast showed little or no changes. However, the inner cell mass continued to undergo modification at a slow pace. Cells within the inner cell mass dissociated and/or disintegrated forming a schizoamniotic cavity (early gastrulation). Later, cells in the outer ectodermal layer flattened and separated from the inner solid sphere of cells. Much of the increase in the size of the embryo during the period of slowed embryonic development was through the formation and expansion of the schizoamniotic cavity. By mid-December, the majority of the bats had embryos showing development of the primitive streak and the formation of mesoderm. Subsequent stages of embryonic development passed quickly suggesting termination of the period of slowed embryonic development. In contrast, during summer pregnancies, the process of gastrulation is relatively shorter and passed quickly (Figs. 2e–h).
Comparison of embryonic development The rate of embryonic development during the winter pregnancy of C. sphinx is clearly slower than during the summer pregnancy resulting in a prolonged gestation length for the winter pregnancy. A comparison of the pattern of embryonic development during two successive pregnancies of the year of C. sphinx showed a marked difference particularly in the early stages of development (Table 2). Preimplantation and implantation stages passed quickly in both pregnancies of the year and showed no noted differences. The gastrulation stage was prolonged during the winter pregnancy as compared to the summer pregnancy. The process of amniogenesis was specifically slowed during the winter pregnancy. The slowed embryonic development terminates at the end of the primitive streak stage. There was no evidence of embryonic development being held or slowed for a prolonged period at the gastrulation or primitive streak stage during the summer pregnancy (Figs. 2e–h). Once the embryo reached the pre-somite stage the diameter of the uterine cornua (about 5.0 mm) began to increase steadily until parturition.
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Comparison of embryonic development for four consecutive years A comparison of the pattern of embryonic development from 1999 to 2002 showed only a marginal variation from year to year. The time of mating showed some variation from one year to another. In 1999 mating was noted as early as mid-October, but in 2001 mating and ovulation were noted as late as early November. The timing of termination of delayed development was usually fixed in a narrow range of time (late December) from one year to another. The time of parturition and post-partum oestrus showed only a limited variation from one year to another.
Discussion The present study shows two periods of mating corresponding to two periods of ovulation in C. sphinx at Varanasi. Two distinct periods of parturition were also observed. The reproductive characteristics such as post-partum oestrus, alternate successive ovulations between the two ovaries and bicornuate uterus as observed in C. sphinx have also been reported in other bats with a seasonally dioestrous breeding pattern (Wimsatt, 1979; Rasweiler, 1982; Krishna and Dominic, 1982a; Heideman, 1989; Heideman and Powell, 1998). Ovulation by alternate ovaries appears to be essential in order for the females to conceive successfully at postpartum oestrus (Rasweiler, 1982). Although sperm transport is inhibited on the recently parturient side of the tract, conception can take place normally in the previously non-gravid uterus horn (Rasweiler, 1982). Similarly, by alternating ovulations the bat can successfully establish a pregnancy on the previously non-gravid side, while involution of the post-parturient horn is still in progress. The progestational reaction is localized in a small segment near the cranial end of the uterine cornua in C. sphinx during the oviductal journey of the embryo. The progestational changes are augmented in the cornu on the side of ovulation, whereas the contralateral cornu reverts to an anoestrous condition after implantation of the blastocyst. Implantation and pattern of early embryonic development in C. sphinx are similar to other Megachiropterans such as H. fischeri (Heideman, 1989), Otopteropus cartilagonodus (Heideman et al., 1993) and Ptenochirus jagori (Heideman and Powell, 1998). The initial attachment of the blastocyst in C. sphinx is superficial, but implantation is secondarily interstitial as is typically described in many chiropterans (Rasweiler, 1979; Heideman and Powell, 1998).
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Female C. sphinx at Varanasi became pregnant as early as late October and early November. However, post-gastrulation embryos were not found until late December. The majority of the females had a uterine swelling between 2 and 4 mm during November and December. This suggests that female C. sphinx either repetitively lose embryos and get reimpregnated or that their embryos were in some form of developmental delay. None of the females examined during November and December showed any degenerating embryos. Additionally, the ovary showed only one functional but no regressing corpus luteum. These observations suggest that C. sphinx undergoes a period of delayed development. The period of delayed development terminates in late December as the majority of females examined in early January contained embryos well past gastrulation. The marked variation in gestation length in the two successive pregnancies of the year in C. sphinx is rare but has been reported previously in Artibeus jamaicensis (Fleming, 1971), Taphozous longimanus (Krishna and Dominic, 1982b) and Carollia perspicillata (Rasweiler and Badwaik, 1997). In C. sphinx, the winter pregnancy has a duration of about 150 days and the summer pregnancy has a duration of about 125 days. The results of the present study suggest that the prolonged gestation length of winter pregnancy in C. sphinx is due to the slowed (delayed) embryonic development during the months of November and December. Because the longer gestation period in C. sphinx is associated with one particular period of the year, the delayed embryonic development in this bat may be due to seasonal factors. Delayed development may be broadly categorized into two types: environmental (temperature)-dependant and environmental (temperature)-independent. In species such as Pipistrellus pipistrellus, Myotis myotis and Corynorhinus rafinesquei, reduced environmental temperature significantly lengthens gestation by slow embryonic development (Eisentraut, 1937; Pearson et al., 1952; Racey, 1969). It is further demonstrated that in temperate latitude vespertilionid bats, food shortages during cold weather may be responsible for a reduced rate of conceptus development (Racey and Swift, 1981). In Artibeus jamaicensis a period of delayed embryonic development occurs during the height of the rainy season (Fleming, 1971). Since this form of embryonic diapause may occur depending upon environmental conditions, it is also referred to as facultative delayed development. In the California leaf-nosed bat, Macrotus californicus, delayed development lasts 4–5 months and is independent of temperature and food supply (Bradshaw, 1962). This is referred to as an obligate form of delayed development. The fruit bat Haplonycteris fischeri from the Philippines exhibits an obligate 8 months’ delay in embryonic development (Heideman, 1989). Delayed embryonic development has also been
