The effect of administration of HCG (Human Chorionic Gonadotropin) on ovulation of Fugu niphobles

was studied. The female fish which exhibited distended abdomen were randomly assigned to four groups and

the treatments for groups 1, 2, 3 and 4 were 0, 100, 200 and 300 I. U of HCG per fish respectively. The

fish were injected immediately after the groups were established and unovulated females were reinjected after

72 hours. The dosage for each fish was calculated based on the body weight and expressed as 1.U/g body weight.

The ovulatory success in fish administered with 0, 1, 2, 3 and 41. U of HCG/g body weight was 40%, 83 %,

70%, 90% and 80% respectively. This indicated that fish in the control group could also ovulate in captivity

and that administration of HCG would enable a higher degree of ovulatory success

Cheah, S. H.

Satoh, H.

Universiti Putra Malaysia Institutional Repository

Pertanika 12(3), 323-328 (1989)Trials on Induced Ovulation of Fugu niphobles(Jordan and Snyder) with Human Chorionic GonadotropinS.H. CHEAHDepartment of Fisheries Biology and Aquaculture,Faculty of Fisheries and Marine Sciences,Universiti Pertanian Malaysia,43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.H. SATOHFisheries Laboratory,Faculty of Agriculture,University of Tokyo,Bentenjima, Shizuoka Prefecture 431-02, Japan.Key words : Induced ovulation, hormone, Fugu niphobles.ABSTRAKKesan penggunaan HCG (Human Chorionic Gonadotropin) untuk ovulasi Fugu niphobles telah dikaji.Ikan betina yang buncit abdomennya dibahagikan kepada empat kumpulan 1, 2, 3 dan 4, dan dirawatdengan hormon HCG pada takaran masing-masing, 0, 100, 200 dan 300 1. U. Selepas ikan betinadikumpulkan mengikut kumpulan, ikan-ikan disuntik dan ikan yang didapati tidak berovulasi disuntiksemula selepas 72 jam. Takaran suntikan dikira selepas berat badan betina diambil kira dan takarandiberikan sebagai 1. U/g berat badan. Data kejayaan ovulasi untuk rawatan 0, 1, 2, 3 dan 41. U/g beratbadan masing-masing, 40%, 83%, 70%, 90% dan 80%. Ini menunjukkan bahawa ikan dalam kumpulankawalan iaitu kumpulan yang tidak menerima suntikan hormon dapat berovulasi dalam keadaan kurungandan penggunaan HCG dapat meninggikan kadar kejayaan ovulasi.ABSTRACTThe effect of administration of HCG (Human Chorionic Gonadotropin) on ovulation ofFugu niphobleswas studied. The female fish which exhibited distended abdomen were randomly assigned to four groups andthe treatments for groups 1, 2, 3 and 4 were 0, 100, 200 and 300 I. U of HCG per fish respectively. Thefish were injected immediately after the groups were established and unovulated females were reinjected after72 hours. The dosage for each fish was calculated based on the body weight and expressed as 1. U/g body weight.The ovulatory success in fish administered with 0, 1, 2, 3 and 41. U ofHCG/g body weight was 40%, 83 %,70%, 90 % and 80% respectively. This indicated that fish in the control group could also ovulate in captivityand that administration of HCG would enable a higher degree of ovulatory success.INTRODUCTIONFugu niphobles locally known as Kusafugu inJapan is toxic and the poison Tetrodotoxin ismainly found in the ovary, liver, blood and skin.In recent years, research on the origins andmetabolism of the poison has been activelypursued in Japan. The changes in the amountof the poison during egg development, growthand maturation are important. The fry of thisfish could be used as food for the more eco-nomically valuable Torafugu (Fugu rubripes) toreduce cannabalism during fry and fingerlingrearing.The earliest reports on breeding of thisfish were those by Uno (1955) and Fujita et al.