Asiatic Herpetological Research

April 1990

Vol. 3, pp. 85-100

Studies of the Early Embryonic Development of Rana rugulosa

jionghuaPan and Danyu Liang1Wiegmann

1^Department of Biology, South China Normal University, Guangzhou, Guandong, China-This paper deals with the early embryonic developmental stages of Rana rugulosa
and with methods of artificial fertilization and experimentally accelerated development.
Developmental stages are distinguished by morphological changes and obvious physiological features. At aAbstract.

Wiegmann,temperature of 25.1 to 27°C it takes 70 hr 20 min to complete development from fertilized eggs to tadpoles
with opercular folds. This course of development is divided into 25 stages, which are standardized withnormal table equivalents.

Key Words: Amphibia, Anura, Ranidae, Rana rugulosa, embryonic development, artificial fertilization,
accelerated development, fertilized eggs, normal table.

Introduction

Rana rugulosa Wiegmann, whose
popular name is the Field Chicken inChina, is strong and big of body. It isfamous for its delicacy of meat and theDomestic and foreign
price is dear.
markets are in great need of it. The growthand development of these frogs arerelatively quick. Hatchlings can become
sexually mature in one year. The average
body length is about 1 10 mm. The weight
is about 0.25 kg.
Consequently, Ranarugulosa farming has been a growing
More papers deal with theenterprise.
embryonic development of R. guentheri
Boulonger and R. catesbeiana. Currently
there are few domestic and foreign studies
on the embryonic development of R.

In order toproduce referencematerial for the captive breeding of thisrugulosa.
frog,

westudiedits

earlyeggs. The water temperature of the six
developing embryos was 25.1 to 27°C; thepH was 6.5-6.8. Several minutes after
oviposition and ejaculation or artificial
fertilization,

we put the oocyte into thelaboratory. Then we used a microscope toobserve the course of development and tomeasurethesizeshape.

TheFor every developmental stage we took
10 to 20 embryos and fixed them in 5%
formalin and Bouin's fluid. This material
was used in drawings, tissue-slices, andphotomicrographs. The figures shown
here were taken from observations of livingmaterial.

Results

embryonic

development, using artificial fertilizationand higher temperatures to accelerate
development experimentally, during themonths of April and June, 1987 and 1988.

anddevelopmental stages were defined asbeginning when half of the embryos in asample showed all of the distinguishing
characters of that stage.

The 25 stages of the early embryonic ofRana

are as follows

tigrinaparentheses

is(time inhours and minutes afterfertilization):

Methods
I) Oocyte stage.

We made1 1

observations ontheembryonic development of Rana rugulosa.
Six of the 11 were artificially fertilized.
The parent frogs were bought inThe description of theGuangzhou.
features of embryonic development

wasmainly based on the six artificially fertilized© 1990 by Asiatic Herpetological Researchmm

Unfertilized eggs are

in diameter (Fig. 26).
spheres 1.4-1.7(00:25-00:30): The egg membrane absorbs
water and expands. The eggs reach 2.6in perivitellic space. (00:50): The
4.0
pigment crown of the animal pole extends
downward upon the gray crescentmm(Fig. la, lb, lc, Id, le, 26).

Asiatic Herpetological ResearchVol. 3, p. 86(01:12): The firstlongitudinal cleavage progresses and acleavage furrow in the animal pole appears,2) 2 cell stage.

April 1990

on the side of the lip are drawn into thefuture archenteron and a semicircle appears(Fig. 11a, lib, 36).

forming 2 equal hemispheres (Fig. 2a, 2b,27).

A cleavagepole appears,
perpendicular to the first cleavage furrow.
It progresses to the vegetal pole and forms3) 4 cell stage.
furrow in the(01:33):

animalfour equal sections (Fig. 3a, 3b, 28).
4) 8 cell stage.

(01:42):The thirdcleavage furrow appears parallelto the(08:05): The12) Late gastrula stage.
blastopore shrinks and closes gradually.
Eventually, the yolk plug is enclosed in theembryo (Fig. 12a, 12b, 12c, 12d, 37)(10:30): Theabecomes
and thefissure,
blastopore
toelongate along theembryo begins
axis,longitudinal
becoming pear-shaped13) Neural plate stage.