demonstrated in Carollia perspicillata (Rasweiler and Badwaik, 1997), Otopteropus cartilagondus (Heideman et al., 1993), Ptenochirus jagori (Heideman and Powell, 1998), Rhinolophus hipposideros (Kolb, 1950), Natalus stramineus (Mitchell, 1965) and Hipposideros caffer (Bernard and Meester, 1982). It is apparent from the present study on C. sphinx that a prolonged delay period is not obligatory in this species, because females do not undergo prolonged developmental delays during the summer pregnancy. The facultative delay in this species may allow females to respond more rapidly to some environmental cue that predicts good conditions in the future (Heideman, 1988). In most mammals, the timing and duration of pregnancy are fixed by genotype and are highly resistant to alteration by environment. The features of embryonic development among Chiroptera, in apparent contrast to those of other mammals, are unique and varied. Most of the bat species with delayed embryonic development also exhibit a highly variable gestation length. A postimplantational delay in embryonic development up to 5 months was detected in young P. jagori females reproducing for the first time. However, adult females of this species showed little evidence of a delay in development. This causes marked variation in gestation length between young and adult P. jagori (Heideman and Powell, 1998). A remarkable degree of variation in gestation length was described in Carollia perspicillata (Rasweiler and Badwaik, 1997). Females caught in the wild have a gestation period that varies between 105 and 178 days (Rasweiler and Badwaik, 1997). Captive-reared females showed a variation in the gestation period of 113–159 days. The prolongation of pregnancy in some captive animals was suggested to be due to stress. So, delayed development in C. perspicillata occurs under two different circumstances: in response to stress in captivity and seasonally in the wild population. In C. sphinx gestation length varies from the winter pregnancy to the summer pregnancy. The histological studies of the genital tract of C. sphinx during early pregnancy indicate that the delay occurred after implantation. The majority of females observed during November and December contained embryos in the stage of gastrulation. At the beginning of the delay the embryos were in the early stage of amniogenesis, but towards the end of the delay embryos were in the stage of primitive streak and formation of mesoderm. In all bat species with delayed embryonic development which have been examined histologically there is evidence that embryonic development is nearly halted for a period of months in an early gastrulation stage, shortly after implantation (Heideman and Powell, 1998). During the delay embryos continue to grow, albeit at a retarded rate. During the delay period in C. sphinx, the length of the embryo increases partly due to growth and partly due to the formation of the amniotic
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cavity as described in H. fischeri (Heideman, 1989). It was recently hypothesized that the developmental delay may be caused by inhibition of one or more genes crucial for gastrulation or post-gastrulation development (Heideman and Powell, 1998). Whether the inhibition of genes is regulated by maternal factors and consists of either the lack of some necessary stimulatory molecular signal or production of an inhibitory signal by the mother needs further investigation. The variation in the length of gestation in C. sphinx may not be attributed to low temperature as the period of delayed development in this bat species does not coincide with the period of lowest environmental temperatures, which is in January. Neither does it coincide with the rainy season. Because the period of peak recruitment of young bats coincides with the period of fruit abundance at Varanasi, this suggests that parturition in C. sphinx may be related to seasonal food supply (Krishna and Dominic, 1983). It is thus possible that a delay period may allow females to time lactation and weaning to the most favorable period of the year. The present study on C. sphinx during four consecutive breeding seasons clearly showed that timing for termination of delay period and timing of parturition are nearly fixed despite much variation of the mating period. This suggests that the delay in this species may function to synchronize parturition among females (Heideman, 1988). A comparison of conditions during the first and second breeding cycles of the year in C. sphinx suggests that the period of delayed embryonic development in this species closely coincides with the period of fat accumulation. Fat deposition in this species was not observed during the summer pregnancy. The delay begins in November when fat also begins to accumulate whereas timing for termination of delay during late December coincides closely with the time when accumulated fat is completely metabolized. Whether the accumulated fat (adipose tissue) retards embryonic development is not yet known. It is well known that fat produces leptin and leptin has been shown to be a ubiquitous feature of pregnant mammals (Zhao et al., 2003). Leptin is known to affect fetal growth and development by binding to its receptor present in fetal organs (Ashworth et al., 2000). Whether leptin receptors are present in the gastrulating embryo and whether it is associated with the delayed embryonic development in C. sphinx needs further study. In summary, the results of this study suggest that the prolonged gestation length of winter pregnancy in C. sphinx was due to the slowed or delayed embryonic development during November and December. Delayed embryonic development occurred between early gastrulation and primitive streak stages. The analysis of the pattern of embryonic development during four consecutive breeding seasons clearly showed that the timing
139
for termination of the delay period and timing of parturition are nearly fixed. This suggests that the delayed embryonic development in C. sphinx may function to synchronize parturition. The present study further noted a close association between the period of delayed embryonic development and the period of fat deposition in C. sphinx. The significance of fat deposition in delayed embryonic development warrants further investigation.
Acknowledgement This research was supported by the grant (DST (SP/ SO/C-33/99)) from the Department of Science and Technology, India.