(1966). The natural breeding season for thisfish in Japan ranges from May to early July. Itordinarily spawns only 1-5 days following eachS.H. CHEAH AND H. SATOHfull or new moon, at rising tide. The males shedmilt everyday during one or three series of hightides in succession whereas the females appearto discharge sticky eggs completely in one spawn-ing period (Uno, 1955).Fujita et al. (1966) successfully ovulated F.niphobles using Synahorin at a dose of 10 RU perfish and it was also reported that ovulationoccurred 24 to 48 hours after the administra-tion of Synahorin.As it is difficult to collect naturally spawnedeggs or fry from the sea, there is a need to beable to spawn this fish in captivity and also beable to culture the fry and fingerlings. In orderto pursue the problem of establishing a con-sistent technique for breeding F. niphobles, anexperiment was conducted to determine theeffect of administration of HCG (HumanChorionic Gonadotropin, commercial name-Puberogen) at different dosages on ovulation.The results of the ovulatory response and eggincubation experiments are discussed.MATERIALS AND METHODSFugu niphobles (Jordan and Snyder) that werecollected from fishermen at Ehima, AtsumiPeninsula, Aichi Prefecture,japan in the monthsof june and july 1985, were packed in 30 litreplastic bags under oxygen and transported backto the University of Tokyo hatchery at Benten-jima, Shizuoka Prefecture. At the hatchery, thebroodfish were immediately separated by sexand the females that had ovulated before thestart of the experiment were separately grouped,then anaesthetized with 1% urethane, weighed,stripped and their eggs fertilized followed byegg incubation.The unovulated females which exhibiteddistended abdomen were randomly divided intofour groups (1, 2, 3 and 4) and immediatelyanaesthetized, weighed and then injected withHCG (Puberogen) intramuscularly at the dos-ages of 0, 100,200 and 300 LU. per fish respec-tively. The amount of HCG administered perfish was then computed based on the bodyweight of the fish and then expressed in termsof LV. of HCGIg body weight of fish. The fishwere then released into 2.0 ton tanks contain-ing 1.5 m 3 ofwater. The tanks had a flow throughsystem and fish within the same treatment werekept together. Fish that did not ovulate in thehormone treated groups after three days wererei~ected with a second and final dose of thehormone according to the specific treatmentdosages.In the first trial, the fish were checked forovulatory response once a day at 1500 hoursbut in the subsequent trials, the fish werechecked twice a day at 1000 hours and 1500hours. When a gentle pressure on the abdo-men resulted in the release of eggs, the femaleswere anaesthetized and weighed. The weight ofeggs that were stripped from each female wasalso determined. The data for ovulatory successand failure were subjected to the Kolmogorov-Srnirnov test.A small fraction of about 100 to 200stripped eggs were then put into three 1 1 bea-kers containing seawater. A semen-water mix-ture was then poured into the beakers to fer-tilize the eggs. The beakers were given a freshchange of seawater after 15 minutes and all thelabelled beakers were put into a water bathwhich had a water temperature of 24°C. Thewater in the beakers were given an 80% changeevery day. In good eggs, hatching occurredabout 5-6 days alter fertilization. The hatchingrate was determined by counting the numberof larvae and number of unhatched eggs.. RESULTS AND DISCUSSIONThe ovulatory response of non hormone andhormone administered fish is summarised andpresented in Table 1. The results showed that40% offish in the control group (non hormonetreated) ovulated under tank conditions andthat ovulation occurred about 48 hours afterthe commencement of the experiments. Ovu-lation and oviposition had already occurred infish number 1 and as such the weight of eggscollected from it was only 1.2 g.The ovulatory success in fish that received1 LV. of HCG/g body weight was 83%. Thenumber of fish that ovulated 24 and 72 hoursafter the first injection were 2 and 1 respec-tively while 2 fish ovulated 24 hours after thesecond injection. In the study reported by Fujitaet al. (1966) on induced spawning of this fish,10 Rabbit Units (RU) of Synahorin were in-jected per fish in the first trial and the fish324 PERTANlKA VOL. 12 NO.3, 1989INDUCED OVULATION OF FUCU NJPHOBLESWITH HUMAJ'J CHORIONIC GONADOTROPINTABLE 1The ovulatory response of Fugu niphobles after administration ofHuman Chorionic Gonadotropin (HeG).HCG Hormone Fish Total Body Ovulatory