Omar, R., Ali Rahman, Z., Latif, M.T., Lihan, T. and Adam J.H. (Eds.

)
Proceedings of the Regional Symposium on Environment and Natural Resources
10-11th April 2002, Hotel Renaissance Kuala Lumpur, Malaysia. Vol 1: 720-728
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SPERM COUNT, HORMONE CONCENTRATION AND HISTOLOGICAL

STUDY OF DOG-FACED FRUIT BAT, Cynopterus brachyotis,
IN AN AGRICULTURAL AREA

Wong C.H., Tay T.W., Marina M.T., Zubaid A.1 and Kamis A.B.

School of Biosciences & Biotechnology,

1
School of Environmental & Natural Resource Sciences,
Faculty of Science & Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor.
E-mail: acu4179@tm.net.my

ABSTRACT

This study was conducted at an agricultural area (Kuala Perlis, Perlis) for 12 months
from April 2000 to March 2001 to gather information on the reproductive cycle of
the dog-faced fruit bat, Cynopterus brachyotis. Sampling on bats was carried out
using mist-nets. The relationship among sperm concentration, changes in
testosterone and progesterone concentration and reproductive organs were used to
assess the reproductive cycle of the dog-faced fruit bat. It was found that Cynopterus
brachyotis in Kuala Perlis is seasonal and continuous bimodal polyestry
corresponded to the fruiting season. Analysis of testosterone concentration revealed
significant difference in spermatogenically active and non-active males bats. The
progesterone concentration increased during pregnant period. Adult females of this
species produce 2 litters per year and 1 neonate per birth. The females are pregnant
twice a year with pregnant seasons from May to July and September to October.

INTRODUCTION

The dog-faced fruit bat or also known as lesser short–nosed fruit bat (Cynopterus
brachyotis) is probably the commonest and most widespread fruit bat in tropical Asia
(Legakul & McNeely 1977; Phua & Corlett 1989). It occupies most available habitats:
coastal, urban, agricultural, riverine and all types of forest up to 1500 m altitude.
Cynopterus brachyotis is a medium-sized bat (body weight mean 35 g) which typically
roosts in small group in trees, particularly under the fronds of palms. It appears to be an
important seed dispersal agent due to it widely distribution and also an important in
pollination as it feeds on nectar (Funakoshi & Zubaid 1997; Phua & Corlett 1989).

Increasing development in industries, urbanization, animal husbandry and agriculture are

affecting bat populations. If these man-made disturbances prevail without any
perturbations, it will lead to bat populations being threatened with extinction due to
habitat loss, decreasing food resources, pollution, deliberate killing and loss of genetic
diversity (Meffe & Carroll 1994; Cox 1997). Bats play an important role in ecosystem
balancing and human activities such as arthropod control, pollination and seed dispersing,
providing fertilizer and food source, and etc. (Hill & Smith 1984). It is therefore
important to document and understand the reproductive biology of bats that may be
crucial in conservation of this diverse group of mammals.

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There have been many studies of bat reproduction in the tropics (Kofron 1997). In
contrast, relatively few papers concerning the comparative of reproduction cycle of
Cynopterus species have been published (Lim 1970; Funakoshi & Zubaid 1997). Besides,
there are limited publications on the histological study of reproductive organ and plasma
steroids of Cynopterus brachyotis (Crichton & Krutzsch 2000). Thus the present study
was carried out to measure sperm concentration, the changes in testosterone and
progesterone concentration, and reproductive organs to study the reproductive cycle in
this bat.

MATERIALS AND METHODS

Cynopterus brachyotis bats were captured from agricultural area (100° 8.5’E, 06° 26.2’
N) in Kuala Perlis, Perlis. Sampling was done monthly from April 2000 to March 2001.
The specimens were captured by mist-nets. The specimens were killed by chloroform,
their reproductive organs – testes and ovaries dissected out. The weight, length (mm) and
width (mm) of the testes and ovaries were measured. Microscopic observation of the
organs was made on sectioned tissues stained with Wiegert’s haematoxylin and
counterstained with eosin.

Spermatozoa were obtained by teasing the epididymides in 1000 µl phosphate buffer

saline (PBS) medium. The concentration of spermatozoa was accounted by using a
haemacytometer. Sperm count method was based on the guidelines of WHO (2000).

Blood samples from bats were obtained from the heart region. Serum or supernatant was
obtained and analyzed with using Testosterone Enzyme Immunoassay Kit and
Progesterone Enzyme Immunoassay Kit (Diagnostic Systems Laboratories, Inc.) for
testosterone and progesterone respectively. Data were expressed in the form of mean ±
standard error. Statistical tests were done using Analysis of Varians and Duncan Multiple
Range Test from Statistical Analysis System (SAS) version 6.12 software. This was done
to detect if there were any significantly differing values of the parameters measured.