References Ashworth, C.J., Hoggard, N., Thomas, L., Mercer, J.G., Wallace, J.M., Lea, R.G., 2000. Placental leptin. Rev. Reprod. 5, 18–24. Bernard, R.T.F., Meester, J.A.J., 1982. Female reproduction and the female reproductive cycle of Hipposideros caffer (Sundevall, 1846) in Natal, South Africa. Ann. Transvaal. Mus. 33, 131–144. Bradshaw, G.V.R., 1962. Reproductive cycle of the California leaf-nosed bat, Macrotus californicus. Science 136, 645–646. Carter, D.C., 1970. Chiropteran reproduction. In: Slaughter, B.H., Walton, D.W. (Eds.), About Bats – a Chiropteran Biology Symposium. Methodist University Press, Dallas, TX, pp. 233–247. Eisentraut, M., 1937. Die Wirkung niedriger Temperaturen auf die Embryonalentwicklung bei Flederma¨usen. Biol. Zentralblatt. 57, 59–74. Fleming, T.H., 1971. Artibeus jamaicensis: delayed embryonic development in neotropical bat. Science 171, 402–404. Gopalakrishna, A., 1964. Post-partum pregnancy in the Indian fruit bat, Rousettus leschenaulti. Curr. Sci. 33, 558–559. Gopalakrishna, A., Choudhari, P.N., 1977. Breeding habits and associated phenomena in some Indian bats. Part I – Rousettus leschenaulti (Desmarest) – Megachiroptera. J. Bombay Nat. Hist. Soc. 74, 1–16. Heideman, P.D., 1988. The timing of reproduction in the fruit bat Haplonycteris fischeri (pteropodidae): geographic variation and delayed development. J. Zool. Lond. 215, 577–595. Heideman, P.D., 1989. Delayed development in Fischer’s pygmy fruit bat, Haplonycteris fischeri, in the Philippines. J. Reprod. Fertil. 85, 363–382. Heideman, P.D., Powell, K.S., 1998. Age-specific reproductive strategies and delayed embryonic development in an old world fruit bat, Ptenochirus jagori. J. Mamm. 79, 295–311. Heideman, P.D., Cummings, J.A., Heaney, L.R., 1993. Reproductive timing and early embryonic development in an old world fruit bat, Otopterus cartilagonodus (Megachiroptera). J. Mamm. 74, 621–630.
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Kolb, A., 1950. Beitra¨ge zur Biologie einheimischer Flederma¨use. Zool. Fb. Abt. Syst. Oekol. Geogr. Tiere 78, 547–573. Koopman, K.F., 1993. Bats. In: Wilson, D.E., Reeder, D.M. (Eds.), Mammalian Species of the World. A Taxonomic and Geographic Reference. Smithsonian Institution Press, Washington. Krishna, A., Dominic, C.J., 1982a. Reproduction in the Indian sheath-tailed bat. Acta Theriol. 27, 97–106. Krishna, A., Dominic, C.J., 1982b. Differential rates of fetal growth in two successive pregnancies in the emballonurid bat, Taphozous longimanus Hardwicke. Biol. Reprod. 27, 351–353. Krishna, A., Dominic, C.J., 1983. Reproduction in the female short-nosed fruit bat Cynopterus sphinx (Vahl). Period. Biol. 85, 23–30. Marshall, A.J., 1947. The breeding cycle of an equatorial bat (Pteropus giganteus) of Ceylon. Proc. Linn. Soc. Lond. 159, 103–111. Mitchell, G.C., 1965. A natural history study of the funneleared bat, Natalus stramineus. M.Sc. Thesis. University of Arizona, Tucson. Moghe, M.A., 1951. Development and placentation of the Indian fruit bat, Pteropus giganteus giganteus. Proc. Zool. Soc. Lond. 121, 703–721. Mutere, F.A., 1967. The breeding biology of equatorial vertebrates: reproduction in the fruit bat, Eidolon helvum, at latitude 01200 N. J. Zool. Lond. 153, 153–162. Okia, N.O., 1974a. Breeding in Franquet’s bat, Epomops franquetti (Tomes), in Uganda. J. Mammal. 55, 462–465.
Okia, N.O., 1974b. The breeding pattern of the eastern epauletted bat, Epomophorus anurus Heuglin, in Uganda. J. Reprod. Fertil. 37, 27–31. Pearson, O.P., Koford, M.R., Pearson, A.R., 1952. Reproduction of the lump-nosed bat Corynorhinus rafinesquei in California. J. Mammal. 33, 273–320. Racey, P.A., 1969. Diagnosis of pregnancy and experimental extension of gestation in the pipistrelle bat, Pipistrellus pipistrellus. J. Reprod. Fertil. 19, 465–474. Racey, P.A., Swift, S.M., 1981. Variations in gestation length in a colony of pipistrelle bats (Pipistrellus pipistrellus) from year to year. J. Reprod. Fertil. 61, 123–129. Rasweiler IV., J.J., 1979. Early embryonic development and implantation in bats. J. Reprod. Fertil. 56, 403–416. Rasweiler IV., J.J., 1982. The contribution of observations on early pregnancy in the little sac-winged bat, Peropteryx kappleri, to an understanding of the evolution of reproductive mechanisms in monovular bats. Biol. Reprod. 27, 681–702. Rasweiler IV, J.J., Badwaik, N.K., 1997. Delayed development in the short-tailed fruit bat, Carollia perspicillata. J. Reprod. Fertil. 109, 7–20. Wimsatt, W.A., 1979. Reproductive asymmetry and unilateral pregnancy in Chiroptera. J. Reprod. Fertil. 56, 345–357. Zhao, J., Kuntz, T.H., Tumba, N., Schulz, L.C., Li, C., Reeves, M., Widmaier, E.P., 2003. Comparative analysis of expression and secretion of placental leptin in mammals. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R438–R446.
