2001

Asiatic Herpetological Research

Vol.9, pp. 1-5

The Reproductive Biology of Rana boulengeri
Wen-Jian Li
Laboratory of Economic Frogs, Changde Teachers College, Changde, Hunan, 415000 China
Abstract.- Maturation of Rana boulengeri Giinther occurs at the age of 24 to 36 months. After reproduction thegonads are in an intermittent period. Gonads begin to develop rapidly from next February to April. After May, theovocytes grow mature in batches. In the wild, the adult frogs often spawn under waterfalls or in shallow pools

where the water flows slowly, with high dissolved oxygen. The reproductive period lasts from May to August.
The ovulation time of females and fertilization time are generally from 5:00 a.m. to 8:00 a.m. The Water
temperature for deposition at the beginning is 15.5°C and the appropriate temperature for most deposition is17.7-22.5°C. The total duration under artificial constant temperature of 23±0.5°C lasts 261 hours. The
appropriate temperature for development is 22-24°C. This species can not develop below 4°C, and it dies at 30°C.

The development of the ovary is divided into 6 stages and the development of the testis is divided into 5 stages.
The morphological and histological studies at different stages of sex cells are described.
Key words.- Amphibia, Rana boulengeri, reproductive biology, China

Introduction

sea level; the lowest, 75 meters above sea level). The

Rana boulengeri is a large frog that is found in theWuling Mountains of Hunan Province. It is an important species for maintaining the ecological balance ofthe region (Yuan and Wen, 1990). Besides,

itcan beused as a kind of medicine (Li et al., 1993). In order tohelp protect this natural resource, these studies on its
reproductive biology have been carried out.

Material and Methods

mountains are covered with evergreen broad-leaf subtropical forest. The mean air temperatures for a wholeyear are 16.10 degree-days. The temperature summation above 10°C is 50320; the mean sun hours peryear are 1292. 7h; frostless period is 270.6 days; themean rainfall per year is 1397.2mm; the mean humidity

per yearisabove

82% (R.H). These conditions

constitute an ideal environment for Rana boulengeri.

Rana boulengeri

frequently

liveinbrooks orponds in mountain forests where there are few people.

Laboratory experiment and artificial breeding
The studies on the natural condition of habitat, growthand ages, characteristics of reproduction, the develop-ment of genital gland and development of the embryoof Rana boulengeri was conducted for four years
from 1989 to 1993.

The water in the brooks or ponds is very clear. During
the day, R. boulengeri often hide at the edge of small
caves and during the night, they go out to feed. From

July to September of 1987, the physical factors in 35observation points were measured. Their values are:the elevation of the habitat

is204-675 meters above

sea level; air temperature 17.2-24°C; water tempera-

Field investigation

The field sites chosen for investigation were places
where Rana boulengeri are common. Field observations were made two or three times a month.

ture 14.9-20.5°C; the depth of water 0.2-1.0 meters;

the transparency of water 0.2-1.0m; the humidity

92.0-97.5%.
Activities of Rana boulengeri and temperatures ofhabitat. Because the air temperature and water tem-Results and Discussion

perature play important roles in the development andHabitat

growth of R. boulengeri; we observed their relationship in a cave (450m above sea level). In autumn,

Natural habitat of Rana boulengeri. The Xiang Xi
mountains are a part of the Wuling Mountains, which
are located on the border of Hunan, Hubei, Sichuan
and Guizhou; between north Latitude 27°44'- 29°48'
and east longitude 109°10'-1 H"20'; with a mean elevation of 686 meters (the highest, 1900 meters above

when the water temperature fall lower than 12.5°C, R.
boulengeri begins hibernation. In spring, when watertemperature is above 12.5°C, they start to feed, andwhen water temperature is higher than 15.5°C, they
enter the reproductive period.

The air temperatures

and water temperaturesmonths are

Table 1.

inalllisted inVol. 9, p. 2

Asiatic Herpetological Research

Table 1 Air and water temperatures of the habitat of.

Plana boulengen (450m above sea level).

