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FISHERIES AND MARINE SERVICE

Translation Series No. 3558

The maturation and spawning of fish (Fundamentals and applications)

Takashi Eibiya, et al. (Editors) Eleven papers by various authors from a symposium of the Japanese Society of Scientific Fisheries, April 5, 1974

Original title: Gyorui no seijuku to sanrar.

From: Nippon Suisan Gakkaishi suisangaku shirizu 6 Koseisha Koseikaku, Tokyo, pp. 1 - 130, October 15, 1974

Translated by the Translation Bureau(JWC) Multilingual Services Division

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Department of the Environment Fisheries and Marine Service

Vancouver Laboratory Vancouver, B.C.

1975

263 pageu typescript

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The maturation and spawning of fish.

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CT 2 1 19 75

Scientific Fisheries, No. 6.

THE MATURATION AND SPAWNING OF FISH.

(Fundamentals and applications).

The Jah -mose Society of Scientific Fisheries.

Published by Koseisha Koseikaku.

UNEDITED TRANSLATION For information only

TRADUCTION NON REVISEE Information seulement

101 .-200..1 0..31

Yutaka MURAKAMI.

Reijiro HIRANO.

Kazunori TAKANO.

Sadaichi KATO.

Fumio-YAMASAKI.

Hiroshi YOSHIOKA.

Teruo HARADA.

Quality of the Y. The assessment of the spawn.

10. Freshwater fish.

11. Marine fish.

Kiyoshi SAKAI.

Michiyasu KIYONO.

2 ••

Contents and Authors.

Foreword , Takashi HIBIYA, Minoru NOMURA, Yutaka MURAKAMI

and Reijiro HIRANO.

I. Present condition and uncertainties.

1. Freshwater fish.

2. Marine fish.

II. Parent fish for egg collecting.

3. The process of maturation of the gonads.

4 • Detailed characteristics of reproduction.

5. Internal secretions, egg maturation and ovulation.

III. Environment, maturation, and spawning.

6. Freshwater fish.

7. Marine fish.

IV. Maturation and metabolism.

8. Maturation and fat metabolism. Fumio TAKASHIMA.

9. The accumulation of yolk protein. Katsumi AIDA.

General discussion.

Takashi HIBIYA, Minoru NOMURA, Reijiro HIRANO.

3

Foreword.

An increased rate of reproduction of fish is of

fundamental importance in the maintenance of continued

stability of the fishing industry, and particularly important

in the planning of any increase of production. Formally, of

course, this is a matter of pisciculture, and active efforts

have recently come to be concentrated on the production of

fry for the cultivation and stocking of the offshore fish

resources.

There are many questions concerned with the production

of fry, such as the food required in the early stages, the

control of the environment and of disease, but the very first

step is the procurement of good quality eggs from superior

parent fish and the preservation of these eggs in a suitable

hatching place to produce the required number of healthy fry.

There have been very few studies of the culture of the parent

fish from the point of view of breeding, or of the feeding of

the parent fish from which eggs are to be collected. The

methods of collecting eggs have a history reaching back to

the last century, and a great deal of basic research has been

accumulated in relation to the maturation of the gonads, the

acceleration of maturation, and the induction of spawning.

There has also been a very large amount of research on the

life history of the fish from the formation of the eggs to

the hatching of the fry.

However the present discussions show that the results

so far obtained by basic research are by no means sufficient

for practical application in the collection of eggs. This is

because basic research is apt to be limited to a narrow range

of phenomena of an essentially scientific ichracter, and it

is considered that there has not been sufficient recognition

of the mutual stimulation which can accompany the expansion

of both basic research and applied technology.

The Japanese Society for Scientific Fisheries therefore

arr.Lnged a symposium, held on 5 April 1974, entitled "Questions

relating to the collecting of eggs and to the parent fish

used for the production of fry". Discussion during this

symposium centred on the female fish and dealt with the

relation between basic knowledge and practical technique in

the collection of eggs.

The present publication is the record of this

symposium, and is the joint work of many contributors.

Nothing can give more pleasure to all those concerned than

to see it contribute to both scientific and technical

progress in this field.

Takashi HIBIYA (Department of Agriculture, Tokyo University)

Minoru NOMURA (Tokyo Maritime University)

Yutaka MURAKAMI (Department of Aquatic Animal Husbandry,

University of Hiroshima)

Reijiro TAUANO (Department of Agriculture,

Tokyo University).

•

The maturation and siawnincr of fish.

(Fundamentals and applications).

Index.

Foreword. Takashi HIBIYA, Minoru SATOMURA, Yutaka MRAKAMI

and Reijiro HIRANO.

I. Present conditions and uncertainties,

1, Freshwater fish

2. Marine fish.

Page

Yukata MURAKAMI 9

Reijiro HIRANO 20

II. Parent fish for eg collecting.

3. The process of maturation of the

gonads. Kazunori TAKANO 32

1. The structure of the ovary and the oviduct.

2 . Genital cavity fluids. 36

3. The process of ripening of ovarian ova. 39

4 . Ovulation.

5. The functions of ovary hormones. 47

4. Detailed characteristics of

reproduction. Sadaichi KATO 58

1. The ripe eggs. 58

2, The gonad index of mature individuals

and the size distribution of eggs

in the ovary. 59

3. The ripe eggs. 62

4. The weight of an egg. 64

5. The spawning season. 69

33

• 6

5. Internal secretions, egq maturation and ovulation. Fumio YAIUSAKI 76

1. The maturation of the egg. 76

2. Ovulation. 85

3. Piscine GTH. 90

Environment, maturation, and spawning.

6. Freshwater fish. Hiroshi YOSHIOKA 105

1, Illumination. 105

2, Temperature. 117

7. Marine fish. Teruo HARADA 128

1. Water temperature, maturation

and spawning. 129

2. Illumination and spawning. 134

3. Other environmental factors,

maturation and spawning. 135

4. The culture of the parent fish

for egg collecting. 136

5. The stimulation and inhibition

of maturation and spaWning by

environmental control.

6. Future prospects. 142

IV. Maturation and metabolism.

8. Maturation and fat metabolism. Fumio TAKASHIMA 152

1. The chemical properties of

fish egg fats. 153 .

2. Changes in body fat content

during maturation of the

ovary. 156

138

•

•

208

211

212

2 15

3. Endocrine control of changes in

fat metabolism during Maturation

of the ovary. 159

4. Fats in the diet of the parent fish. 163

9. The accumulation of yolk

protein. Katsumi AIDA 176

The appearance and endocrine •

control of female specific

proteins during maturation.

2. The connection between FSSP and

yolk protein. 181

3. The site of synthesis of FSSP. 183

4. The introduction of FSSP into

the maturiag egg. 191

•V. The evaluation of egg quality in spawn.

10. Freshwater fish. Kiyoshi SAKAI 200

1. The Quality of the spawn and

its internal and external

morphological characteristics. 202

2, Change of egg quality with time. 208

3. Hormone administration and the

process of maturation ofthe

ovarian egg.

4. Assessment of the Quality of eggs

spawned near the upper limit of

spawning temperature.

5. Egg quality and egg fractionation

by centrifuging.

6. Egg quality and other criteria.

1.

17 8

• 8

Michiyasu KIYONC 225

1. The characteristics used in

determining egg quality. 226

2. Discussion of methods of

appraisal of egg quality. 232

Questions.

General discussion. 246

•

11. Marine fish.

238

•

• 9 Present condition and uncertainties.

1. Freshwater fish. Yutaka MURAKAMI.*

In discussions of pisciculture and the stocking of

lakes, marshes, and rivers, it is usual to start with the

preservation of the fry. Ideally, the parent fish are

cultured in a pond, the eggs are artificially collected and

fertilized and healthy fry are reared to the required size

so that the production of the quantity needed to meet the

demt.nd can be planned. However methods which will come close

to guaranteeing fry production in this way are known for only

a few species. They are limited to rainbow trout Salmo

gairdneri irideus, Salmo macrostoma, Oncbrhynchus rhodeus,

the carp Cyprinus carpi°, the crucian carp and the goldfish

Carassius auratus.

For the cultivation of such fish as the eel Anguilla

japonica, the sweet smelt Plecoglossus altivelis, and of

Ctenopharyncodon idellus and Hypophthalmichthys molitrix,

it is normal to rely mostly, or even completely, on young

fish or eggs which have been naturally produced. It has

recently become difficult to obtain sufficient quantities of

these fish to meet the demand because the number of natural

fry is diminishing, and there has been a great deal of

experimental study of fry production through artificial egg

collection. In one such experimental study, made with

P. altivelis, it was possible to produce the large quantity

of approximately 10 million fry for stocking.