ZOOLOGY Zoology 108 (2005) 131–140 www.elsevier.de/zool
Delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx Karukayil J. Meenakumari, Amitabh Krishna Department of Zoology, Banaras Hindu University, Varanasi 221 005, India Received 14 August 2004; received in revised form 28 January 2005; accepted 10 February 2005
Abstract The unusual feature of the breeding cycle of Cynopterus sphinx at Varanasi is the significant variation in gestation length of the two successive pregnancies of the year. The aim of this study was to investigate whether the prolongation of the first pregnancy in C. sphinx is due to delayed embryonic development. The first (winter) pregnancy commences in late October and lasts until late March and has a gestation period of about 150 days. The second (summer) pregnancy commences in April and lasts until the end of July or early August with a gestation period of about 125 days. Changes in the size and weight of uterine cornua during the two successive pregnancies suggest retarded embryonic growth during November and December. Histological analysis during the period of retarded embryonic development in November and December showed a slow gastrulation process. The process of amniogenesis was particularly slow. When the embryos attained the early primitive streak stage, their developmental rate suddenly increased considerably. During the summer pregnancy, on the other hand, the process of gastrulation was much faster and proceeded quickly. A comparison of the pattern of embryonic development for 4 consecutive years consistently showed retarded or delayed embryonic development during November and December. The time of parturition and post-partum oestrus showed only a limited variation from 1 year to another. This suggests that delayed embryonic development in C. sphinx may function to synchronize parturition among females. The period of delayed embryonic development in this species clearly coincides with the period of fat deposition. The significance of this correlation warrants further investigation. r 2005 Elsevier GmbH. All rights reserved. Keywords: Cynopterus sphinx; Short-nosed fruit bat; Embryonic development; Pteropodidae; Bats
Introduction The order Chiroptera includes 916 species, about one quarter of all known mammalian species and ranked second only to rodents (Koopman, 1993). Two suborders of bats have long been recognized: MegachirCorresponding author.
E-mail address: [email protected] (A. Krishna). 0944-2006/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2005.02.002
optera (Megabats, old world fruit bats) and Microchiroptera (Microbats, echolocating bats). The suborder Megachiroptera includes a single family, Pteropodidae with 42 genera and 166 species (Koopman, 1993). The members of the family Pteropodidae are largely distributed throughout the tropics and subtropics of the old world from Africa to South-East Asia, Australia, Samoa and the Carolines (Koopman, 1993). Despite this widespread distribution, patterns of
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reproductive timing and embryonic development of old world fruit bats (Megachiroptera) are poorly known as compared to those of other mammals. Reproductive biology of certain Megachiropterans is particularly interesting because of an unusual adaptation that extends the period between implantation and parturition. One of the longest gestation periods, including a period of about 8 months of delayed development is reported in Haplonycteris fischeri, a member of the family Pteropodidae (Heideman, 1989). Mutere (1967) noted the occurrence of delayed implantation in the tropical African fruit bat, Eidolon helvum. Our knowledge of the reproductive pattern in members of the family Pteropodidae is restricted to records of breeding in a few species of the genera Pteropus, Epomops, Rousettus, Eidolon, Epomophorus, Cynopterus, Haplonycteris, Otopterus, Micropteropus, Macroglossus etc. (Marshall, 1947; Gopalakrishna, 1964; Mutere, 1967; Carter, 1970; Okia, 1974a, b; Krishna and Dominic, 1983; Heideman, 1989). Marshall (1947) reported that Pteropus giganteus from Sri Lanka has a sharply defined annual breeding season despite the climatic uniformity. However, the report on the breeding cycle of P. giganteus from central India differed from Marshall’s report in gestation length and also regarding the timing of embryo development and parturition (Moghe, 1951). Studies of the reproductive cycles of pteropodid bats indicate that breeding in most of the species is restricted to one or two sharply defined seasons. The larger pteropodid bats, e.g. Pteropus breed once in a sharply defined season and smaller pteropodid bats, e.g. Rousettus leschenaulti breed twice a year (Gopalakrishna and Choudhari, 1977). The short-nosed fruit bat, Cynopterus sphinx is found throughout India. C. sphinx breeds twice a year in quick succession at Varanasi (Krishna and Dominic, 1983). Mating in October is followed by pregnancy, which terminates in March. Within a short period, females mate again and deliver young in late July. The unusual feature of the breeding cycle of C. sphinx at Varanasi is the variation of gestation length in those two successive pregnancies (Krishna and Dominic, 1983). The aim of the present study was to investigate whether the prolonged gestation length of the winter pregnancy in C. sphinx is due to delayed embryonic development.
captured alive between 1998 and 2004 on and around Banaras Hindu university premises and in Ramnagar, Varanasi, India. They were then transported to the laboratory immediately. Body weight of bats was recorded as soon as they were brought to the laboratory (within 2 h of capture). Females weighing 43 g or more with a wing-span exceeding 46 cm were sexually mature (Krishna and Dominic, 1983).
Study site Cynopterus sphinx is available in Varanasi and adjacent sites in a reasonable number throughout the year. Varanasi is located in Northeast India (251N, 831E), where the cold and dry season lasts from November to February and the warm and dry season from March to June. Rainfall is seasonal. The rainy season normally begins in July and lasts until September. Temperature in Varanasi ranges from extremely hot in May and June (mean high 4074 1C and mean low 2674 1C) to cold during mid-December and late January (mean high 1874 1C to a mean low of 874 1C).
Histological procedures Usually twice a month (about 1st and 3rd week) between 15.00 and 16.00 h four female bats were killed by decapitation, blood was collected and their reproductive tracts were immediately dissected out. Serum was separated out within 1 h and stored at 20 1C until assayed. Reproductive tracts were fixed in Bouins fluid for 24 h, dehydrated through a graded series of ethyl alcohols, cleared in xylene and embedded in paraffin wax. After fixation of the reproductive tract, the greatest diameter of the uterine horn, which normally carried the conceptus, was measured. The uteri with diameters o5.0 mm were sectioned and examined histologically to determine the status of early pregnancy. Bats with uterine diameters 45.0 mm were in mid-pregnancy. Some of the reproductive tracts were sectioned in a longitudinal plane whereas some others were serially sectioned transversely at a thickness of 6–7 mm. The histological sections were stained with hematoxylin and eosin or by the periodic acid-Schiff (PAS) technique.