RESULTS

The weight of bats in Kuala Perlis increased from June (25.50 ± 0.87 g), peaked in
August (34.75 ± 1.36 g) and decreased in September (31.29 ± 2.62 g) and seemed to
increase again in December (28.67 ± 5.46 g) (Table 1). The highest testes weight value
for Cynopterus brachyotis in the Kuala Perlis was 0.2708 ± 0.0918 g in August. The
changes of sperm concentration are showed in Figure 1. The highest value was in June
(10.29 ± 7.25 x104 cell/ml). There were no presence of sperm detected in April, May,
September, November and December.

Table 2 shows the measurements of female bats. The highest body weight recorded was
in December (41.83 ± 1.58 g). The ovary weight of female bats in Kuala Perlis increased
from May (0.0039 ± 0.0010 g), peaked in July (0.0097 ± 0.0014 g) and decreased in
August (0.0043 ± 0.0013 g) and seemed to increase again in October (0.0050 ± 0.0007g).

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Table 1 also showed the changes of testosterone concentration in males. The highest
value was in July (6.73 ± 2.55 ng/ml). Analysis of variance showed significant difference
in spermatogenically active and non-active male bats. For females, the highest
progesterone concentration was in January (59.20 ± 8.46 ng/ml) (Table 2).

Figure 1 shown an active stage of the testes obtained in January. There were no
spermatozoa in the seminiferous tubules (Figure 2). The lumen of the tubules can hardly
be seen as they were covered with spermatozoa remnants. This abrupt change in the
absence of spermatozoa occurred throughout the sampling period on the inactive male.

Figure 3 showed an ovary with maturing primary follicles (PF) and secondary follicles
(SF). In Figure 4, a corpus luteum (CL) was observed. Based on the observation of
dissected pregnant females bats, it was found that all implantations were occurred on
either side of the uterus. In Kuala Perlis, most of the captured females were pregnant
during March, April and May, and in November, December and January high percentage
of females were pregnant.

DISCUSSION

Body and testes weight of Cynopterus brachyotis fluctuates throughout the year. In some
species fluctuation of body weight can indicate times of food abundance and scarcity
(Medway 1972). According to McGuckin & Blackshaw (1991a), assessment of changes
in testicular size in individuals provided information on the cycle of seasonal testicular
changes. The total weight of the testes showed peaks in August. The increased of weight
was about 1-2 months after the higher food availability. Then, the weight of the testes
increased again after November. Thus, there is a seasonal testicular change in males. This
finding was consistent with Churchill (1994), who found that during the period of high
food availability in the wet season, male Rhinolophus aurantius accumulate fat,
particularly around the testes. For some frugivorous bats (e.g. Eldon helvum, Pteropus
giganteus), the time of testis enlargement has been shown to be the time of higher sperm
production (Mutere 1967).

The range of sperm concentration was between 1.70-10.30 x 104 cell/ml and the highest
value was in June. This species had a much smaller minimum and maximum sperm range
compared to sheath-tail bat Molossus fortis. M. fortis had a minimum range of 3 x 104
cell/ml in spring and a maximum of 463 x 104 cell/ml in autumn (Jolly & Blackshaw
1987). Hosken (1998) found that maximum testes size was associated with maximal
spermatogenic activity.

Males C. brachyotis experienced two high spermatogenic activities in January to

February and second one was between June to August. Pregnant female Cynopterus
brachyotis were captured during every month in Kuala Perlis suggesting that breeding is
non-seasonal (Lim 1970; Medway 1983; Funakoshi & Zubaid 1997). These data also
indicated there were two distinctive birthing seasons which one about May to July and a
second about September to October. Kofron (1997) found that the C. brachyotis in Brunei
gave birth in two seasons, one about mid January to mid April and a second about mid
June to early October. Funakoshi and Zubaid (1997) found that C. brachyotis in Ulu
Gombak may produce two or three young each year.

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Table 1: Measurements and Duncan Multiple Range Test of body and testes weight,
sperm count and testosterone of male Cynopterus brachyotis in Kuala Perlis
from April 2000 to March 2001. (Mean ± S.E with the same letter are not
significantly different for each column).

Sperm Testosterone
Month Body weight (g) Testes weight (g) concentration concentration
(X104 cell/ml) (ng/ml)

April 33.17 ± 2.21ab 0.0600±0.0209c - 2.59 ± 0.44bc

May 30.00 ± 1.69cd 0.0529±0.0264c - 2.81 ± 0.58bc
June 25.50 ± 0.87bc 0.1346±0.0410abc 10.29±7.25a 3.84 ± 0.46abc
July 26.73 ± 1.41bcd 0.1744±0.0254abc 3.18±1.02ab 3.86 ± 0.26abc
August 34.75 ± 1.36a 0.2708±0.0918a 7.14±1.64ab 3.37 ± 0.65bc
September 31.29 ± 2.62abc 0.0744±0.0280c - 3.25 ± 0.36bc
October 29.29 ± 1.55abcd 0.1038±0.0390c 1.71±0.61b 3.22 ± 0.58bc
November 25.33 ± 0.33bc 0.0798±0.0466c - 2.80 ± 0.71bc
December 28.67 ± 5.46abcd 0.1856±0.0883abc - 3.64 ± 1.02abc
January 28.50 ± 1.88abcd 0.1972±0.0189abc 4.87±1.29ab 4.89 ± 0.15ab
February 27.60 ± 0.75abcd 0.2476±0.0405ab 8.63±3.78ab 5.31 ± 0.34a
March 23.00 ± 4.00d 0.1297±0.1040abc - 3.57 ± 0.87abc