(Tokyo University of Fisheries).

1 0

In present day production of freshwater fish fry both

cultivated and wild-caught parent fish are used. In some

species, hormone administration is needed for artificial egg

collection. There are two methods of artificial egg collection.

In one method, which is used with carp, goldfish and bluegills,

mature male and female parent fish are kept in the same pond

and they spawn naturally in nests in a man-made spawning bed.

In the second or "stripping" method, the parent fish are

inspected and when it is judged that they have ovulated the

eggs and mut are taken from the body either by pressure on

the abdomen (the squeezing out method) or by cutting (the

incision method). We will here refer to the first method as

the "carp" method of collection, and to the latter as the

"rainbow trout" method.

Table 1.1 shows the methods of egg collection from the

parent fish normally used for the production of fry of the

species most often stocked or cultivated. The table also shows

those which are in the experimental stage. It shows that

various experimental studies are in progress with species for

which the production of fry by artificial egg collection has

not yet been commercially established. The following summary

of present conditions and uncertainties about the production

of fry from parent fish is limited to the topics of parent

fish and egg collection. •

Table 1.1.

Parent fish and methods used for the collection of eggs of freshwater fish.

Parent fish used Cultivated Wild Eggs or fry ob-tained

Hormone use Not used Used Not used Used from the . wild

Method of collection* Rainbow Carp Rainbow Carp Rainbow Carp Rainbow Carp trout type trout type trout type trout type type type type type

Salmo gairdneri X

Salmo macrostoma X Oncorhynchus rhodurus -

Cyprinus carpio X

Carassius carassius X

Carassius auratus X

Lepomis macrochirus X

- Basilichthys bonariensis

Hi lmesus transpacificus X. 1 i..

li Y Y X

. , • Hypomesus transpacificus X . • . ; '

; Plecoglossus altivelis Y . Y Y X

L ,

: Misgurnus anguillicaudatus Y Y , y Y

Anguilla japonica v y X .

Gnathopogon elongatus I Y ' y .

,

Tribolodon hakonensis Y Y Y i X

Ctenopharyncodon and Y . y Y Y X Hypophtalmichthys

Parasilurus azotus Y Y

Zacco platypus Y

Black bass

X: Methods . most usually employed commercially.

Y: Methods in experimental use or in process of transfer to commercial use.

See page 10 of the present article. 1-■ • b-‘

}o^,r

12

In species in which the collection of eggs from

cultured fish is difficult, use is made of artificial

collection from mature parent fish which have assembled

in their r.latural spawning places, and of immature fish which

are approaching the age of natural maturation. Eggs can be

collected from some fish immediately after capture, and others

can be cultured and brought to complete maturation by the

administration of hormones, after which the eggs can be

collected. At the present time the list of such fish, taking

account of those in the experimental stage, includes the

eel Anmilin_inmni.22, the sweet smelt Plecoglossus altivelis,

the loach Misgurnus anguillicaudatus, the roach Gnathopogon

elongatus caerulescens, the dace Tribolodon hakonensis

hakonensis, the Chinese carps Ctenopharyncodon idellus and

Hypophtalmichthys molitrix, the pond smelt Hypomesus

transpacificus nipponensis, the freshwater catfish Parasilurus

azotus and Zacco platypus. When fish captured in the wild

are used, and more particularly if they are captured already

mature at the spawning places, it is known that the results

of egg collection may be greatly influenced by the method of

transportation from the place of capture to the place of

collection, and by the length of the period of cultivation

between capture and collection. For example, with

Ctenopharyncodon and Hypophthalmichthys the results of egg

collection are increased if the fish are anaesthetized during

• 13

transport. Good eggs can be obtained from Misçurnus if

hormones are administered immediately after capture, but

the yield from egg collection is lowered if there is a long

interval.. Good results can be obtained with Tribolodon if

the eggs are collected immediately after capture, but if they

are kept for as long as 24 hours, the eggs become difficult

to extract, and the yield is lowered.

If the required quantity of parent fish can easily be

caught and the price is not high, there is little difficulty

in the use of wild fish. However when, as has recently become

the case with the production of fry of P. altivelis, the

demand becomes large it becomes difficult to maintain a

sufficient quantity of parent fish to meet the demand for this

large production. The price then rapidly rises and it is

better to rely on artifial collection of eggs from parent

fish cultivated in ponds.

With present day techniques of pond cultivation, the

parent fish can be divided into three groups according to the

relative difficulty of attaining maturation and egg collection.

(1) Species which can easily be brought to the size and

age for maturation, and can be matured and made to produce

eggs by the normal methods of pond feeding and rearing.

Examples are Salmo gairdnerii, Salmo macrostoma, Oncorhynchus

rhodurus, Cyprinius _carpi°, Goldfish, Carassius auratus, the

Leyomis macrochirus and the pejerry

(Odontesthes) bonariensis.

• 14

(2) Species in which parent fish can be reared to become

mature even without hormone treatment, although in many cases

hormone treatment is necessary at the time of egg collection,

and the results of egg collection are inferior to those

obtained from wild fish. Examples are Plecoglossus altivelis,

Misgurnus anguillicaudatus, and Tribolodon hakonensis hakonensis.

(3) Species which in cultivation can reach the age and pi°

size of maturation of wild fish, and which can be made to

mature and to produce eggs by the administration of hormones,

but with which this is very difficult. Examples are Anguilla

jamoniu, Ctenopharyncodon idellus and Hypophthalmichthys

molitrix.

Species of the first type •are by no means lacking in

problems with regard to rearing and egg collection, but on the

whole it is those of types 2 and 3 Which present the most

difficulties.

Among the problems which these species have in common,

though to different extents, are

(a) The proportion of the cultured fish from wIlich eggs

can be collected is low.

(h) Only a fraction of the ripe eggs can be collected,

or if all the eggs can be collected, the fraction which

develops is low.

(c) The condition of the eggs ovulated or spawned may

vary from one parent fish to ;another, or even between eggs from

the same parent, so that the results of collection are poor.

15

(d) The fraction which die during development or hatching

is high, or the fraction which are abnormal or feeble when

hatched is high, so that it is difficult to get healthy fry.

The conditions in which the parent fish are raised and

the methods of egg collection are factors in these difficulties.

The environment in which the parent fish are raised

influences maturation and ovulation, and the principal

environmental factors normally considered are illumination and

temperature. The relations between these factors and the

maturation and ovulation of freshwater fish have recently been

clarified, and techniques have been developed whereby fish

maturation and spawning can be controlled by me=s of

artificial variations of temperature and illumination. Using

these methods with goldfish, S. gairdnerii, O. rhodurus,

S. macrostoma, and P. altivelis, it has become possible to

make them spawn outside the natural spawning season, and this

can be of practical utility in the production of fry. When

illumination and temperature are within an approximately

limited range, normal maturation and spawning occur, but when

they are close to the limits, maturation and ovulation may be

greatly hindered. Temperature differs from illumination in

that there may be great differences between different places

of rearing, and it is particularly necessary to take note of

this. The upper limits of temperature at which there is some

hindrance of maturation and ovulation in S.J2,-airdnerii,

• 16

•

•

Cyprinius carpio and P. altivelis have recently been

experimentally determined, and it is considered to be important

to establish such temperature limits for the rearing of parent

fish of many species.

The nutrition of the parent fish (the quality and

quantity of feed) not only influences the maturation of the

fish with regard to the amount of spawn and the quality of

the eggs, but also controls the growth of the fish, and

there is a close relation between growth and the maturation

of the gonads. It is known that egg collection is easy and pll

that good quality eggs can be obtained from P. altivelis if

it is reared in uncrowded conditions. The good results of

egg collection are due to the fact that they ingest not only

the artificial food, but also large quantities of diatoms

from the edges and the bottom of the pond. • Experimental

studies have recently been made of the nutrition of several

types of freshwater fish such as S. gairdnerii and P. altivelis,

but many of them have not obtained any definite results. One

reason for this which can be offered is as follows. The

assessment of the quality of the feed is made by means of many

criteria such as the fraction of the experimental fish from

which eggs can be collected, the quantity of eggs collected,

the fraction of eggs laid which are fertile, the fraction

which hatch, the health of the fry after hatching, and the

fraction of the parent fish which survive spawning. These

•

•

• 17

criteria can be influenced by many factors other than the

quality of the feed occurring during the process of rearing.