Determination of gestation length
Materials and methods Source of animals All experiments were conducted in accordance with principles and procedures approved by Banaras Hindu University, Departmental Research Committee. About 276 female bats (C. sphinx) utilized in this study were
Because all females collected at any time from the same locality had embryos at nearly the same stage of development, it appears that ovulation and fertilization are nearly synchronous in the colony. Thus the gestation period can be calculated as the interval between the first recorded ovulation (presence of corpus luteum in the ovarian sections) and the first recorded parturition (appearance of lactating young one). For calculating
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abdominal region by mid-November prior to the severe winter season. Adults show a decrease in the deposited fat as winter passes and the accumulated fat is completely utilized by late December.
80 Body weight (g)
133
75 70 65 60
Reproductive seasonality
55 50 45
O
N D
J
F M-1 M-2 A
M
J
J
A
S
Months M-1 = March Pre-partum M-2 = March Post-partum
Fig. 1. Seasonal changes in the body weight of Cynopterus sphinx.
the age of the conceptus, day 1 of pregnancy was taken as the mid-point between first recorded ovulation and last recorded non-pregnant female.
Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA). Where appropriate, student t-tests were applied. Differences were considered significant if po0:05. Data were expressed as mean7SEM.
Results Body weight Changes in the body weight of female bats (Fig. 1) were primarily related to the seasonal cycle of fat deposition and during pregnancy to increase in fetal mass. The bat accumulates fat in the posterior Table 1.
Ovulation and fertilization Ovulation in C. sphinx was observed between late October to mid-November and then again in April (Table 1). In exceptional cases ovulation may be observed in late November or early January also. A single corpus luteum was always found in the ipsilateral ovary suggesting that polyovulation or unsuccessful pregnancy is rarely found in the species. Spermatozoa were found in uteri of females captured during late October to early November and April suggesting that mating occurred only during the periovulatory period. Females with sperm in their uteri were not found during other times of the year. The presence of spermatozoa in uteri of females having mature preovulatory follicles suggests that mating is closely tied to ovulation in C. sphinx. Pregnant females had only a single corpus luteum and ovulation was found to have occurred from the ovary ipsilateral to the side of the pregnant uterine cornu. The number of ovulations from right and left ovaries did not differ significantly. Postpartum ovulation occurred in the ovary contralateral to the ovary containing the corpus albicans. Post-partum oestrus In late March, the majority of bats captured were in advanced stages of pregnancy and/or with suckling young indicating recent parturition. By mid-April, most of the females had delivered and were with suckling young. By late April, several females were found with
Reproductive seasonality of Cynopterus sphinx at Varanasi, India
Month
August September October November December January February March April May June July
Number of animals collected
14 14 20 35 20 25 24 24 28 26 18 28
Pregnant females EP
MP
LP
— — 12 26 17 4 3 3 20 3 — —
— — — — 3 21 4 — 2 21 1 —
2 4 — — — — 15 13 4 2 14 5
Non-pregnant
Lactating
— — 8 9 — — — — 2 — 2 1
12 10 — — — — — 8 20 17 4 22
Early Pregnancy (EP) ¼ Uterine diameter p4 mm. Mid Pregnancy (MP) ¼ Uterine diameter ranges from 5–10 mm. Late Pregnancy (LP) ¼ Uterine diameter X10 mm.
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recently ovulated follicles in the ovary and spermatozoa in the genital tracts. Blastocysts were also seen in the uteri of some females and in a few, implantation was already completed. The ovary ipsilateral to the parturated uterine horn contained a single corpus luteum in an advanced stage of regression. Almost all the lactating females examined in the last week of April had an implanted embryo and well-developed corpus luteum in the ovary ipsilateral to the pregnant uterine horn. The occurrence of simultaneously pregnant and lactating females indicates the incidence of a post-partum oestrus. The pregnancies follow one another in quick succession.
Early embryonic development and implantation Morphology of early embryonic development in C. sphinx was similar to that of other cynopterine fruit bats. The embryo’s stay in the oviduct was relatively short in C. sphinx. Females collected during early November and/or early April generally had embryos in different stages of cleavage in the oviduct. The embryos in the oviduct were enclosed in a zona pellucida. The latest embryonic stage observed in the oviduct was a morula or early blastocyst. The earliest stage of embryo found in the uterus was a blastocyst (Fig. 2a). Blastocysts shed their zona pellucida soon after reaching the uterus. All preimplantation and implanting blastocysts were centered within the progestational (swollen) region of the uterus, where hypertrophy of the uterus and proliferation of uterine glands were most extreme. The preimplantational early blastocyst consists of an undifferentiated inner cell mass surrounded by a single layer of columnar trophoblast. Initial contact of the blastocyst was superficial and at one side of the uterus. Soon it expanded and attached around its entire circumference. The uterine endometrium was either missing or degraded at the site where the developing trophoblast and endometrium were in contact. Following implantation, the embryo and its trophoblast were completely embedded within the uterine wall and a decidua capsularis had developed suggesting that implantation in C. sphinx is secondarily interstitial. During early implantation, the embryo was a roughly spherical mass of cells (Fig. 2a). The embryo at this stage was composed of an outer layer of hypoblast along Reichert’s membrane and an inner layer of columnar epiblast surrounding a central region of undifferentiated cells. The hypoblast cells subsequently formed the yolk sac membrane lining the yolk sac cavity. Prior to gastrulation, two primary schizoamniotic cavities developed in the inner core by a process of cavitation (Fig. 2b). The primary cavity in the center of the inner core of cells expanded as the cells at the cavity borders degenerated. The secondary cavity expanded as the
Fig. 2. Early embryonic stages of Cynopterus sphinx during the winter pregnancy (a–d) and the summer pregnancy (e–h). (a) Bilaminar blastocyst stage prior to gastrulation (11 November), 75. (b) Mid-gastrula stage showing the primary (1) and secondary (2) schizamniotic cavities (28 November), 75. (c) Late gastrula stage (11 December), 75. (d) Post-gastrula presomite stage embryo (28 December); three embryonic germ layers are seen, 34. (e) Oviductal embryos; note the presence of zona pellucida (arrow) (2 April), 300. (f) Implanting blastocyst in uterine lumen, trophoblast cells (arrow) are closely apposed to the uterine endometrial cell lining (8 April); ICM ¼ inner cell mass, 250. (g) Late gastrula stage (22 April), 75. (h) Post-gastrula pre-somite stage embryo (5 May), 34.