Month1

Mean air

Mean watertemperaturetemperature

2001

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Asiatic Herpetological ResearchVol. 9, p. 3

Late stage 4. Ovocyte cells filled with yolk: When

BW of 79.7-125.8 g and BL of 87.3-98.0

frogs havemm, the ovum cells are fully filled with yolk and thenuclei are isolated in the middle of the cells. The twolayers of follicle cells>
oand zona pellucida are wellmarked.
7

•BBW

are
Stage 5. Slanting of nucleus of oocyte: When
86.3-161.5 g and BL are 89.4-1 12.3 mm, the nucleus

9?

oeinclines to the side of animal pole. At thisstage, thea>oofemale can ovulate under the action of gonadotrophic<Uhormone.

o5Stage6.

Ovary

after ovulation:

When frogs bodyweights decrease to 70.6-102.5 g and body length
decrease to 82.1-100.2 mm. In this stage, corpora

3-lutea appear in ovary.

J10Figure 1.

LI

11121

J
2

Development of testis. The development of testis canl_L

III

345

67

89 month

The seasonal change of Mature coefficient

Eggs. The mean number of eggs spawned in eachbatch is about 218. The egg of R. boulengeri is bigwith a mean diameter of 3.98mm, enveloped by 3layers of gum membrane (The diameter of egg includ-membrane ismm). The outer layer is thethickest, the inner layer the thinnest. The outer layer is
15.85

very sticky, so thatStage 1. Multiplication period of spermatogonium: Atspermatogonium aggregates densely, andthis stage,of ovary (1991-1993)ing

be divided into 5 stages.

many eggs connect together toform a long cluster, the end of egg cluster is stuck onranges irregularly.

Stage 2. Formation of sperm-tube: In this stage, thespermatogonium is separated into sperm-tube by connective tissue of testis and part of spermatogonium insperm-tube becomes spermatocyte of the first order.

Stage 3. Differentiation of spermatocyte: In this stage,thenumberdecreases.

ofspermatogonium

insperm-tube

On the wall of sperm-tube, there are pri-mary sperm mother cells, secondary sperm motherand spermatophore formed by spermoblast

the wall or to ratty plants under water.

cells

Development of genital glands
Development of ovary. The

respectively.

developmentof ovary

can be divided into 6 stages.
Multiplication period of ovogonium: The
body weights (BW) of young frogs are 7.3-12.5 g andStage1.

Stage 4. Formation of sperm: This is the ripe stage ofthe testis. The spermatogonium and the spermatocyteof the first order are few on the wall of sperm-tube
instead of spermatocyte of the secondary order andnium cells, aggregating densely with the ability ofspermatophore formed by spermoblast respectively.
After the forming of spermatozoon, the spermatozoon

division, have not been surrounded by follicle cells.

aggregates densely first, then disperses.

the body lengths (BL) are 37.2-43.6 mm. The ovogo-Stage 2. Growth of ovocyte (oocyte): When theof young frog are 10.2-19.5 g and BL are 47.2-56.2

Stage 5. Post-ejecting: Most spermatozoons havebeen ejected and only a few could be found in themm, ovogonium cells develop into ovocyte cells. Thesperm-tube.

ovocyte clusters are divided by connective tissue of

Female order of ovary development and age at sex-

the ovary and surrounded by follicle cells.

ual maturity. After metamorphosis, the ovaries of theBW

Stage 3. Transition growth of ovocyte:

When BW ofyoung frog are 36.5-74.2 g and BL are 71.7-84.3 mm,thevolume of ovocyte cells increases. The follicle

cells increasefrom1to 2 layers

and the zona pellu-

young frogs reach stage

1in 2-3 months; stage 2 in 68 months; stage 3 in 10-13 months; early stage 4 in

21-24 months; late stage 4 and stage 5 in 33-36
months (if ecological conditions are very suitable, incida becomes clear.

only 22-24 months), when the females can spawn nat-

Early stage 4. Formation of yolk in ovocyte: When
young frogs weigh 58.7-80.8 g and their BL are 81.5-

reaches stage 6.

89.7 mm, oocytes begin rapid growth and particles ofMale order of testis development and age at sexual

yolk begin to form.

urallyorartificially.

After

spawning,theovaries

Vol. 9, p. 4

Asiatic Herpetological Research

2001

The whole development procedure is divided into 25
stags on the basis of both the morphological and thephysiological changes during this period (Pollister

1.2and Moore, 1937; Shumway, 1940). The temperaturesummation above 0°C for the embryo to complete thewhole development is 6004.15 degree-hours.