Response WeightNumber Length Weight response Time of eggs(I.U./g) (cm) (g) (Hours) (g)0 1 1l.5 63.0 5 48 1.22 11.0 51.5 5 48 13.93 1l.8 64.9 5 48 14.94 13.0 84.8 5 48 15.95 11.2 54.5 F6 14.0 90.3 F7 11.4 46.1 F8 12.6 62.9 F9 14.2 94.9 F10 14.0 84.0 FII 11.8 78.4 51 24 19.212 12.2 71.7 51 24 17.513 13.1 82.5 51 72 23.514 13.4 89.8 52 24 26.915 12.6 75.6 52 24 20.016 13.0 77.7 F2 17 10.5 41.7 51 24 0.318 1l.8 58.1 51 48 18.019 13.4 84.6 51 72 24.220 12.2 61.9 52 24 12.121 11.6 53.7 52 24 1.222 14.9 117.5 52 48 32.123 14.4 96.0 52 48 17.324 11.3 54.9 F25 11.2 49.0 F26 13.3 83.8 F3 27 13.3 96.4 51 24 21.128 12.7 77.0 51 24 18.329 1l.8 69.3 51 24 19.330 13.3 96.4 51 24 21.131 11.8 60.4 51 24 10.732 13.9 116.9 51 48 30.533 13.2 75.2 51 48 24.834 12.3 64.1 52 24 12.035 13.3 78.4 52 48 19.236 14.5 89.0 52 48 20.537 12.2 62.5 F4 38 13.0 81.5 51 24 15.439 12.1 71.4 51 24 11.440 13.0 73.5 Sl 48 14.441 13.3 74.5 S2 48 19.842 12.7 83.8 FNote: 5 Ovulatory success without hormone administration51 - Ovulatory success after the first injection52 - Ovulatory success after the second injectionF Ovulatory failurePERTANll<A VOL. 12 NO.3, 1989 325S.H. CHEAH AND H. SATOHovulated after 24 hours. However, in their occurred prior to stripping and fertilization thussecond trial, some fish that did not have well causing zero hatching of egg samples collectedenlarged abdomens 24 hours after the first from fish numbers 1, 18, 19,23,34,39, and 41.injection required a second injection for suc- In future studies, the fish should be checked atcessful ovulation. more frequent intervals especially in the eve-The ovulatory success in fish which were ning and at night. There was also zero or lowadministered with 2 LV. ofHCG/g body weight hatching rates in fish numbers 3, 4, 12, 27,31,was 70%. Three fish ovulated between 24 -72 and 38. The plastic bag that was used to packhours after the first injection while four fish them under oxygen leaked very badly and asovulated between 24 and 48 hours after the such the stressful conditions in the low oxy-second injection. genated water may have caused some damageThe highest percentage of ovulatory suc- to the eggs.cess i.e. 90% was recorded from fish that had When Fugu niphobles were injected withbeen administered with 3 LV. of HCG/g body Synahorin at a dosage of 10 RV per fish, theweight. Seventy percent of the fish that ovu- hatching rates ranged from 91.3 to 98.6%lated did so after a single injection of HCG whereas those in the control fish were 94.6%(Puberogen). However, three fish could only (Fujita et al. 1966). As there was a time lapseovulate after the administration of the second of at least 1 day from the time the broodstockinjection. When the ovulatory success and fail- was caught till the time of hormone administra-ure rates were subjected to the Kolmogorov- tion in the present study, the onset of atresiaSmirnov test, there were no significant differ- may have commenced and as such the timeences in the success rates for the different dosage interval between capture and hormone admini-rates (P > 0.05). stration should be preferably reduced. AIn Limanda yokohamae, ovulation was suc- comparative study on the use of Synahorincessful when the females were injected with (pituitary gland of horse) versus HCG would beHCG (Puberogen) at the rate of 3.4 to 8.6 LU. beneficial considering the high hatching rates/g body weight (Hirose et al. 1979 in Lam obtained when Synahorin was administered.1982) but in the case of Plecoglossus altivelis In Macquaria ambigua, all females injectedhigher dosages ranging from 12.7 to 25.0 LV./ with a spawning threshold dosage of 500 LU.g body weight were required for successful per kg of HCG or dosages of 1000 and 2000ovulation (Hirose et al. 1977 in Lam 1982). I.U. per kg of HCG spawned completely. How-The ovulatory success in fish that were ever the mean hatching rate of 87.4% of eggsadministered with 4 LU. ofHCG/g body weight spawned by females injected with 500 LU. perwas 80%. As the sample size was rather small, kg (0.5 LU./g) of HCG was significantly higherthis result should be viewed with