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presumptive ectoderm at its boundaries elongated, enclosing an increasingly larger volume. Finally, prior to gastrulation the two schizoamniotic cavities fused to form a single proamniotic cavity filled with cell debris. Following fusion of the two schizoamniotic cavities, presumptive mesoderm appeared between the presumptive hypoblast and epiblast (Fig. 2c). In the advanced gastrula stage, the mesoderm completely lined the yolk sac, and a small head fold and notochord were found, but the neural groove and somites had not yet appeared (Fig. 2d).
Delayed development At Varanasi, C. sphinx breeds twice a year in rapid succession. The winter pregnancy in C. sphinx commences in late October and terminates in March, with a duration of about 150 days. The summer pregnancy commences in early April and terminates in late July and
Table 2. The range of days post ovulation during which different developmental stages are found during the winter and summer pregnancies of Cynopterus sphinx Stage of embryonic development Implanting blastocyst Gastrula Somite stage Limb bud stage
Winter pregnancy (days post ovulation)
Summer pregnancy (days post ovulation)
9–13
9–13
13–50 48–64 64–71
13–25 26–40 40–47
135
has a duration of about 125 days. A difference of about 25 days in gestation length between the two successive pregnancies of the year suggests delayed embryonic development during the winter pregnancy in C. sphinx (Table 2). Differences in embryonic development during winter and summer pregnancies are also reflected in highly significant differences in fetal weight, size and placental weight. Interestingly, late embryos from winter pregnancies were significantly heavier than late embryos from summer pregnancies, while crown-rump length was the same (Table 3). The changes in the size and weight of uterine cornua of bats killed at regular intervals throughout the two pregnancies are shown in Figs. 3a and b. The data clearly suggest a slow increase in the size (diameter) of the uterus at the site of implantation during November and December. The uterine swelling ranged between 2 and 4 mm for about 6–8 weeks during this period. By the end of December a marked increase in the size of uterine swelling was noted. Subsequently a regular increase in uterine swelling (diameter) was found in all succeeding months until parturition. Similarly, the weight of uterine cornua also showed only a slow increase during November and December. No such retardation in uterine swelling during the early stage of embryonic development was noticed during the summer pregnancy. A gradual increase in the size of uterine swelling and weight of uterine cornua was observed throughout the summer pregnancy (Table 4). Histological analysis of embryos during the period of delayed embryonic development of the winter pregnancy (November and December) showed that the embryos were mainly undergoing the process of gastrulation (Figs. 2a–d). The majority of females collected during
Table 3. Changes in the fetal mass, crown-rump length and placental mass of Cynopterus sphinx during winter and summer pregnancies Month
Pattern of pregnancy
Fetal weight (g)
Crown-rump length (cm)
Placental weight (g)
Winter pregnancy November December January February March
EP EP MP LP LP
a
a
a
a
a
a
0.3470.05 6.6871.021,4,5 8.7770.781,4,5
2.7270.24 6.5270.431,4,5 7.8270.361,2,3,6
0.34970.05 0.88270.081,4,5 0.77870.081,4
Summer pregnancy April EP May MP June LP July LP ANOVA
a
a
a
0.9870.21 2.0970.59 7.6371.271,4,5 Po0:001
3.3270.353 4.4670.391,3 7.9670.601,2,4,5 Po0:001
0.38170.05 0.63870.031,4 1.05070.061,2,3,4,5 Po0:001
Values are mean7SEM of at least four animals. Values are statistically significant (Po0:05) by Duncan’s multiple range test as compared with January (1), February (2), March (3), May (4), June (5) and July (6). Early Pregnancy (EP) ¼ Uterine diameterp4 mm. Mid Pregnancy (MP) ¼ Uterine diameter ranges from 5–10 mm. Late Pregnancy (LP) ¼ Uterine diameter X10 mm. a Data not available.
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uterine diameter (mm)
25
Summer pregnancy
20 15 10 5 0
0-15
15-30
30-45
45-60
60-75
75-90
90-105 105-120 120-135 135-150
Days post coitum
(a) 12
Uterine weight (g)
10
Winter pregnancy Summer pregnancy
8 6 4 2 0 1-15
15-30
30-45
45-60
60-75
75-90
90-105 105-120 120-135 135-150
Days post coitum
(b)
Fig. 3. (a) Changes in the uterine diameter (mean of 4 years) of Cynopterus sphinx during winter and summer pregnancies. (b) Changes in uterine weight (mean of 4 years) of Cynopterus sphinx during winter and summer pregnancies.
Table 4. Changes in the uterine diameter (mm) and mean uterine weight of Cynopterus sphinx during the winter and summer pregnancies Days postcoitumb
Winter pregnancy
Summer pregnancy
t-test (P value)
Winter pregnancy
Uterine diameter (mm) 0–15 15–30 30–45 45–60 60–75 75–90 90–105 105–120 120–135 135–150
1.0370.05 2.0270.17 2.6770.12 3.0670.15 3.8670.35 9.7370.79 11.9370.66 19.7571.04 24.0070.58 28.5071.08
1.0170.05 2.1970.18 4.1170.29 8.5770.76 14.3270.55 18.2471.41
NS NS Po0:001 Po0:001 Po0:001 Po0:001
a
a
28.2271.38
Po0:001
a
a
a
a
Values are mean7SEM of at least four animals. NS ¼ Non-significant. a Data not available. b Determination of days post-coitum is described in Materials and methods.