(Fig. 13a,.13b, 38).

equator, approximately bisecting the animal

hemisphere, forming four small animalcells and four large vegetal cells (Fig. 4a,4b, 29).
(01:48): Observed fromthe animal pole, the fourth cleavage forms5) 16 cell stage.

eightblastomeres in two circular or

elliptical tiers (Fig. 5a, 5b, 30).

6) 32 cell stage.
cleavage is horizontal.

(02:05): The fifth
The animal pole has

eight small cells; the vegetal pole has 8large cells (Fig. 6a, 6b, 31).
(02:23): The
the large cell blastula stage,

7) Early blastula stage.

embryois at

but the cells are still clearly distinguished.

The blastocoel begins to appear in themiddle part of the embryo near the animalA(11 :50):
14) Neural fold stage stage.
fold appears on both sides of the neuralplate. The neural groove forms the mediansinus of the embryo. The front of the plateis bigger, and the neural fold gradually
approaches the dorsal axis from both sides.

The embryo elongates to 1.9 mm (Fig. 14a,14b, 39).

15) Cilial movement stage.
(12:40): The
neural folds are joining.
The embryo
rotates within the vitelline membrane (Fig.
15a, 15b, 40).

16) Nerve tube stage. (13:45): The neuraltube has formed and the gill plate and thecement gland can be seen (Fig. 16a, 16b,41).
(14:55): Two outergill buds protrude on the side of each gill17) Tail bud stage.

pole (Fig. 7a, 7b, 32).

many small blastomeres. The blastocoel

The total length is 3.0-3.6 mm, andthe length of the tail bud is 1/10 to 1/7 ofcontinues to amplify (Fig. 8a, 8b, 33).

this (Fig. 17a, 17b, 17c, 42).

Late blastula stage.
(03:18): Theblastomeres are the color of a red bayberry.

Muscular

The cell boundary becomes indistinct andindividuals.

8) Mid-blastula stage.

(02:50): There are9.

the blastocoel

expands to its maximum(Fig. 9a, 9b, 34)

plate.

18) Muscle effect stage.

mostbegins
The olfactory organ appears,

response

the cement gland is complete. Total length
3.3-3.8 mm., and the length of the tailis

bud is10) Early gastrula stage.

(15:55):inWof this (Fig. 18a, 18b, 18c, 43).

(05:56): The

pigmented crown epiboly of the animal cap
occurs, extending to cover over 75% of the19) Hatching stage. (16:45):

The embryo

embryo. The dorsal lip forms. Involution

hatches from the egg membrane and twoouter gill budlets with 3-5 branchesof cells begins (Fig. 10a, 10b, 35).

protrude obviously (Fig. 19a, 19b, 19c,19d, 44).

11) Mid-gastrula stage.
(07:00): The
dorsal lip continues to amplify. The cells20) Heart beat stage.

(29:25):The heartAsiatic Herpetological Research

April 1990

begins to move, the eyes protrude the pairof otoliths can be seen, and the 3rd external
gill matures (Fig. 20a, 20b, 20c, 45).
,TABLE

1.

Normalandembryo. Body length is 5.8-6.2 mm. Tailfin length is half of the body length (Fig.
21a, 21b, 21c, 46).

22) Tail fin blood circulatory stage.
The cement gland begins to(32:00):
degrade, circulation begins in the tail bud,and tadpoles are able to swim in a straightline (Fig. 22a, 22b, 22c, 47).

23) Gill opercular fold stage. (35:55):
The opercular fold appears in the base of
the external gill and the intestines form abow (Fig. 23a, 23b, 23c, 48).
24) Right side operculum closed stage.
The opercular fold stretches(55:30):
toward the right side, the right external gillis covered and forms the right internal gill.

The left external gill is exposed.
intestines have 2-3 twists.

6-9TheTotal length ismm (Fig. 24a, 24b, 24c, 49).