caution. (P <0.01) than the mean hatching rate of 44.2%The effect of the dosage of HCG adminis- in fish that were injected with 2000 LV. per kgtered to Fugu niphobles on hatching rates is of HCG. Thus the use of HCG at the two dos-presented in Table 2. In this study it was ob- ages above the spawning threshold level resultedserved that normal developing eggs turned grey in apparent deterioration of egg quality in M.usually after 3 days of incubation and' hatched ambigua (Rowland 1983) .out between 5 to 7 days at 24°C. Generally this During the preparation of the fish groupsstudy was unable to demonstrate any correla- for HCG administration, some females weretion between the dosage of hormone adminis- observed to have ovulated. The eggs were thentered and the hatching rate. Egg samples from stripped, fertilized and incubated. However thefish numbers 14, 20, 35 and 40 had high hatch- eggs became opaque, white or light brown anding rates indicating that hatching failure was did not develop the characteristic grey colournot due to the stripping, fertilization nor incu- of developing eggs by day 3 at 24°C. Generallybation techniques but was probably due to the there is a certain length of time after ovulationtiming of egg stripping. In this study, the fish when the eggs remain viable in the ovarianwere checked for ovulation only twice a day and cavity. In this case, it was inferred that the eggsit was possible that overripening- of egg-s had that were collected from females which had326 PERTANlKA VOL. 12 NO.3, 1989INDUCED 0\ LATION OF FUGU NIPHOBLES'NITH HUMAN CHORIONIC GONADOTROPINTABLE 2The relationship between the dosage of HCG administered andhatching rate of Fugu niphobles eggsHCG Hormone Fish Hatching rate of eggs (%) in replicate Mean hatchingDosage number rate(I.U./g) 2 3 (%)0 1 0 0 0 02 34.4 26.7 32.0 31.03 0 0 0 04 0 0 0 011 14.3 3.2 6.4 8.012 0 21.3 3.9 8.413 11.8 8.6 14.5 11.614 82.8 88.5 84.7 85.315 62.8 60.4 65.9 63.02 17 0 2.4 1.6 1.318 0 0 0 019 0 0 0 020 91.3 89.8 82.5 87.922 73.7 74.9 58.7 69.123 0 0 0 03 27 4.2 2.1 7.4 4.628 1.3 1.6 1.9 1.629 47.2 39.0 49.1 45.130 4.2 2.1 7.4 4.631 0 0 0 032 41.9 42.4 23.0 35.833 77.1 66.7 78.9 74.234 0 0 0 035 79.2 91.7 95.9 88.936 80.2 47.1 36.6 54.64 38 0 0 0 039 0 0 0 040 60.9 71.1 81.2 71.141 0 0 0 0ovulated prior to the commencement of thetrials had exceeded this critical period of timefor successful fertilization and as a result eggsamples from 17 fish had zero hatching withthe exception of egg samples from 1 fish.CONCLUSIONEven though administration of Human Chori-onic Gonadotropin (HCG) to female Fuguniphobles increased the ovulatory success rates,there were no significant differences in the suc-cess rates which were the result of administer-ing different dosage rates (P > 0.05).ACKNOWLEDGEMENTSThe authors would like to express their appre-ciation to the Japan International CooperationAgency for funding this experiment and forproviding financial support to the senior au-thor during his stay inJapan. The senior authorwould also like to express his appreciation toUniversiti Pertanian Malaysia, Serdang, Selan-gor, Malaysia for permitting him to be atJapan.The authors would like to thank Dr. YK. Lawfor making a brief English summary of thereport by Dr. Y Fujita, T. Komiyama andYokata. Finally, the authors would like to thankPERTANIKA VOL. 12 NO.3, 1989 327S.H. CHEAH AND H. SATOHAssoc. Prof. Dr. KJ. Ang for his comments onthe manuscript.REFERENCESFuJlTA, Y, T. KaMIYAMA and N. YOKATA. 1966. Arti-ficial Egg Taking and High Density HatchingMethod of Fugu niphobles. The Aquiculture 14(1) :31-36. (In Japanese).LAM, T J. 1982. Applications of Endocrinology toFish Culture. Can.J. Fish. Aquat. Sci. 39: 111-137.ROWLAND, SJ. 1983. The hormone-induced Ovu-lation and Spawning of the Australian FreshwaterFish Golden Perch, Macquaria ambiqua(Richardson) (Percichthyidae). Aquaculture 35 :221-238.UNO, Y 1955. Spawning Habit and Early Deve-lopment of a Puffer, Fugu (Torafugu) nipholesGordan et Snyder).J. Tokyo Univ. Fish. 41 (2) : 169-183.(Received 23 June, 1987)328 PERTANlKA VOL. 12 NO.3, 1989