1.1

to&Experiments have shown

1.0thattemperature canaffect the developmental rate of the embryo of R. bou-clengeri. Under conditions of 13°C, 16°C, 22°C, 24°C,0.9

26°C and 28°C, the time for the embryo to completeCD'o0)

o0.8

development is 937.97h, 687.78h, 321.61h, 304.76h,
300. 18h and 295. 17h. In 2°C and 4°C water the0.7

embryo can not develop. In 7°C water the embryo
develops very slowly. In 30°C water, the embryo cannot complete development. The optimum tempera-0.6-tures for embryonic development are 22°-24°C (Li etoa)

gtiraal.,1994).

0.5

Acknowledgments
AJ

10 11 12Figure 2.

12III

345L
6 7I8I.

9 monthThe seasonal change of Mature coefficientof testis (1991-1993)

maturity. After metamorphosis, the testes of males
develop into stage 1 in 2-4 months; stage 2 in 8-10months; stage 3 in 14-18 months; stage 4 in 24-26

months. At

this stage themales can mate with theMy research has been supported by the Asiatic Herpetological Research Society. I would like to thank Pro-fessor Er-mi Zhao (Chengdu Institute of Biology) forguiding my research and revising my paper. Theauthor wishes to thank Changde Teachers' College forits

financial support.

Literature Citedfemales.

Hu-Ming, Wen-Jian Li, Xing-Guo Gong, andMing-You Ma. 1993. Studies on the nutritional compositions and energy content of the flesh of Rana bouLi,

Type of spawning. In the sexually mature females, 34 grades of oocyte can be seen. This is the cytological
proof of multi-oviposition of Rana boulengeri.

lengeri. Zoological Research, Kunming, 14(1 ):96.

Seasonal change of ovaries. The period from May toisAugust

the reproductive time of R.

boulengeri.
Then the ovaries develop slowly and the mature coefficientof ovary(MCO) decreases to3.10-3.35%.

From February to April of the second year, the ovaries
develop fast and the(5.73-14.5%).

8.47%

MCO reaches

The mean(Fig.l). After

value ofits

highest value

MCO in May isMay, the ovocytes mature

inbatches.

Seasonal change of testes. ThetesteschangelittleLi, Wen-Jian,

Hu-Ming Li, and Ming Li. 1993. Pre-liminary studies on the artificial breeding of the young
frog of Rana boulengeri Giinther. Pp. 255-259. InZhao, Chen and Papenfuss (eds.), Proceedings of theFirst Asian Herpetological Meeting. China Forestry
Press, Beijing.
Li, Wen-Jian and Ming Li. 1993. The early embryonicdevelopment and stages of Rana boulengeri. Pp. 124133. In Zhao, Chen, and Papenfuss (eds.). Proceedingof the First Asian Herpetological Meeting. China For-during the different seasons. The range of change ofmature coefficient of testis (MCT) in a year is 0.55-

estry Press, Beijing.

1.14%. From April to July, testes develop faster thanature on the earlyin other months. In July theber theMCT is 1.14%. In Octo-

MCT decreases to 0.55% (Fig.2).

Li, Wen-Jian and Hu-Ming Li. 1994. Effect of temper-eth anniversary of the founding of China ZoologicalSociety.

Embryonic development
Li et al. (1993) reported in detail on the embryonicdevelopment of R. boulengeri. Under temperature
conditions of 23±0.5°C, 261.05 ± 0.54 hours wererequired for the embryo to complete its development.

embryonic development of Ranaboulengeri. Pp. 508-512. In Proceedings of the sixti-ChinaScienceandTechnologyPress,Beijing.
Pollister, A.

W andJ. A.

Moore. 1937. Tables for thenormal development of Rana sylvatica.. The Anatomical Record 68:489-496.

2001

Asiatic Herpetological Research

Shumway W. 1940. Stages in the normal developmentof Rana pipiens. The Anatomieal Reeord 78: 39- 47.
1

1Yuan. Feng-Xia and Xiao-Bo Wen. 1990. A preliminary study on living and feeding habits of Rana boulengeri in western Hubei Province. Chinese Journal ofZoology. Beijing 25(2)17-21.

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