Table 2: Measurements and Duncan Multiple Range Test of body and ovary weight,
and progesterone concentration of female Cynopterus brachyotis in Kuala
Perlis from April 2000 to March 2001. (Mean ± S.E with the same letter are
not significantly different for each column).

Progesterone
Month Body weight (g) Ovary weight concentration
(ng/ml)

April 25.50 ± 1.85c - -

May 32.20 ± 3.06bc 0.0039±0.0010c 22.40±9.30b
June 31.75 ± 2.60bc 0.0075±0.0010abc 12.06±3.12b
July 33.63 ± 2.30b 0.0097±0.0014ab 39.20±9.98ab
August 28.40 ± 1.50bc 0.0043±0.0013c 21.60±10.99b
September 33.80 ± 1.91b 0.0054±0.0020bc 14.10±6.74b
October 30.20 ± 2.35bc 0.0050±0.0007c 23.81±2.86b
November 27.40 ± 1.94bc 0.0083±0.0007abc 25.58±2.06b
December 41.83 ± 1.58a 0.0102±0.0017a 10.80±1.87b
January 30.60 ± 2.36bc 0.0073±0.0006abc 59.20±8.46a
February 29.67 ± 0.67bc 0.0066±0.0006abc -
March 25.67 ± 0.84c 0.0077±0.0010abc 28.80±7.30b

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Figure 1: Section of a testis (Cynopterus brachyotis) some lumens filled with

spermatozoa (Mag: 10x10), H + E staining.
Lumen (L), seminiferous tubule (ST), spermatozoa (S)

Figure2: Section of a testis (Cynopterus brachyotis) showing lumens filled with

spermatozoa remnants, testis not active (Mag: 10x10) H + E staining.
Lumen (L), seminiferous tubule (ST)

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Figure 3: Section of an ovary (Cynopterus brachyotis) showing primary follicles (PF)

and secondary follicles (SF) (Mag: 10x10), H + E staining

Figure 4: Section of an ovary (Cynopterus brachyotis) showing a corpus luteum (CL)

(Mag: 10x4), H + E staining

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According to McGuckin and Blackshaw (1991b), the testosterone is important for

testicular development and mating activity and accessory development. The large increase
in plasma testosterone during the mating appears to be due to increased testicular
production. The hormone concentration of spermatogenically active bat was around 2.92
ng/ml and while for the inactive individuals was around 1.42 ng/ml.

The two major sources for progesterone in bats are the corpus luteum and the placenta
(Crichton & Krutzsch 2000). Thus, the increase of progesterone concentration would
indicate breeding season. From the study, progesterone concentration of females
Cynopterus brachyotis reached higher value in January, March, May, July and November.
The high hormone concentrations in April and May were coincided with the peak
pregnant seasons of the bats which were between March to May and as well as December
to January from observation.

The increase in plasma progesterone concentration after fertilization reported in the

present study is not unusual and has been reported in the intermediate-roundleaf bat
(Hipposideros larvatus: Marina 2001), Schreibers’s long-fingered bat (Miniopterus
schreibersii: Bernard et al. 1991). In Schreibers’s long-fingered bat (Miniopterus
schreibersii), plasma progesterone concentration was low during non-pregnancy (1.54
ng/ml) and during delayed implantation (1.67 ng/ml), and thereafter increased to reach a
peak mean of 64.82 ng/ml in late pregnancy (Bernard et al. 1991).

Histological examination of the testes showed that male Cynopterus brachyotis was in a
spermatogenic stage throughout the sampling period. During spermatogenesis activity,
presence of spermatozoa was observed in the seminiferous tubules and ductus
epididymides.

Based on observation, female bats of both species in this study had a bilaterally functional
reproductive system and pregnant may occur on either side of the uterus. According to
Ramakrishna (1950), they found that the bat C. sphinx also showed no dominance of
either ovary. In contrast, the ovaries were equal but alternate in successive cycles.
Wimsatt (1979) however, described this as a “pteropid cycle” which involved a non-
random alternation of ovulations between right and left ovaries in successive cycles.
Some species displaying this cycle are seasonally polyoestrous.

ACKNOWLEGEMENTS

Financial support for this study was provided by the Ministry of Science, Technology and
Environment through IRPA 09-02-02-0148.

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