Summer pregnancy
t-test (P value)
Uterine weight (g) 0.01370.001 0.01970.001 0.02470.002 0.02970.002 0.07270.016 0.91970.209 1.43070.172 5.04670.429 5.83071.302 9.61070.574
0.0270.001 0.0270.002 0.1370.028 0.6970.039 1.8470.188 3.5170.488
NS NS Po0:004 Po0:003 Po0:001 Po0:001
a
8.1970.764 a a
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mid-November had an embryo in early gastrulation showing the beginning of amniogenesis. The embryos were composed of a layer of endoderm surrounded by ectoderm and surrounding an inner sphere of cells. Embryos attained the primitive streak stage at a later phase of delay. Since relatively few specimens were found with embryos in either preimplantation or implantation stages between early to mid-November, the development up to this stage may be rapid and possibly took less than 13 days. The evidence suggests that the developmental rate suddenly slowed down considerably in the embryos that completed the implantation process. During the period of slowed embryonic development the trophoblast showed little or no changes. However, the inner cell mass continued to undergo modification at a slow pace. Cells within the inner cell mass dissociated and/or disintegrated forming a schizoamniotic cavity (early gastrulation). Later, cells in the outer ectodermal layer flattened and separated from the inner solid sphere of cells. Much of the increase in the size of the embryo during the period of slowed embryonic development was through the formation and expansion of the schizoamniotic cavity. By mid-December, the majority of the bats had embryos showing development of the primitive streak and the formation of mesoderm. Subsequent stages of embryonic development passed quickly suggesting termination of the period of slowed embryonic development. In contrast, during summer pregnancies, the process of gastrulation is relatively shorter and passed quickly (Figs. 2e–h).
Comparison of embryonic development The rate of embryonic development during the winter pregnancy of C. sphinx is clearly slower than during the summer pregnancy resulting in a prolonged gestation length for the winter pregnancy. A comparison of the pattern of embryonic development during two successive pregnancies of the year of C. sphinx showed a marked difference particularly in the early stages of development (Table 2). Preimplantation and implantation stages passed quickly in both pregnancies of the year and showed no noted differences. The gastrulation stage was prolonged during the winter pregnancy as compared to the summer pregnancy. The process of amniogenesis was specifically slowed during the winter pregnancy. The slowed embryonic development terminates at the end of the primitive streak stage. There was no evidence of embryonic development being held or slowed for a prolonged period at the gastrulation or primitive streak stage during the summer pregnancy (Figs. 2e–h). Once the embryo reached the pre-somite stage the diameter of the uterine cornua (about 5.0 mm) began to increase steadily until parturition.
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Comparison of embryonic development for four consecutive years A comparison of the pattern of embryonic development from 1999 to 2002 showed only a marginal variation from year to year. The time of mating showed some variation from one year to another. In 1999 mating was noted as early as mid-October, but in 2001 mating and ovulation were noted as late as early November. The timing of termination of delayed development was usually fixed in a narrow range of time (late December) from one year to another. The time of parturition and post-partum oestrus showed only a limited variation from one year to another.
Discussion The present study shows two periods of mating corresponding to two periods of ovulation in C. sphinx at Varanasi. Two distinct periods of parturition were also observed. The reproductive characteristics such as post-partum oestrus, alternate successive ovulations between the two ovaries and bicornuate uterus as observed in C. sphinx have also been reported in other bats with a seasonally dioestrous breeding pattern (Wimsatt, 1979; Rasweiler, 1982; Krishna and Dominic, 1982a; Heideman, 1989; Heideman and Powell, 1998). Ovulation by alternate ovaries appears to be essential in order for the females to conceive successfully at postpartum oestrus (Rasweiler, 1982). Although sperm transport is inhibited on the recently parturient side of the tract, conception can take place normally in the previously non-gravid uterus horn (Rasweiler, 1982). Similarly, by alternating ovulations the bat can successfully establish a pregnancy on the previously non-gravid side, while involution of the post-parturient horn is still in progress. The progestational reaction is localized in a small segment near the cranial end of the uterine cornua in C. sphinx during the oviductal journey of the embryo. The progestational changes are augmented in the cornu on the side of ovulation, whereas the contralateral cornu reverts to an anoestrous condition after implantation of the blastocyst. Implantation and pattern of early embryonic development in C. sphinx are similar to other Megachiropterans such as H. fischeri (Heideman, 1989), Otopteropus cartilagonodus (Heideman et al., 1993) and Ptenochirus jagori (Heideman and Powell, 1998). The initial attachment of the blastocyst in C. sphinx is superficial, but implantation is secondarily interstitial as is typically described in many chiropterans (Rasweiler, 1979; Heideman and Powell, 1998).