25) Operculum completion stage. (70:20):
The external gill in entirely enclosed in thegill cavity, the posterior of which has anexhalent pore to the left side. The yolk has
almost been absorbed, and the tadpoles
begin feeding. Total length is 6.8-10.5(Fig. 25a, 25b, 25c).

mmshows N & F normal tableequivalents (Nieuwkoop and Faber 1967)
Table

1of our results.

Discussion

The circumstances of the earlyembryonic development of Rana tigrina, R.
catesbeiana, R. Umnochiris, and R.
1.

nigromaculata basically identical, but R.
rugulosa has some minor differences indevelopmental timing.

tableequivalentsFaber 1967) of theofRana rugulosa as listeddevelopmental stages(Nieuwkoop
in this paper.

The21) Open mouth stage. (31:10):
membrane covering the mouth splits toshow the mouth cavity. The alimentary
canal is complete and body segments areobvious.
The inverted "V" shape
myomeres appear on the side of theVol. 3, p. 87

N & F stages

Asiatic Herpetological ResearchVol. 3, p. 88

minutes after fertilization,crescentof Rana rugulosa
greyThis
isidenticalto the timing ofappears.
R. catesbeiana, but different from that of R.

April 1990

References

3. Fifty-fivethenigromaculata, in which the grey crescentcan barely be seen, or is not visible.
4. Thedevelopment of Rana rugulosa

from the eight cell stage to the 16 cell stage
takes six min, while the same period takes

59 min in R. catesbeiana.
5. In

Rana rugulosa,external

by Rana rugulosa adhere

mass.

Zoology, Beijing. 2:49-50. (In Chinese).

CHENGDU INSTITUTE OF BIOLOGY 1977. [KeytoAmphibians of China].

Science Press,Beijing, China. 93 pp. (In Chinese).

GU, S. AND J. LE.

1986.

[Animal Embryology].
(IngillA

reason for low hatching success is thatoxygen levels can be low in the center ofegg[Hasten parturition and artificial

method of frogs]. Chinese Journal ofChinese).

in a pile or on the surface of the water.

the1980.

fertilize

People's Education Press, Beijing, China.

develops 3-5 branches during the period ofhatching, while in R. catesbeiana, theexternal gill develops 2-3 branches during
the tail bud circulatory stage. The third
external gill appears sooner in R. rugulosa
than in R. catesbeiana or R. guentheri.
6. Eggs laid

CAI, M.

Accordingtoourobservations, eggs at the edge of a mass areeasy to hatch. If we separate the masses
early enough, adequate dispersion can beachieved. However, the operation must be
a careful one, or the egg membranes will be

broken and the proper development of theembryo can be affected, causing deformityand death.

LIU, C. ANDS.

HU.

Salientia in China].

1961.
[The TaillessScience Press, Beijing,China. 364 pp. (In Chinese).

NIEUWKOOP, P. D. AND J. FABER.

1967.

Normal table oiXenopus laevis. North Holland
Publishing Co. Amsterdam.

SHANDONG FISHERIES INSTITUTE.

1966. [TheRanaofcatesbeiana.]
embryonic development
Chinese Journal of Zoology 1966(4): 182-185.
(In Chinese).

ZHANG, J., J. LIU, AND M. CAI.

1985. [A study
on the seasonal variation in the ovary and thereproductive frequency in Rana limnochiris].
Acta Herpetologica Sinica 1985, 4(4):276-281.
(In Chinese).

Asiatic Herpetological Research

April 1990

Fig. 1AFig.

IDIHI»

Fig. 2 Bmmmm

Vol. 3, p. 89

Fig. 1 B

Fig. 1 E

Fig. 2A1Fig. 3A

Fig. 3 B

FIG. la, lb, lc, Id, le, 2a, 2b, 3a, 3b. Embryonic development of R ana rugulosa. See text fordetails.

Asiatic Herpetological ResearchVol. 3, p. 90

Fig. 4A

Fig. 4 BlFig. 5 B

Fig. 7A

April 1990

Fig. 5A

1Fig. 6A

Fig. 7 B

Fig. 6 B

Fig. 8A

FIG. 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a. Embryonic development of Rana rugulosa. See text fordetails.