These may include individual differences in the reproductive

ability Of the experimental fish, and variations in the

techniques of egg collection, of fertilization and of

incubation, and these may introduce such a range of error

that the experiments are difficult. It will be necessary to

establish satisfactory methods of assessing the quality of

the eggs after laying, of measuring the reproductive quality

of the parent fish, and of standardizing the methods of

egg collection.

There have been many experimental studies of the

artificial collection of eggs by means of the administration

of hormones, and general techniques are being established by

experience. However with some species or in some states of

maturation the reproductive efficiency may be low, and there

are many circumstances in which the administration of hormones

may be considered to be a factor in the death or damaged

health of the parent fish.

In some fish, eggs are to be collected by the "rainbow

trout" method and it may be found that ovulation occurs a

number of times. Since the hormone is thought to have a bad

influence on ovulation and on the health of the parent fish,

it is difficult to determine the appropriate time for egg

collection. In many species, satisfactory results are obtained

• 18

by the collection and fertilization of the eggs immediately

after ovulation. In S. gairdnerii a satisfactory development

ratio of more than 80% is found up to 10 days after ovulation,

but with species in which the temperature best suited to

rearing is high, the fertilizability of the eggs drops off

shortly after ovulation. In such species, the "carp" method

of collection gives better results than the "rainbow trout"

method. In the planned production of fry, the "rainbow trout"

method is superior to the "carp" method. However, in general,

the "carp" method gives satisfactory results with many of the

fish species under discussion. This is because there is

gl› • little increase in artificial handling in the 'carp" method,

and ovulation and spawning are allowed to occur in the natural

way. It is known that the water flow, the water temperature,

the physical and chemical properties of the bottom, and the

mutual stimulation between the sexes all take part in ovulation

and spawning, and all of these are used in the "carp" method.

However it is considered necessary that the relations between

these external stimuli and ovulation and spawning should be

better known, so that they can be applied positively to the

production of fry.

• We have here mentioned a number of the problems

involved in the production of freshwater fish fry, particularly

those concerned with the rearing of the parent fish and the

techniques of egg collection. In order to solve these problems,

p12

•• • 19

it will be of importance to study the process of maturation

of the eggs, the mechanism of ovulation (and its induction),

the connection between nutrition and metabolism of the parent

fish, and the relation of these to the environment in which the

fish are reared. It is necessary to plan for many stages of

study from basic research to practical application so

that the results may be used as a foundation for the rearing

of parent fish and for egg collection. It is also considered

that increasing importance should be given in future to studies

of genetic breeding in order to utilize parent fish of valuable

reproductive quality.

•

• 20

•

2. Marine fish. Reijiro HIRANO*. p13

Studies of the production of marine fish fry have

gradually increased since the last half of the 1950s. At

first, they relied on the collection of eggs from fish caught

in the wild. This method of egg collection involved very

strenuous efforts, and it was very soon recognized that it

was necessary that marine parent fish should be cultured.

Since the beginning of the 1960's progress has been made in

the techniques of culture of marine fish, and as the culture

industry has spread it has become possible to collect eggs

from a number of species. This is at present being done with

the species shown in ?able 2.1. However there are still many

basic questions to be answered about the effects of feed and

environmental conditions on the spaWning of marine fish, and

it is to be hoped that there will in the future be further

fundamental research.

However it will continue to be convenient with some

species or in some regions to keep wild-caught fish for a p14

short period before collecting the eggs, and it is important

that progress should be made in studies in this area. At the

present time, two methods are used for collecting eggs from

marine fish. They are.the so-called "artificial collection"

method, using pressure on the abdomen, and the "natural spawning

collection" method. The "artificial" method may be used to

* Department of Agriculture Science, University of Tokyo.

•

•

21

Table 2.1.

Examples of the collection of eggs from cultured parent fish.

Species Research agency ' Culture facility

Chrysophrys Hiroshima maritime Shore pond (75t) major experimental station Small

Oita maritime Small experimental station

Kanagawa maritime Embankment, 8400m3 experimental station

Tokushima maritime Aquarium (100t) experimental station

Kumamoto maritime Small experimenta7. station

Ishikawa Small

Mie, Owase maritime Shore pond 12.5mi expelimental station

_ . Kinki University Small

. Tokyo University • Shore pond, culture

' pond

Mylio macro- Hiroshima maritime Shore pond (75t) cephalus experimental station

Aichi maritime Shore pond (75t) experimental station

Nagasaki nui7ritional Small laboratory

Kinki University Small

Tokyo University Shore pond, culture pond

Rhabdosaurus Shizuoka maritime Small sarba experimental station

•

• 22

•

Table 2.1 (continued)

Oplegnathus Kumamoto maritime Small fasciatus experimental station

Nagasaki maritime Small . experimental station

Wakayama maritime Small ._ _ _ _ _ experimental - station , _ _

Kinki University Small

Erynnis Kinki University Small . -j,aPanica

Oplegnathus Kinki University Small _ . _ 'plunctata

'Parapristipoma Oita maritime Small 1•- .trilineaturi - --6kpériderital station

1 . _ , Tokushima maritime -e.. (111ari-um (100t)

- experimental station

lac. rack- Oita maritime Small - -- -Ugh - ' experiMental station ._ , ,

nexagrammas Hyoga maritime • q_P-917P pond otaki experimental station

Seriola Nagasaki maritime Small _ quinquera7 experimental station ._ diata

Kochi maritime Small experimental station

Mie Owase maritime Small experimental station

Kinki University Small

Seriola Kinki University Small aureovattata

Seriola Kinki University Small purpurascens

• 23

•

•

obtain eggs from fish caught in the wild, but the period which

is usable for fertilisation is believed to be short, and many

satisfactory results have been obtained with the "natural"

method in species which lay separately floating eggs1 .

When cultured fish are used as parents, it is usual

to employ the "natural spawning" method of egg collection.

To do this the male and female parent fish are housed in the

same tank, the spawn and the fertilized eggs are collected

and used for rearing. The ratio of males to females for this

purpose is usually about unity, but further investigation on

this point is required for each species. With appropriate

environment and management of the rearing, there will be little

difficulty if the place for rearing and the place for egg

collection are the same, but when the parent fish have to be

moved from the place in which they were reared to some other

place for egg collection, or when fish caught in the wild are

cultured for egg collection, consideration must be given to

the following points.

Careful attention must be given to the method and

season of capture and transport of the fish, and also the size

and construction of the tank used for collection. In general

a round tank gives more satisfactory results than a square

tank of the same surface area. In these circumstances, it is

also often effective to use hormones which stimulate the

gOnads, but when hormones are used it is evidently important

24

to consider the type of hormone to be used and its relation

to the state of maturation of the parent fish, and also the

quantity and time for its administration.

If the fish are to be removed from a roomy place of

rearing to a narrow tank for egg collection, the hormone

must be used as soon as possible after the nove. It has

often occurred - with Mylio macrocephalus that the hormone

became ineffective after the lapse of a week. It is believed p1 .5

that this is strongly connected to stress, and further studies

are considered to be needed. •

The hormone which is at present generally used is '

synaholin, but the studies of the relation betwem the state

of maturation of the fish and the amount of hormone to be

administered to a fish of a given weight are not yet complete.

Many experiments have been made in Which 4o to 120 R.U. of

synholin per kilogram of fish body weight are injected into

the dorsal muscles, but there is scope for further investigation.

It is also to be remembered that when wild-caught

fish are to be used, they may not be sufficiently mature when

caught, and it is often found that the administration of

hormones is then completely ineffective.

Another important point to remember with marine fish

is the so-called "successive spawning". A number of studies 2

have already made it well known that in Chrysophorus major

the same parent fish will spawn a number of times. Hibiya et al3

• 25

Table 2.2.

Collection of eegs from mylio microcephalus.

(Hibiya and Sato, 1973).