Trials on Induced Ovulation of Fugu niphobles (Jordan and Snyder) with Human Chorionic Gonadotropin

ABSTRAK Kesan penggunaan HCG (Human Chorionic Gonadotropin) untuk ovulas

S H Cheah

H Satoh

CiteSeerX

Pertanikal2(3), 323-328(1989)
Trials on Induced Ovulation of Fugu niphobles
(Jordan and Snyder) with Human Chorionic Gonadotropin
S.H. CHEAH
Department of Fisheries Biology and Aquaculture,
Faculty of Fisheries and Marine Sciences,
Universiti Pertanian Malaysia,
43400 UPM Serdang, Selangor Darul Fhsan, Malaysia.
H. SATOH
Fisheries Laboratory,
Faculty of Agriculture,
University of Tokyo,
Bentenjima, Shizuoka Prefecture 431-02, fapan.
Key words : Induced ovulation, hormone, Fugu niphobles.
ABSTRAK
Kesan penggunaan HCG (Human Chorionic Gonadotropin) untuk ovulasi Fugu niphobles telah dikaji.
Ikan betina yang buncit abdomennya dibahagikan kepada empat kumpulan 1, 2, 3 dan 4, dan dirawat
dengan hormon HCG pada takaran masing-masing, 0, 100, 200 dan 300 I.U.. Selepas ikan betina
dikumpulkan mengikut kumpulan, ikan-ikan disuntik dan ikan yang didapati tidak berovulasi disuntik
semula selepas 72 jam. Takaran suntikan dikira selepas berat badan betina diambil kira dan takaran
diberikan sebagai LU./g berat badan. Data kejayaan ovulasi untuk rawatan 0, 1, 2, 3 dan 4 I.U./g berat
badan masing-masing, 40%, 83%, 70%, 90% dan 80%. Ini menunjukkan bahawa ikan dalam kumpulan
kawalan iaitu kumpulan yang tidak menerima suntikan hormon dapat berovulasi dalam keadaan kurungan
dan penggunaan HCG dapat meninggikan kadar kejayaan ovulasi.
ABSTRACT
The effect of administration of HCG (Human Chorionic Gonadotropin) on ovulation o/Fugu niphobles
was studied. The female fish which exhibited distended abdomen were randomly assigned to four groups and
the treatments for groups 1, 2, 3 and 4 were 0, 100, 200 and 300 I.U. of HCG per fish respectively. The
fish were injected immediately after the groups were established and unovulated females were reinjected after
72 hours. The dosage for each fish was calculated based on the body weight and expressed as I. U./g body weight.
The ovulatory success in fish administered with 0, 1, 2, 3 and 4 I.U. ofHCG/g body weight was 40%, 83%,
70%, 90% and 80% respectively. This indicated that fish in the control group could also ovulate in captivity
and that administration of HCG would enable a higher degree of ovulatory success.
INTRODUCTION
Fugu niphobles locally known as Kusafugu in
Japan is toxic and the poison Tetrodotoxin is
mainly found in the ovary, liver, blood and skin.
In recent years, research on the origins and
metabolism of the poison has been actively
pursued in Japan. The changes in the amount
of the poison during egg development, growth
and maturation are important. The fry of this
fish could be used as food for the more eco-
nomically valuable Torafugu {Fugu rubripes) to
reduce cannabalism during fry and fingerling
rearing.
The earliest reports on breeding of this
fish were those by Uno (1955) and Fujita et al
(1966). The natural breeding season for this
fish in Japan ranges from May to early July. It
ordinarily spawns only 1-5 days following each
S.H. CHEAH AND H. SATOH
full or new moon, at rising tide. The males shed
milt everyday during one or three series of high
tides in succession whereas the females appear
to discharge sticky eggs completely in one spawn-
ing period (Uno, 1955).
Fujita^a/. (1966) successfully ovulated F.
niphobles using Synahorin at a dose of 10 RU per
fish and it was also reported that ovulation
occurred 24 to 48 hours after the administra-
tion of Synahorin.
As it is difficult to collect naturally spawned
eggs or fry from the sea, there is a need to be
able to spawn this fish in captivity and also be
able to culture the fry and fingerlings. In order
to pursue the problem of establishing a con-
sistent technique for breeding F. niphobles, an
experiment was conducted to determine the
effect of administration of HCG (Human
Chorionic Gonadotropin, commercial name-
Puberogen) at different dosages on ovulation.
The results of the ovulatory response and egg