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Female C. sphinx at Varanasi became pregnant as early as late October and early November. However, post-gastrulation embryos were not found until late December. The majority of the females had a uterine swelling between 2 and 4 mm during November and December. This suggests that female C. sphinx either repetitively lose embryos and get reimpregnated or that their embryos were in some form of developmental delay. None of the females examined during November and December showed any degenerating embryos. Additionally, the ovary showed only one functional but no regressing corpus luteum. These observations suggest that C. sphinx undergoes a period of delayed development. The period of delayed development terminates in late December as the majority of females examined in early January contained embryos well past gastrulation. The marked variation in gestation length in the two successive pregnancies of the year in C. sphinx is rare but has been reported previously in Artibeus jamaicensis (Fleming, 1971), Taphozous longimanus (Krishna and Dominic, 1982b) and Carollia perspicillata (Rasweiler and Badwaik, 1997). In C. sphinx, the winter pregnancy has a duration of about 150 days and the summer pregnancy has a duration of about 125 days. The results of the present study suggest that the prolonged gestation length of winter pregnancy in C. sphinx is due to the slowed (delayed) embryonic development during the months of November and December. Because the longer gestation period in C. sphinx is associated with one particular period of the year, the delayed embryonic development in this bat may be due to seasonal factors. Delayed development may be broadly categorized into two types: environmental (temperature)-dependant and environmental (temperature)-independent. In species such as Pipistrellus pipistrellus, Myotis myotis and Corynorhinus rafinesquei, reduced environmental temperature significantly lengthens gestation by slow embryonic development (Eisentraut, 1937; Pearson et al., 1952; Racey, 1969). It is further demonstrated that in temperate latitude vespertilionid bats, food shortages during cold weather may be responsible for a reduced rate of conceptus development (Racey and Swift, 1981). In Artibeus jamaicensis a period of delayed embryonic development occurs during the height of the rainy season (Fleming, 1971). Since this form of embryonic diapause may occur depending upon environmental conditions, it is also referred to as facultative delayed development. In the California leaf-nosed bat, Macrotus californicus, delayed development lasts 4–5 months and is independent of temperature and food supply (Bradshaw, 1962). This is referred to as an obligate form of delayed development. The fruit bat Haplonycteris fischeri from the Philippines exhibits an obligate 8 months’ delay in embryonic development (Heideman, 1989). Delayed embryonic development has also been
demonstrated in Carollia perspicillata (Rasweiler and Badwaik, 1997), Otopteropus cartilagondus (Heideman et al., 1993), Ptenochirus jagori (Heideman and Powell, 1998), Rhinolophus hipposideros (Kolb, 1950), Natalus stramineus (Mitchell, 1965) and Hipposideros caffer (Bernard and Meester, 1982). It is apparent from the present study on C. sphinx that a prolonged delay period is not obligatory in this species, because females do not undergo prolonged developmental delays during the summer pregnancy. The facultative delay in this species may allow females to respond more rapidly to some environmental cue that predicts good conditions in the future (Heideman, 1988). In most mammals, the timing and duration of pregnancy are fixed by genotype and are highly resistant to alteration by environment. The features of embryonic development among Chiroptera, in apparent contrast to those of other mammals, are unique and varied. Most of the bat species with delayed embryonic development also exhibit a highly variable gestation length. A postimplantational delay in embryonic development up to 5 months was detected in young P. jagori females reproducing for the first time. However, adult females of this species showed little evidence of a delay in development. This causes marked variation in gestation length between young and adult P. jagori (Heideman and Powell, 1998). A remarkable degree of variation in gestation length was described in Carollia perspicillata (Rasweiler and Badwaik, 1997). Females caught in the wild have a gestation period that varies between 105 and 178 days (Rasweiler and Badwaik, 1997). Captive-reared females showed a variation in the gestation period of 113–159 days. The prolongation of pregnancy in some captive animals was suggested to be due to stress. So, delayed development in C. perspicillata occurs under two different circumstances: in response to stress in captivity and seasonally in the wild population. In C. sphinx gestation length varies from the winter pregnancy to the summer pregnancy. The histological studies of the genital tract of C. sphinx during early pregnancy indicate that the delay occurred after implantation. The majority of females observed during November and December contained embryos in the stage of gastrulation. At the beginning of the delay the embryos were in the early stage of amniogenesis, but towards the end of the delay embryos were in the stage of primitive streak and formation of mesoderm. In all bat species with delayed embryonic development which have been examined histologically there is evidence that embryonic development is nearly halted for a period of months in an early gastrulation stage, shortly after implantation (Heideman and Powell, 1998). During the delay embryos continue to grow, albeit at a retarded rate. During the delay period in C. sphinx, the length of the embryo increases partly due to growth and partly due to the formation of the amniotic
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cavity as described in H. fischeri (Heideman, 1989). It was recently hypothesized that the developmental delay may be caused by inhibition of one or more genes crucial for gastrulation or post-gastrulation development (Heideman and Powell, 1998). Whether the inhibition of genes is regulated by maternal factors and consists of either the lack of some necessary stimulatory molecular signal or production of an inhibitory signal by the mother needs further investigation. The variation in the length of gestation in C. sphinx may not be attributed to low temperature as the period of delayed development in this bat species does not coincide with the period of lowest environmental temperatures, which is in January. Neither does it coincide with the rainy season. Because the period of peak recruitment of young bats coincides with the period of fruit abundance at Varanasi, this suggests that parturition in C. sphinx may be related to seasonal food supply (Krishna and Dominic, 1983). It is thus possible that a delay period may allow females to time lactation and weaning to the most favorable period of the year. The present study on C. sphinx during four consecutive breeding seasons clearly showed that timing for termination of delay period and timing of parturition are nearly fixed despite much variation of the mating period. This suggests that the delay in this species may function to synchronize parturition among females (Heideman, 1988). A comparison of conditions during the first and second breeding cycles of the year in C. sphinx suggests that the period of delayed embryonic development in this species closely coincides with the period of fat accumulation. Fat deposition in this species was not observed during the summer pregnancy. The delay begins in November when fat also begins to accumulate whereas timing for termination of delay during late December coincides closely with the time when accumulated fat is completely metabolized. Whether the accumulated fat (adipose tissue) retards embryonic development is not yet known. It is well known that fat produces leptin and leptin has been shown to be a ubiquitous feature of pregnant mammals (Zhao et al., 2003). Leptin is known to affect fetal growth and development by binding to its receptor present in fetal organs (Ashworth et al., 2000). Whether leptin receptors are present in the gastrulating embryo and whether it is associated with the delayed embryonic development in C. sphinx needs further study. In summary, the results of this study suggest that the prolonged gestation length of winter pregnancy in C. sphinx was due to the slowed or delayed embryonic development during November and December. Delayed embryonic development occurred between early gastrulation and primitive streak stages. The analysis of the pattern of embryonic development during four consecutive breeding seasons clearly showed that the timing
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for termination of the delay period and timing of parturition are nearly fixed. This suggests that the delayed embryonic development in C. sphinx may function to synchronize parturition. The present study further noted a close association between the period of delayed embryonic development and the period of fat deposition in C. sphinx. The significance of fat deposition in delayed embryonic development warrants further investigation.
Acknowledgement This research was supported by the grant (DST (SP/ SO/C-33/99)) from the Department of Science and Technology, India.
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