Date. 1 1 y 2 July 3 July 3 July 4 July 5 July

'Experiment number

B - 2 4o 30,00 0 60 ___ Few 100,000 units units

H - 2 syna- 500,000 syna- --- Few 400,000 holin holin

3 in- in- •600,000 --- 400,00 0 jected jected

7 300,00 0 --- 700,000

8 100,00 --- Few

Table 2.4

(Hibiya, Sato, 1973). DATE t

TEsT. tio • t i 12. 7 12. 10 12. 11 12. 11 12. 12 12. 13 12. 14

1 • 43g• • • 42g —g —.—g ---1;

2 20 40 -:-- 40 — 43• UNITS 'UNITS - 3 SYNA•• — SYNA -• — 15 26

HOE I A HOL IN 43 .71 . 4 INJECT

65 INJECT

5 En o ■ -7-- ED., _____ 66

29 —

6 • _____ '

_____ 52 31 —

g. This note is nowhere explained. (Translator).

•

• • •

Table 2.3

Number of ova spawned by Sillago sihama. (Sato, Hibiya, Kiyono and Hirano, 1973).

Test fish Date and Expti time of First Second Third Fourth

!Female Male hormone Injection night night night night ! injection 1 1

10 July 72 30 to 40 40 to 50 10 to 20 1 5 (1800) Syn 40 thousand thousand thousand

2 ; 5 i 12 July it 50 to 60 50,000 20,000 i (1800) thousand 60,000

•

3 1 3 2 13 July t, 100,000 10,000 1,000 1 (evening)

3 2 • 4 August ti Small 70 to 80 5,000 2 to 3

(1800) quantity thousand thousand

2 4 8 August vi 100,000 40 to 50 (1600-1700) thousand

i

2 3 " " " Syn 100 40 to 50 10,000 thousand

7 2 3 15 August 10,000 80,000

(1400) Syn 40 , I

2 7

have recently shown that when eggs are collected in a tank,

Mylio màcrocephalus, Sillago sihama, and Kareius bicoloratus

show successive spawning. (Tables 2.2, 2.3, and 2.4). It

will therefore be necessary to make further studies of each

species from which it is proposed to collect eggs by means of

the administration of hormones, in order to determine whether

or not their normal spawning habit includes "successive pU;

•

•

spawning". It has been found that when hormones are administered

to Limanda yokohamae "successive spawning" does not occur.

As is of course to be expected, it is difficult to

obtain a large number of eggs by means of pressure on the

abdomen in species in which successive spawning occurs. It

is with such species that rearing of the parent fish is

particularly important.

It has been shown with freshwater flsh that the

external environment, in particular the illumination and the

temperature, are important in the control of maturation

and spawning.

There has been little study of this in relation to

marine fish, but studies of the early egg collection from

Chrysophorus major through the use of warm water have been

in progress at the Mie-Owase marine experimental station p i7

since 1972. It was found that by keeping the water temperature

at 19° C to 20 °C during the winter of 1972 to 1973, it was

possible to maintain normal mature condition for 65 days

• 59

28

Table 2.S ••

Spawning by Chrysophrys major.

(Owase experimental station 1973)

Test Spawning period Number Total Ova per Ova per group of ova fish fish per

females day

(x104 ) (x104 ) (x104 )

,

,

A : 15 Feb - 12 Apr 6* 1662.5 305 5.6 (warmed

B 31 Mar - 15 Apr 5 254 50.8 3.2 (control)

*. Including one which died on 18 March.

Figure 2.1.

Spawning by Chrysophrys major.

(Owase experimental station 1973)

12 1 2 3 4 Date 1973

›

cd 501

0 4

CL..• . 3

NH 2 Q) ><

e n

10/11 20 inu io 2'0 . io L I:1111 Date.

Figure 2.2.

Water temperature during the period of culture.

Water temperature for group A.

0---• Water temperature in the test station rearing facility.

•

2 9

after 15 February. This was one month sooner than in the

control group, and the number of eggs produced per fish was

3 million, which was 6 times the number from the control group.

These experiments have shown that as with fresh-

water fish, the temperature of the water is a very important

factor of the external environment for marine fish. However

we still have insufficient information about the period when

the temperature should be increased and about the limits of

suitable temperature which will result in early spawning.

Further basic studies of this will be necessary.

There is also an intimate connection between the age

of the parent fish and the results of egg collection, and

from what little is known it is clear that there is much

which awaits further study. Individual future studies which

are considered important include the conditions in which the

parent fish are to be reared, the fodder to be given, the

acceleration or stimulation of maturation by the administration

of sex hormones 3 and the physical and chemical properties of

the eggs, especially those which float.

•

References.

1. Reijiro HIRANO.

Kurodai no chigyo shi iku.

Nichi sui shi .15 567 - 569 (1969).

Rearing of the young of the black porgy,

Mylio microcephalus.

Bulletin of the Japanese Society of Scientific

Fisheries, 15 567 - 569 (1969).

2. Hiroshimaken suisan shiken jo.

Madai no oyauo yosei ni kansuru kenkyu.

Showa 46 nendo shubyo seisan kenkyu hohokusho 1971.

Hiroshima prefectural maritime test station.

A study of the nurture of red sea bream çnEunrima2

• major.

Reports of research on the production of fish fry. 1971.

3. Takashi HIBIYA.

Horumon no seijuku sanran no jinko togyo ni

kansuru kenkyu.

Showa 47 nendo florin suisangyo shiken kenkyu

hihojokin ni yoru kenkyu hohokusho 1973.

Studies related to the artificial control of

maturation and spawning by hormones.

Report for 1972 of research subsidized by the Maritime

Industrial test station of the Ministry of

Agriculture and Forests. (1973).

•

• 3 1

4. Mie ken Owase suisan shiken jo.

Onkaisui shiiku ni yoru madai soki sanran ni tsuite.

Gyorui shubyo seisan kenkyu II. 1 - 6 1973.

Mie prefectural maritime test station, Owase.

On the early spawning of the red sea bream,

plirmonrionnnui2E due to rearing in warm sea water.

Studies of the production of fish fry. II 1-6, 1973.

•

32 •

Parent fish for ege collection.

3. The process of maturation of the gonads..

Kazunori TAKANO.

(Faculty of Fisheries,

University of Hokkaido).

The process of maturation of the gonads extends from

the formation of the gametes to their release, and includes

the morphological and functional changes of the gonads and

of many other related organs which accompany this maturation.

Thus this process of maturation begins as the peritoneal

region is formed during the embryonic developmert of the

original gamete, continues with the formation of the gonadal

primordium, and with the attainment of maturity passes on to

the first arrival at a functionally mature condition. This

takes a long time, up to the whole lifetime of fishes which

spawn only once. However in many fish which are able to

breed two or more times, and which after spawning can once

again pass from a sexually inactive condition to funutional

maturity, the process can be properly considered as that of

the ripening of the gonads. Moreover, since the process of

arriving at maturity for the first time involves morphological

changes of the gonads and of the related organs, it is

necessary to distinguish it from ihe so-called reproductive

cycle of the fully grown fish.

• 33

Since the endocrinological processes which take part

directly or indirectly in the maturation of the gonads are

dealt with under a separate heading, the morphological

development will form the main subject of the present outline

of the development of the gonads of female Teleosts.

1. The structure of the ovary and the oviduct.

The ovaries of many Teleostà develop as a pair to

left and right. However in the viviparous swordtail

Xiphophorus helleri 1 , and in the oviparous killifish Oryzias

latipes 2 a simple Dvary only is present. In these fish a

double gonad primordium is produced, but as the organs develop

the ovaries fuse and become a single organ. Th 3 process of

fusion of the left and right lobes depends on the species,

and in fish such as the surf fish Ditrema temmincki the simple

ovary retains traces of the original bilobate structure 3.

Hoar4 recognizes two types of connection between the

fish ovary and oviduct, the cystovarian type and the

gymnovarian type. The ovaries of teleosts are usually of

the cystovarian type. This type of ovary is almost completely

composed of a large number of thin sheets (lamellae) which

contain the gametes, and the ripe eggs which have matured in

the ovary break away into the ovarian cavity. The location

of the ovarian cavity depends on the species. For example

in Oryzias latipes and in the goldfish Carassius auratus it

extends dorsally from the somatic layers of the multi-lamellate

34

•

structure of the ovarian stroma, whereas in Cottus bairdii

it extends ventrally 5, and in the perch Lateolabrax japonicus 6

it passes through the middle of the ovary. However the

cystovarian type of ovary is in all cases connected at the

tip of the ovarian cavity to a short genital tube (oviduct),

and the ripe eggs ovulated to the ovarian cavity directly from

the ovary are discharged to the outside of the body by way of

this oviduct. In contrast to this, the eggs in the ovaries

of the Chondrostei, and in the Holostei family Amia, are all

exposed in the body cavity, and the ripe eggs are released into

the peritoneal cavity. Such a structure is called an ovary

of the gymnovarian type.