incubation experiments are discussed.
MATERIALS AND METHODS
Fugu niphobles (Jordan and Snyder) that were
collected from fishermen at Ehima, Atsumi
Peninsula, Aichi Prefecture, Japan in the months
of June and July 1985, were packed in 30 litre
plastic bags under oxygen and transported back
to the University of Tokyo hatchery at Benten-
jima, Shizuoka Prefecture. At the hatchery, the
broodfish were immediately separated by sex
and the females that had ovulated before the
start of the experiment were separately grouped,
then anaesthetized with 1 % urethane, weighed,
stripped and their eggs fertilized followed by
egg incubation.
The unovulated females which exhibited
distended abdomen were randomly divided into
four groups (1, 2, 3 and 4) and immediately
anaesthetized, weighed and then injected with
HCG (Puberogen) intramuscularly at the dos-
ages of 0, 100, 200 and 300 I.U. per fish respec-
tively. The amount of HCG administered per
fish was then computed based on the body
weight of the fish and then expressed in terms
of I.U. of HCG/g body weight of fish. The fish
were then released into 2.0 ton tanks contain-
ing 1.5 m3 of water. The tanks had a flow through
system and fish within the same treatment were
kept together. Fish that did not ovulate in the
hormone treated groups after three days were
reinjected with a second and final dose of the
hormone according to the specific treatment
dosages.
In the first trial, the fish were checked for
ovulatory response once a day at 1500 hours
but in the subsequent trials, the fish were
checked twice a day at 1000 hours and 1500
hours. When a gentle pressure on the abdo-
men resulted in the release of eggs, the females
were anaesthetized and weighed. The weight of
eggs that were stripped from each female was
also determined. The data for ovulatory success
and failure were subjected to the Kolmogorov-
Smirnov test.
< A small fraction of about 100 to 200
stripped eggs were then put into three 1 1 bea-
kers containing seawater, A semen-water mix-
ture was then poured into the beakers to fer-
tilize the eggs. The beakers were given a fresh
change of seawater after 15 minutes and all the
labelled beakers were put into a water bath
which had a water temperature of 24°C. The
water in the beakers were given an 80% change
every day. In good eggs, hatching occurred
about 5-6 days after fertilization. The hatching
rate was determined by counting the number
of larvae and number of unhatched eggs.
RESULTS AND DISCUSSION
The ovulatory response of non hormone and
hormone administered fish is summarised and
presented in Table 1. The results showed that
40% offish in the control group (non hormone
treated) ovulated under tank conditions and
that ovulation occurred about 48 hours after
the commencement of the experiments. Ovu-
lation and opposition had already occurred in
fish number 1 and as such the weight of eggs
collected from it was only 1.2 g.
The ovulatory success in fish that received
1 I.U. of HCG/g body weight was 83%. The
number of fish that ovulated 24 and 72 hours
after the first injection were 2 and 1 respec-
tively while 2 fish ovulated 24 hours after the
second injection. In the study reported by Fujita
et al (1966) on induced spawning of this fish,
10 Rabbit Units (RU) of Synahorin were in-
jected per fish in the first trial and the fish
324 PERTANIKA VOL. 12 NO. 3, 1989
INDUCED OVULATION OF FUGU NIPH0B1JLS WITH HUMAN CHORIONIC GONADOTROPIN
TABLE 1
The ovulatory response of Fugu niphobles after administration of
Human Chorionic Gonadotropin (HCG).
HCG Hormone
(I.U./g)
0
•
1
2
3
4
Fish
Number
1
2
5
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Total
Length
(cm)
11.5
11.0
11.8
13.0
11.2
14.0
11.4
12.6
14.2
14.0
11.8
12.2
13.1
13.4
12.6
13.0
10.5
11.8
13.4
12.2
11.6
14.9
14.4
11.3
11.2
13.3
13.3
12.7
11.8
13.3
11.8
13.9
13.2
12.3
13.3
14.5
12.2
13.0
12.1
13.0
13.3
12.7
Body
Weight
(g)
63.0
51.5
64.9
84.8
54.5
90.3
46.1
62.9
94.9
84.0
78.4
71.7
82.5
89.8
75.6
77.7
41.7
58.1
84.6
61.9
53.7
117.5
96.0
54.9
49.0
83.8
96.4
77.0
69.3
96.4