Asiatic Herpetological Research

April 1990

Fig. 8 B

Fig. 9I

Fig. 10A

Fig. 1 1 BA

Vol. 3, p. 91

Fig. 9 B

mm

Fig. 10 B

Fig. 12A

Fig. 1 1A

Fig. 12 B

FIG. 8b, 9a, 9b, 10a, 10b, 11a, lib, 12a, 12b. Embryonic development of Rana rugulosa. Seetext for details.

Asiatic Herpetological ResearchVol. 3, p. 92

Fig. 12C

Fig. 12I

HFig. 13Fig. 14AA

April 1990

Dmm
1Fig. 13 B

Fig. 14 B

FIG. 12c, 12d, 13a, 13b, 14a, 14b. Embryonic development of Rana rugulosa. See text for details.

Asiatic Herpetological ResearchApril 1990

Fig. 15A

Fig. 15 BImm

IFig. 16

1

A

Fig. 16 B

Fig. 17I

Fig. 17 B

Vol. 3, p. 93

iA

mm<Fig. 17C

FIG. 15a, 15b, 16a, 16b, 17a, 17b, 17c. Embryonic development of Rana rugulosa. See text fordetails.

Asiatic Herpetological ResearchVol. 3, p. 94

Fig. 18 B

Fig. 19

A

Fig. 19

C

Fig. 182mm

mm

Fig. 20

Fig. 19

DFig. 20

CA

Fig. 20 B

FIG. 18a, 18b, 18c, 19a, 19b, 19c, 19d, 20a, 20b, 20c.
rugulosa. See text for details.

CFig. 19 B

1iApril 1990

Embryonic development of Rana

AsiaticApril 1990

Herpetological Research

Fig. 21

Fig. 21Vol. 3, p. 95

AB

Fig. 21m m

Fig. 22

CA

Fig. 22 B
Fig. 22

C

Fig. 23 B

Fig. 23

FIG. 21a, 21b, 21c, 22a, 22b, 22c, 23a, 23b, 23c.
See text for details.

CEmbryonic development of Rana rugulosa.

Asiatic Herpetological ResearchVol. 3, p. 96

Fig. 24

April 1990

AFig. 24 B

Fig. 242mmiC«

Fig. 25A

Fig. 25 B

Fig. 25C

FIG. 24a, 24b, 24c, 25a, 25b, 25c. Embryonic development of R ana rugulosa. See text for details.

April 1990

Asiatic Herpetological Research

FIG. 26 (X30)

FIG. 27 (X30)

FIG. 28 (X30)

FIG. 29 (X30)

FIG. 30 (X30)

FIG. 31 (X30)

FIG. 26, 27, 28, 29, 30, 31. Embryonic development of Rana rugulosa. See text for details.

Vol. 3, p. 97

Vol. 3, p. 98

Asiatic Herpetological Research

April 1990

FIG. 32 (X30)

FIG. 33 (X30)

FIG. 34 (X30)

FIG. 35 (X30)

FIG. 36 (X30)

FIG. 37 (X30)

FIG. 32, 33, 34, 35, 36, 37. Embryonic development of Rana rugulosa. See text for details.

April 1990

Asiatic Herpetological Research

FIG. 38 (X30)

FIG. 39 (X30)

FIG. 40 (X30)

FIG. 41 (X30)

FIG. 42 (X30)

FIG. 43 (X20)

FIG. 38, 39, 40, 41, 42, 43. Embryonic development oiRana rugulosa. See text for details.

Vol. 3, p. 99

Vol. 3, p. 100

Asiatic Herpetological Research

April 1990

FIG. 44 (X20)

FIG. 45 (X15)

FIG. 46 (X15)

FIG. 47 (X15)

FIG. 48 (X10)

FIG. 49 (X10)

FIG. 44, 45, 46, 47, 48, 49. Embryonic development oiRana rugulosa. See text for details.