In the eel Anguilla japonica the ovary of the immature

fish develops from a long narrow ridge formed on the dorsal

peritoneal wall. It protrudes into the body cavity, and during

maturation swells up into a fimbria. The median side of this

ovary is covered by the ovarian wall, but the body wall side

lacks a covering membrane and the ovarian lamellae are

directly exposed to the body cavity. In salmon and trout the

ovary generally develops a triangular cross-section, and

apart from a portion of each tip the boundaries of the upper

edges on the body wall . lack a covering membrane. Thus in

these fish the ripe eggs formed in the lamellae of the ovary

are all ovulated into the body cavity. In the eel they pass

directly from the body cavity into the genital pore, in salmon

35

•

and trout they pass through an oviduct to the genital papilla - and are discharged to the exterior710 . In these cases in

which there is no genital organ attched to the oviduct, the

body cavity has a temporary role to fulfil, and even though

the ovary is of the cystovarian type in that one part of the

ovarian wall is covered, it would be rational to class it as

of the gymnovarian type.

We turn now to a histological examination of the ovary.

The smooth section of the ovarian wall of the cystovarian type

in Oryzias latipes are well developed and cytoplasmic

protuberances develop into a villus structure only on the

epithelial cells bordering on the ovarian cavity. In the

goldfish, the muscles of the ovarian wall are missing and the

epithelial cells bordering on the ovàrian cavity develop not

only the villi but also a typical 9 plus 2 pattern of ciliae6

According to the descriptions which can be gathered from

published reports, the ovaries of adult teleosts may be

classified on the basis of such structural characteristics

in the following way.

I. Ovary gymnoform, villi developed on epithelial

cells of the body cavity.

Anguilla japonica , Salmo trutta 11 , Oncorhynchus

nerka .

Unpublished.

•

•

• 3, lia. Ovary cystoform, no muscle developed in the

ovarian wall, villi present on the epithelial

cells of the ovarian cavity.

Rhodeus amarus 12 , Rhodeus ocellatus 13, goldfish14 ,

Misgurnus anguillicaudatus .

IIb. Ovary cystoform, muscle developed in the ovarian

wall, villi absent from the epithelial cells of

the ovarian cavity.

Fundulus heteroclitus 15 , Mugil cephalus16 , Pungitius

tymensis 17 , Oryzias latipes 18 and Tilapia

massambica

This classification of the ovaries of adult fish is

closely connected with their organogenesis, and more particularly

with the development of the ovarian cavity. If more of these

observations of the structure of the gonads of adult fish are

obtained, it may become possible to deduce the way in which

the organs are formed and the manner in which fish should be

ree?.red, and to understand the relations of the organs to their

breeding behaviour. This would lead to an increase in utility.

2. Genital cavity fluids.

It is well known that when ova are stripped from

rainbow trout or chum salmon a large amount of liquid is

expelled with the ripe ovà. In species with cystovarian

ovaries such as Oryzia latipes 19 and goldfish these liquids

are secreted by the epithelium of the ovarian cavity wall,

37

and in species such as Oncorhynchus nerka with gymnovarian

ovaries they are secreted from the inner epjAheirld ef the

body cavity and from the mesovarium. In either case, the

epithelium in the sexually immature stage is formed of a

simple flat layer of epithelial cells, but at about the time

that the ova begin to accumulate yolk, a morphological

specialization into a ciliate form begins. As maturity is

approached these cells develop either a cubic or a cylindrical p21

shape. At the time of ovulation the vertices show very

remarkable morphological changes and secrete liquids from an

excretory apparatus 20 . The Golgi bodies of the epithelial

cells have an importan:; role in the detailed structure of

these liquid producers, and water soluble substances are

accumulated in the interior of the cells, mostly by a process

of absorption. The liquids obtained in this way at the time

of o\-ulation have been called "coelomic fluids" or "peritoneal

fluids" according to the structure of the genital organs of

the fish in which they are produced but Ginzburg21 advocates

the use of the generalized name "cavity fluids". Even though

there are variations in the structure of the genital organs,

the place in which the ovulated ova are temporarily accumulated

can be thought of as a "genital cavity", and since the

excreted fluids have similar origins a distinctive term for

general use will be "genital cavity fluids". •

• 38

•

•

The composition, properties and functions of these

genital cavity fluids are not yet sufficiently well known.

It is known that the liquid obtained during the stripping of

ova from trout and salmon is normall y. a rather viscous serum-

like substance. Its properties have been fairly well

investigated in salmonids. The pH is 7.9 to 8.65 21

is relatively rich in Na, K, and Cl, it contains carbohydrates

and proteins23 , and the depression of the freezing point

shows that it is somewhat hypotonie to the contents of the

ripe ova and to the blood serum 21 It is not difficult to

imagine that the liquid has functions related to the retention

of the ripe ova after Dvulation, and to their lubrication and

protection during transport. It is believed that in the • stickleback lumitimensi.s17 this secretion takes part

in the formation of a jelly-like substance in which the ova

are wrapped. It has recently been found that this secretion

in the rainbow trout contains substances which enhance the

motility of the spermatozoa and prolong their life 23 , and it

has been suggested that they may possibly contain an essential

participant in the interaction of the sperm and the ova of

Oncorhynchus keta24 . It has also been found that mammalian

gonad stimulating hormones and fish pituitary extracts could

cause an admittedly incomplete ovulation of in vitro cultures

of rainbow trout, and it is suggested that the genital cavity

fluid may contain substances which control ovulation25 . It

22 . it ,

•

39

is therefore possible that the genital cavity fluids may not

only have physical functions at the time of discharge of the

ripe ova, but may also have physiologically important roles

in the whole process from ovulation to fertilization.

3. The process of ripening of ovarian ova.

The most important role of the ovary is the formation

of functional female gametes. The process of oogenesis can

be divided into a period of proliferation, a period of growth

and a period of maturation.

The period of proliferation is one of repeated mitodis

in the oogonium. In adult fish the period during which this

proliferation occurs depends on the species. In Gasterosteus

aculeatus26 , Fundulus heteroclitus 15 , Phoxinus laevis 27 and

Pleuronectes platessa 28 it is limited to a definite period

after spawning. However in Salmo gairdnerii 29 and in Rhodeus

ocellatus 3° oogonium division can occur throughout the year

and in the guppy Lebistes reticulatus (Poecilia reticulata)31

it occurs in a cycle of ovogenesis seasons. In the interphase

oogonium, a thin cytoplasm surrounds a round nucleus with

uniformly distributed, clear chromonemata and from one to

several karyosomes.

When the period of proliferation by division is

finished the germ cells enter the growth phase. The germ

cells of this phase are called oocytes. The growth phase can

further be divided into primary and secondary phases. The

• 40

•

primary phase starts with nucleolation of the prophases

of maturation division and with chromosome synapsis, and is

characterized by the formation of plasmosomes and an increase

of the egg cytoplasm. This phase can be called the karyosome

or the peri-nucleolus stage, and the oocyte, being without

yolk is in no way affected even by removal of the pituitary.

It next enters the secondary phase. The distinctive character

of the oocyte which is seen in this phase is the accumulation

of yolk materials in the cytoplasm. The yolk materials of

fish eggs are made up of three kinds, yolk vesicles, yolk

globules and fatty droplets 32 The yolk vesicles normally

appear around the periphery of the cytoplasm, and in the next

stage they increase in number and size, but their formation

ceases relatively early, and in the mature egg they are lined

up inside the cortical protoplasm. These yolk vesicles are

homologous to the cortical alveoli, and when the eggs are

fertilized they play an important part role in the formation

of the perivitelline space. It is known that these yolk

vesicles are formed of glycoproteins and contain mucopoly-

saccharides 32 . Thus although the yolk vesicles and the fatty

droplets are simultaneously present in fish ova, they can

both be easily identified by using histochemical methods to

test for polysaccharides.