60.4
116.9
75.2
64.1
78.4
89.0
62.5
81.5
71.4
73.5
74.5
83.8
Ovulatory
response
S
S
S
S
F
F
F
F
F
F
SI
SI
SI
S2
S2
F
SI
SI
SI
S2
S2
S2
S2
F
F
F
SI
SI
SI
SI
SI
SI
SI
S2
S2
S2
F
SI
SI
SI
S2
F
Response
Time
(Hours)
48
48
48
48
24
24
72
24
24
24
48
72
24
24
48
48
24
24
24
24
24
48
48
24
48
48
24
24
48
48
Weight
of eggs
(g)
1.2
13.9
14.9
15.9
19.2
17.5
23.5
26.9
20.0
0.3
18.0
24.2
12.1
1.2
32.1
17.3
2L1
18.3
19.3
21.1
10.7
30.5
24.8
12.0
19.2
20.5
15.4
11.4
14.4
19.8
Note : S - Ovulatory success without hormone administration
51 - Ovxilatory success after the first injection
52 - Ovulatory success after the second injection
F - Ovulatory failure
PERTANIKA VOL. 12 NO. 3, 1989 325
S.H. CHEAH AND H. SATOH
ovulated after 24 hours. However, in their
second trial, some fish that did not have well
enlarged abdomens 24 hours after the first
injection required a second injection for suc-
cessful ovulation.
The ovulatory success in fish which were
administered with 2 I.U. of HCG/g body weight
was 70%. Three fish ovulated between 24-72
hours after the first injection while four fish
ovulated between 24 and 48 hours after the
second injection.
The highest percentage of ovulatory suc-
cess i.e. 90% was recorded from fish that had
been administered with 3 I.U. of HCG/g body
weight. Seventy percent of the fish that ovu-
lated did so after a single injection of HCG
(Puberogen). However, three fish could only
ovulate after the administration of the second
injection. When the ovulatory success and fail-
ure rates were subjected to the Kolmogorov-
Smirnov test, there were no significant differ-
ences in the success rates for the different dosage
rates (P > 0.05).
In Limanda yokohamae, ovulation was suc-
cessful when the females were injected with
HCG (Puberogen) at the rate of 3.4 to 8.6 I.U.
/g body weight (Hirose et al 1979 in Lam
1982) but in the case of Plecoglossus altivelis
higher dosages ranging from 12.7 to 25.0 I.U./
g body weight were required for successful
ovulation (Hirose et al 1977 in Lam 1982).
The ovulatory success in fish that were
administered with 4 I.U. of H C G / g body weight
was 80%. As the sample size was rather small,
this result should be viewed with caution.
The effect of the dosage of HCG adminis-
tered to Fugu niphobles on hatching rates is
presented in Table 2. In this study it was ob-
served that normal developing eggs turned grey
usually after 3 days of incubation and hatched
out between 5 to 7 days at 24°C. Generally this
study was unable to demonstrate any correla-
tion between the dosage of hormone adminis-
tered and the hatching rate. Egg samples from
fish numbers 14, 20, 35 and 40 had high hatch-
ing rates indicating that hatching failure was
not due to the stripping, fertilization nor incu-
bation techniques but was probably due to the
timing of egg stripping. In this study, the fish
'were checked for ovulation only twice a day and
it was possible that overripening of eggs had
326 PERTANIKA VOL.
occurred prior to stripping and fertilization thus
causing zero hatching of egg samples collected
from fish numbers 1, 18, 19, 23, 34, 39, and 41 .
In future studies, the fish should be checked at
more frequent intervals especially in the eve-
ning and at night. There was also zero or low
hatching rates in fish numbers 3, 4, 12, 27, 31 ,
and 38. The plastic bag that was used to pack
them under oxygen leaked very badly and as
such the stressful conditions in the low oxy-
genated water may have caused some damage
to the eggs.
When Fugu niphobles were injected with
Synahorin at a dosage of 10 RU per fish, the
hatching rates ranged from 9 L 3 to 98.6%
whereas those in the control fish were 94.6%
(Fujita et al 1966). As there was a time lapse
of at least 1 day from the time the broodstock
was caught till the time of ho rmone administra-
tion in the present study, the onset of atresia
may have commenced and as such the time
interval between capture and ho rmone admini-
stration should be preferably reduced. A
comparative study on the use of Synahorin
(pituitary gland of horse) versus HCG would be