The yolk globules form an important part of the yolk

substances in most fish ova, and are an important source of

energy during the development of the embryo. In all species

•• • of fish, the yolk globules are at first deeply stained with

haematoxylin and appear as minute granules in the cytoplasm

between the yolk vesicles, and as their number increases

they gather together centripetally. The completed yolk

globules are composed of a central portion wrapped in a

surrounding membrane and a peripheral layer, and it has been

found by means of the electron microscope that the central

part has a crystalline structure'. In the herring

Clutea pallasi135 , the goldfish14 and the bitterling Rhodeus

ocellatus 13 the individual yolk glr)bules do not fuse together

even in the ripe ova. However in the flounder Liopsetta

obscura36 and in the rainbow trout29 , the yolk globules

rapidly fuse together after the germinal cell (nucleus) is

displaced by the progress of vitellogenesis, and in the ripe

egg they form a single yolk mass. In Oryzias latipes37 and

Lebistes reticulatus 38 the period during which this yolk mass

begins to form is quite early, and consequently the nucleus

is also displaced early. Many histochemical studies have

shown that the yolk globules are complex, and consist mostly

of lipoproteins with an admixture of many polysaccharides and

other carbohydrates 32 . In addition to these yolk vesicles and

yolk globules there are also fatty droplets which take

individual shapes which depend on the species. The fatty

droplets principally contain neutral fats, in particular 32 glycerides . The time at which the fatty droplets appear

•• • 42

in the oocytes depends on the species, and in Lebistes

reticulatus 38 , Lateolabrax japonica 6 , and Anguilla japonica39

they appear earlier than the other yolk substances. However

in Oryzias latipes4o and rainbow trout 29 they appear

simultaneously with or somewhat later than the formation of

the yolk vesicles and in Hypomesus àaponicus41 they appear

quite late, after the formation of the yolk globules.

The secondary growth phase can be divided into several

phases like the primary phase. Thus the period during which

the yolk vesicles appear and are formed can be taken as the

yolk vesicle period and divided into first, second and third

stages of the accumulation of yolk globules. Or the process

of oogenesis may be divided into phases characterized by

stages of formation of the fatty droplets. Since the process

of oogenesis is dynamic, these distinctions may be made in

any convenient way, and will depend on the species and on the

objectives of the study. Various methods of listing the

stages and the degree of maturation of fish ova have in the

past been proposed, but the most accurate is the histological

method. When the maturation of the ovarian egg is expressed

in terms of its cytological properties the details are made

as comprehensible as is possible, and comparative studies can

be made more useful by the use of a common terminology for

ail species.

• 43

When the accumulation of yolk is completed the egg

enters a stage of maturation and Cleavage. In many species

other than Oryzias latipes and Lebistes reticulatus the

nucleus, which up to now has been centrally established,

migrates to the animal pole in the direction of a previously

formed micropyle. This stage is called the stage of migration

of the blastula. The mechanism by which the nucleus is

displaced is as yet unknown, and the role played by the cells

of the micropyle presents a problem for the future. Soon

after the nucleus reaches thu surface layer of cytoplasm in

the neighbourhood of the micropyle, its profile becomes

unclear, and a stage of dissolution of the blastula beginsc

During this prematuration stage thread-shaped nucleolus

material, deeply coloured with haemotoxylin appears in the

region surrounding the chromosomes. When this once more

disappears, the chromosomes become clearly distinguishable 39 '

Next comes the first maturation cleavage in which the first

polar body is discharged from the chorion, and then after

the second stage of maturat'..on cleavage is reached ovulation

occurs8 . This phase is known as the phase of maturation.

In Figure 3.1 four species of teleosts are used as

examples to show how the changes in the degree of maturation

of the ovary can be based on the development of the egg,

right up to the first time that functional sexual maturity is

reached. Immediately after birth, the viviparous guppy

42.

- iiV3A ZI II 01 6 j 11 01 6 g L 9 S InJ 1 u cn 6 8 L 9 I H1NOGI

7

(S961

(1961 niaup.tp23 mains iv la Van s')

vy.t au grupu8t1ioDu0

11;0111.11134 . 1113e0,1

3srls sr. lo nonwt u 3d

3D Y1S 3N YUSW3W )410A

—peq. -em-p_se . auTT paq.q.op , poTaed Ge2JeAy 'CD

tenxas unj eiTu -ree eaojact uoTq.uanm /Ç.JUAO UT saetreqo

ave aanzu

35 %US 3ingo1a ' xlcu

3SY1 $ Si S3N3b01,111A

39Y St S3N3SC;13111A

3UYIS SI S3N 30011314

NO I irunivw IsAoolsvm

35 us 3nin›.3idd

35 VIC 3nsv47

3S Y1S

N0 11Yell VW AW 'MO

•

45

Poecilia reticulata contains oocytes from •the karyosome stage

to the peri-nucleolus stage, and within 70 to 120 days after

birth they pass beyond the third yolk droplet stage. Early

individuals . may contain fertilized eggs after 80 days. In

contrast, the ova of the rainbow trout Salmo gairdneri reach

the first yolk stage only in July after one or two years of

maturation, and after this stage egg development proceeds

rapidly, the ripe stage being reached in December29 . The eggs

in the ovary of the sockeye salmon Oncorhvnchus nerka

similarly wait till the year of maturation, and pass from the

'fatty droplet stage to the first yolk droplet phase during the p25

period from mid-April to mid-May, and then grow rapidly to

become ripe in the middle of 0ctober43 . When the eel Anguilla

japonica is artificially brought to maturity, ripe ova are

obtained in less than three months from the first accumulation

of yolk. Thus the length of time after birth needed for the

attainment of functional maturity depends on the species, but

in all cases the second phase of growth, the period needed

for the accumulation of yolk is several months. That is to

say that fish which require a long time for maturation remain

in an infantile condition of sexual immaturity for most of

the time, but once the "trigger is pulled" they reach the

functionally mature condition in a short time. The period

of transformation from the sexually immature state to the

mature state will be characterized by the commencement of

secretion of gonad stimulating hormones, but the physiological

mechanism by means of which the action is triggered is not

• 46

•

•

yet known. There are extra problems in those fish which need

an extremely long time to become mature, and in the many

species which divide into groups, one of which reaches

maturity within a year while the other remains immature.

4. • Ovulation.

Ovulation is the phenomenon of the discharge of the

mature oocyte from the ovarian lamellae into the ovarian

cavity or the body cavity. Before ovulation the oocyte is

wrapped in two layers of theca cells from the interior of the

gra-ulosa membrane cells (the epithelial cells of the

follicles). There is a chorion between the oocyte and the

granulosa cells. The chorion can usually be distinguished at

about the time of the peri-nucleolus stage, and as the oocyte

enters the second stage of growth it can be seen to be

composed of an internal and an external layer44 . The light

microscope shows that the zona radiata is pierced by many

pore canals, through which villi protrude in both directions

from the oocyte and the granulosa cells. These villi are

believed to have an important role in the interchange of

metabolic materials.

As the maturing oocyte approaches ovulation, the villi

are cut off from the pore canals of the chorion and the

adhesion between the chorion and the granulosa cells, or

between the inner and outer thecal cells is weakened. The

granulosa cells take part in producing a fluid filling the

gap which forms44,45 . Soon afterwards the cell layers

•• •

•

•

surrounding the oocyte rupture and the oocyte separates from

the ovarian lamellae. In the chum salmon and the rainbow

trout it is ovulated from a previously cracked portion of

the internal thecal cells 8 , but this does not happen in

goldfish, in which the oocyte continues to be protected by

the upper part of the lamellae.

The morphological changes which accompany ovulation

are preceded by a remarkable absorption of water into the

ovarian egg, and it is suggested that this is one of the

factors which cause ovulation46 ' 47

It is very important to be able to estimate precisely

the degree of maturation of the fish, especiall7 if ovulation

is to be artificially controlled. Methods which are being

tried out on an experimental scale include the insertion of

a polythene tube into the ovary in circler to suck out some of the

ova48 , and the control of the external environment so as to

cause all fish to reach the reproductive phase together49 .

Desirable conditions for such methods include simplicity of

operation, rapidity of determination, and little damage to

the body of the fish. This question of the determination of

the degree of maturation remains one of the principal problems

for the future.

5. The functions of ovarian hormones.

In addition to producing germ cells, the ovary also

has the important function of producing and secreting ovarian

50 hormones. The sex hormones estrone, estradio1-17, and

p26

•• • 11-ketotesto sterone 51 are known to be present in teleost ovaries.

Reactions have been observed in the eranulosa cells52 ' 53 , in

the thecal cells 54 and between them53 ' 55 , and also in the

cells between the granulosa membrane and the follicle56. It

has been suggested that the sex hormones contribute to

maintaining the development of the shape and secreting ability

of the epithelial cells of the ovarian cavity wall", and of

the production of ova58 , and that they indirectly participate,

through the liver cells, in the production and supply of yolk

material 59,60 . Recently 11-deoxycorticosteroids61 have been

extracted from teleost ovaries, and they are supposed to be

concerned in the maturation and ovulation of the egg in the

ovary. In general, if the ovary is surgically removed from

a teleost, the source of the hormone and the tissue on which

it acts are lost at the same time, so that it is difficult to

elucidate the action of the ovarian hormones by physological

means62 . This leaves many problems of very great interest

for the future.

In order to locate the presence and activity of the hormone

producing cells in the ovary, enzyme histochemical tests have

been made with 1.3 -HSD, (z \:5-3p -hydroxysteroid dehydrogenase). 1

References.

1)

8)

10)

J. M. ESSENDERG : Sex-differentiation

in the viviparous teleost Xiphophorus

helleri HECKEL. Biol. Bull., 45, 46

--970923).

2) K.ONITAxE :Morphological studies of

normal sex-differentiation and indu-

ced sex-reversal process of gonads in

the medaka, Oryzias latipes. Annot.

Zool. Japon. 45, 159.-169 (1972).

3) SE E PAGE 53

4) W. S. Ho.ta : The gonads and repro-

duction, in "The Physiology of Fish-

es" (M. E. Buown ed.), Vol. 1, 287—

321, Academic Press, New Yorl • C1957).

5) H. W, HANN : The history of the germ

cells of Cottus bairdii GIRARD. J.

Morph., 43, 427-497 (1927).

6) I. HAYASHI : On the ovarian matura-

tion of the Japanese sea bass, Lateo-

labrax japonicus. Japan. J. Ich-

thyol., 19, 243---254 (1972).

7) D, HEY : The fertility of brown trout

eggs at the Jonkershock Inland Fish

Hatchery. Trans. Am. Fish. Soc.,

77, 65-40 (1949).

SE E PAGE 53

9) V. D. VLADYKOY : Fecundity of wild

speckled trout (Salvelinus fontinalis)

isi Quebec Lakes. J. Fish. Res. Ba.

Canada, 13, 799--841.

SEE PAGE 53

11) K. R. Asumr : The effect of steroid

hormones on the brown trout (Salmo

trutta L.) during the period Of

gonadal differentiation. J. Embryo!.

Exp. Morph., 5, 225-249 (1957).

12) L.H. BRETSCHNEIDER and J.J.Durvstriz

DE WIT : Sexual Endocrinology of

Non-mammalian Vertebrates, 146 pp.,

Elservier Publ. Co., Baltimore (1947).

1.1) K. Sarast : Correlation between the

growth of the ovipositor and ovarian

conditions in the bitterling, Rhodeus

ocellatus. Bull. Fac. Fish. Hokkaido

Univ., 13, 137-151 (1962).

14) K. YAMAMOTO and F. YAILAZAM

' Rhythm of development in the oo-

cytes of the goldfish, Carassius

auratus.. Bull. Fac. Fish. Hokkaido

Univ.,12, 93-410 (1961). • .

15) S. A. MATTHEWS : The seasonal cycle

in the gonads of Fundulue. Biol.

Bull., 75, 66.--.74 (1938).

16) A. H. STENGER A study of the

structure and development of certain

reproductive tissues of Mugil ceph-

alus LINNAEUS. ZOO/OgiCG, 44, 53--70

(1959),

17) T. S. Ysussioro : Eggs and ovaries

of the stickleback, Pungitius tymen-

sis, with a note on the formation

of a jelly-like substance surrounding

the egg. J. Fac. Sci. Hokkaido Univ.

VI, 15, 190,...,201 (1963).

18) K. YAMAMOTO : Cyclical changes in

•

• 5 0

the wall of the ovarian lumen in the medaka, Oryzias latipes. Annot.

Zool. Japon., 36, 179-186 (1963).

19) K. TAEANO : Fine structure of the wall of the ovarian lumen in the teleost, Oryzias latipes. Bull. Fac.

Fish. Hokkaido Univ.,19,76-82(1968).

SE E PAGE 54

21) A.S. Grxzentos : Fertilization in Fishes and the Problem of Poly-

sPerinY. 36 51V., Izdatel'stvo "Nauka", Moskva (1968).

22) SEE PAGE 54

23) T. YOSHIDA and M. Nounne. : A substance enhancing sperm motility in the ovarian fluid of rainbow trout. Bull. Jap. Soc. Sci. Fish., 38, 1073 (1972).

24) SE E PAGE 55

25) B. JALABERT, B. BRETON and C. BRY

Maturation et ovulation in vitro des ovocytes de l'a Truite Arc-en-Ciel Salmo gairdnerii. C. R. Acad. Sci. D, 275, 1139-4142 (1972).

26) A. CRAIO•BENNETT : The reproductive cycle of the t h ree- spi ned stickleback, Gasterosteus aculeatus LINN. Phil.

Trans. roy. Soc. London, 13, 219, 197--279 (1930).

27) W. S. BI:mu:won : Gametogenesis and some endocrine factors affecting it in the adult minnow (Phoxinus laevis

L.).J. Endocrinol., 3, 211---219 (1942).

28) W. A. BARR : The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (L). I. Cyclical changes in the normal ovary. Gen.

Comp. Endocrinol., 3, 197-204 (1963).

29) SEE PAGE 56

30) K. 7'Am/dim.° and K. SHIRAI : Origin of the yearly crop of eggs in the bitterling, Rhodeus ocellatus. Annot.

Zool. Japon., 35, 218-222 (1962). 31) K. TAXA» : Origin of the oocytes in

the adult guppy, Lebistes reticulatus.

Bull. Fac. Fish. Hokkaido Univ.,18,

137,-142 (1965).

32) S' EL PAGE 56

33) K. YAIIAMOTO and I. 00Tà. : Fine structure of yolk globules in the oocyte of the zebrafish, Brachydanio

rcrio. Annot. Zool. Japon., 40, 20---27 (1967).

34): N. N. GUPTA and K. YAMAMOTO:

Electron microscope study on; the fine structural changes in the oocytes

- of goldfish. Carassius auratus, during

yolk formation stage. Bull Fac. Fish.

Hokkaido Univ., 22, 187-205 (1971).

35) K. YAMAMOTO : Studies on. the formation of fish eggs. VII. The fate of the yolk vesicle in the oocytes of the herring, Clupea

20)

SEE PAGE 56

SEE PAGE 57

• 51

•

during vitellogenesis. Annot. Zool.

Japon., 29, 91 ,-96 (1956).

36) . K. YAMAMOTO : Ditto. XI. The

formation of a continuous mass of

yolk and the chemical nature of lipids

contained in it in the oocyte of the

flounder, Liopsetta obscura. J. Fac.

Sci. Hokkaido Univ., VI, 13, 344-351

(1957). 37) K. YAMAMOTO and H. Yosutou. :

Rhythm of development in the oocyte

of the medaka, Oryzias latipes. Bull.

Fat. Fish. Hokkaido Univ.,15, 5-49

(1964). 38) K. Tars»: On the egg formation

and the follicular changes in Lebist es

reticulatus. Bull. Fac. Fish. Hokkaido

Univ., 15, 147-155 (1964).

39)

40)

41) K. YAMAMOTO : Studies on the for-

:nation of fish eggs. VIII. The fate of the yolk vesicle in the oocyte of

smelt,../iypomesus japonicus, during

vitellogenesis. Embryologic, 3, 131-- 138 (1956).

42) K. YAMAMOTO : Ditto. II. Changes in

the nucleus of the oocyte of Liopsetta

obscura, with special reference to the

activity of the nucleolus. J. Fac. Sci.

Hokkaido Univ., VI, 12, 375-499 (1956).

43) R. IsuinA, K. TArlot and S. ARITA :

Criteria for the differentiation of

mature and immature forms of chum and sockeye salmon in northern seas.

In!. North. Pat. Fish. Corn. Bull., 5, 27-47 (1961).

44) K. HiaosE : The ultrastructure of

the ovarian follicle of medaka, Oryzias latipes. Z. Zellforich., 123.:

316-.329(1972). • 45) K. YememoTo and F. YAmAzearr

Hormonal control of ovulation and spermiation in goldfish. Gunmcr

SymPosia on Endocrinol., 4, 131.-145 . (1967).