beneficial considering the high hatching rates
obtained when Synahorin was administered.
In Macquaria ambigua, all females injected
with a spawning threshold dosage of 500 LU.
per kg of HCG or dosages of 1000 and 2000
I.U. per kg of HCG spawned completely. How-
ever the mean hatching rate of 87.4% of eggs
spawned by females injected with 500 LU. per
kg (0.5 I.U./g) of HCG was significantly higher
(P < 0.01) than the mean hatching rate of 44.2%
in fish that were injected with 2000 I.U. per kg
of HCG. Thus the use of HCG at the two dos-
ages above the spawning threshold level resulted
in apparent deterioration of egg quality in M.
ambigua (Rowland 1983).
During the preparation of the fish groups
for HCG administration, some females were
observed to have ovulated. The eggs were then
stripped, fertilized and incubated. However the
eggs became opaque, white or light brown and
did not develop the characteristic grey colour
of developing eggs by day 3 at 24°C. Generally
there is a certain length of time after ovulation
when the eggs remain viable in the ovarian
cavity. In this case, it was inferred that the eggs
that were collected from females which had
12 NO. 3, 1989
INDUCED OVULATION OF FUGUNIPHOBIJCS WITH HUMAN CHORIONIC GONADOTROPIN
TABLE 2
The relationship between the dosage of HCG administered and
hatching rate of Fugu niphobles
HCG Hormone
Dosage
(I.U./g)
0
1
2
3
4
Fish
number
1
2
3
11
12
13
14
15
17
18
19
20
22
23
27
28
29
30
31
32
33
34
35
36
38
39
40
41
Hatching
1
0
34.4
0
0
14.3
0
11.8
82.8
62.8
0
0
0
91.3
73.7
0
4.2
1.3
47.2
4.2
0
41.9
77.1
0
79.2
80.2
0
0
60.9
0
rate of eggs (%)
2
0
26.7
0
0
3.2
21.3
8.6
88.5
60.4
2.4
0
0
89.8
74.9
0
2.1
1.6
39.0
2.1
0
42.4
66.7
0
91.7
47.1
0
0
71.1
0
in replicate
3
0
32.0
0
0
6.4
3.9
14.5
84.7
65.9
1.6
0
0
82.5
58.7
0
7.4
1.9
49.1
7.4
0
23.0
78.9
0
95.9
36.6
0
0
81.2
0
Mean hatching
rate
(%)
0
31.0
0
0
8.0
8.4
11.6
85.3
63.0
1.3
0
0
87.9
69.1
0
4.6
1.6
45.1
4.6
0
35.8
74.2
0
88.9
54.6
0
0
71.1
0
ovulated prior to the commencement of the
trials had exceeded this critical period of time
for successful fertilization and as a result egg
samples from 17 fish had zero hatching with
the exception of egg samples from 1 fish.
CONCLUSION
Even though administration of Human Chori-
onic Gonadotropin (HCG) to female Fugu
niphobles increased the ovulatory success rates,
there were no significant differences in the suc-
cess rates which were the result of administer-
ing different dosage rates (P > 0.05).
ACKNOWLEDGEMENTS
The authors would like to express their appre-
ciation to the Japan International Cooperation
Agency for funding this experiment and for
providing financial support to the senior au-
thor during his stay in Japan. The senior author
would also like to express his appreciation to
Universiti Pertanian Malaysia, Serdang, Selan-
gor, Malaysia for permitting him to be at Japan.
The authors would like to thank Dr. Y.K. Law
for making a brief English summary of the
report by Dr. Y. Fujita, T. Komiyama and N.
Yokata. Finally, the authors would like to thank
PERTANIKA VOL. 12 NO. 3, 1989 327
S.H. CHEAH AND H. SATOH
Assoc. Prof. Dr. KJ. Ang for his comments on
the manuscript.
REFERENCES
FUJITA, Y., T. KOMIYAMA and N. YOKATA. 1966. Arti-
ficial Egg Taking and High Density Hatching
Method of Fugu niphobles. The Aquiculture 14(1) :
31-36. (In Japanese).
LAM, TJ. 1982. Applications of Endocrinology to
Fish Culture. Can.}, Fish. AquaL Sci. 39 : 111-137.
ROWLAND, SJ. 1983. The hormone-induced Ovu-
lation and Spawning of the Australian Freshwater
Fish Golden Perch, Macquaria ambiqua
(Richardson) (Percichthyidae). Aquaculture 35 :
221-238.
UNO, Y. 1955. Spawning Habit and Early Deve-
lopment of a Puffer, Fugu (Torafugu) nipholes
(Jordan et Snyder)./ Tokyo Univ. Fish. 41(2): 169-
183.
(Received 23 June, 1987)
328 PERTANIKA VOL. 12 NO. 3, 1989


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Trials on Induced Ovulation of Fugu niphobles (Jordan and Snyder) with Human Chorionic Gonadotropin

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