SEE P A GE 57

47) K. HIROSE, T. HIRANu and R. !man :- Effects of salmon gonadotropin in

the ayu, Plecoglossus altivelis, with

special reference to water balance. Conde. Biochem. Physiol., 47A, 283 .--,289 (1974).

48) Z. H. SLIEHADEll, C. M. Coo and K.

K. MILISEN: Validation of in vivo

method for monitoring ovarian de-

velopment in the grey mullet (Mugit

cephalus L.). J. Fish. Biol., 5, 469 —496(1973).

49) SEE PAGE 57

50) Y. KATZ, 13. ECKSTEIN, R. 'tux and

R.GermEn: Estrone and estradiol-

na in the ovaries of Tilapia aurez

(TELEOSTEI, CICHLIDAE). Camp. Bi--

ochem.Physiol., 40B,1005--1009(1971).

51) U. EYLATU and B. ECKSTEIN :Isolation

of 11-ketotestosterone and dehydro-epiandrosterone from ovaries of the

common mullet, Mugit capito. Gen.

Camp. Endocrinol.,12, 58-62 (1969).

46)

•

• 52

•

52) J. G..D. LAMBE R T: The ovary of

guppy Poecilia reticulata. The

granulosa cells as sites of steroid

biosynthesis. Ibid., 15, 464-476(1970).

53) Z. YARON : Observations on the

granulosa cells of Acanthobrama

terraesanctae and Tilapta nilotica

(TELEoszst). Ibid., 17, 247-252(1971).

K. YexAmoTo and H. ONOZATO :

Steroid-producing cells in the ovary

of the zebrafish. Brachydanio rerio.

Annot. Zool. Japon., 41, 119-128 (19

68).

55) G. BAHL : Histochemical localization

of ' 413-31?-hydroxysteroid dehydro-

genase in the ovaries of a teleost

fish, Scornber scomber L. Gen. Comp.

Endocrinol., 5, 284.--296 (1965).

56) Y. Iv/mum : Histochemical detection

of 45-3a-hydroxysteriod dehydro-

genase in the ovary of medaka,

Oryzias • latipes, during annual «

reproductive cycle. Bull. Fac. Fish.

Hokkaido Univ., 23, 177-484(1973).

57) H. TAKAHASHI and K. Dace» : Sex

hormone-induced precocious hyper-

trophy and ciliation of epithelial cells

in the ovarian lumen of the goldfish.

Annot. Zool. Japon., 44, 32-41(1971).

58) N. E. STACEY and N. R. Loam :

Regulation of spawning behaviour

in the female goldfish. Nature, 247,

71--72 (1974).

59) K. AIDA, PHAN-VAN-N(3ex and T.

HIBIYA : Physiological studies on

gonadal maturation of fishes-I Sexual

difference in composition of plasma

protein of Ayu in relation to gonadal

maturation. Bull . JaP. Foe. Sci. Fish.,

39, 1091-1106 (1973).

60) K. AIDA, K. HIROSE, M. YOKOTE and

T. HISIYA : Ditto. II Histological

changes in the live:. cells of Ayu

following gonadal maturation and

estrogen administration. Ibid., 39,

1107-1115 (1973). •

61) L. COLOMBO, H. A. BERN, J. PIEPRZYI

and P. W. JOHNSON : Biosynthesis

of 11-deoxycorticosteroids by teleost

ovaries and discussion of their

possible role in oocyte maturation

and ovulation. Gen. Comp. Endo-

crinol., 21, 168-478 (1973).

62) R. REINBOTH : Hormonal control of

. the teleost ovary. Am. Zoologist, 12,

307-424 (1972).

54)

•

• 53

References.

3. MIZUNOE Kazuhiro.

Umitanago no kenkyu III.

Umitanago no ranso no seijuku narabi ni kisetsuteki

junkan ni kansuru kenkyu.

Nagasaki dai suisan ken ho 11 1 -17 (1961)

K. Mizunoe.

Studies of Ditremma temmincki III.

Studies of the maturation of the ovary of, Ditremma

temmincki and of its seasonal cycle.

Reports of the Fisheries research laboratory,

Nagasaki University, 11 1-17 (1961).

8. YAMAMOTO Tadashi.

Nishin, nijimasu oyobi yatsume no hairan katei.

• Gyo zatsu • 182-192 (1955).

Ti Yamamoto.

The process of ovulation in Clupea pallasii, Salmo

gairdnerii and Entosphema japonicus.

Japanese Journal of Ichthyology. 182-192 (1955).

10. NOMURA Minoru.

Nijimasu no jinko sairan ni kansuru kiso kenkyu I.

Seijuku ni tomonau seishokuso no keitai henka to

hairan katei.

Nichi sui shi 28 409-416 (1962).

• ••

•

10. M.Nomura.

Basic research related to the artificial collection

of eggs from Salmo gairdnerii.I.

Morphological changes accompanying maturation of

ovaries and the process of ovulation.

Bulletin of the Japanese Society of Scientific

Fisheries, 28 409-416 (1962).

20. KUROZUMI Kazumasa.

Bunpitsu no keitaigaku ni kansuru denshi

kenbikyot3ki kenkyu.

Denkengakkai ho 14 12-26 (1965).

K. Kurozumi.

Electron microscope studies of the morphology of

internal secretion.

Journal of the electron microscope society,

14 12-26 (1965).

22. NOMURA Minoru.

Nijimasu no jinko sairan ni kansuru kiso kenkyu VI.

Tansui, tochoeki, taikoeki, nyo no kishaku ni yoru

seishi no katsudosei to seieki no chozo ni tsuite.

Nichi sui shi 723-733 (1964).

55

22. M. Nomura.

Basic research on the artificial collection of

eggs from rainbow trout. VI.

The effect on the storage of mut and the activity

of sperm produced by dilution with fresh water,

isotonic fluid, coelomic fluid or urine.

Bulletin of the Japanese Society of Scientific

Fisheries .3..2 723-733 (1964).

24. TAKANO Kazunori, HIROI Osamu, YASUKAWA Masao,

SUETAKE Toshio.

Sake, masu rui no tamago oyobi seishi no hozon ni

kansuru kenky,. I.

Sake (Oncorhynchus keta) mi jusei tamago no hozon

ni tsuite.

Sake, masu fuka basho ho .2.Z 31-37 (1973).

K, Takano, O. Hiroi, M. Yasukawa, T. Suetake.

Studies of the preservation of the roe or mut

of salmonids I.

Preservation of unrertilized eggs of. Oncorhynchus keta.

Salmon and trout hatchëry reports,

• ?I 31-37 (1973).

56

•

29. YAMAMOTO Kiichiro, OOTA Isao, TAKANO Kasunori,

ISHIKAWA Tetsuji.

Nijimasu no seijuku ni kansuru kenkyu I.

Ichinengyo no ranso no hattatsu ni tsuite.

Nichi sui shi 11 123-132 (1965).

K. Yamamoto, I. Oota, K. Takano, T. Ishikawa.

Studies of maturation of the rainbow trout I.

On the development of the ovary in first-year fish.

Bulletin of the Japanese Society of Scientific

Fisheries. 21 123-132 (1965).

32. YAMAMOTO, Kiichiro.

Gyoran ni oke:Ju rano keisei.

Saibo kagaku shinposhiumu 8 119-134 (1958).

K. Yamamoto.

The formation of yolk in fish eggs.

.Cytochemical symposia, 8 119-134 (1958).

39. YAMAMOTO Kiichiro, 6MORI Masaaki, YAMAUCHI Kohei.

Nihonsan unagi (Anguilla japonica) no rankeisei

ni tsuite.

Nichi sui shi 40 9-15 (1974).

K. Yamamoto, M. Omori, K. Yamauchi.

On the development of the ova of the Japanese

eel (Anguilla japonica).

Bulletin of the Japanese Society of Scientific

Fisheries 40 9-15 (1974).

• 57

40. YAMAMOTO Tadashi.

Medaka no ranshi keisei, toku ni sono saibo

kagaku teki kenkyu.

Gyo zatsu 4 170 - 181 (1955).

T. Yamamoto.

Study of the development of the ovum in Oryzias

latipes, with special reference to its cytochemistry.

Journal of Ichthyology 4 170 - 181 (1955).

46. HIROSE Keichi.

Gyorui no hairan no naibunpitsu

To kai sui ken ho /Lk 67 - 81 (1973).

K. Hirose.

Endocrine control of ovulation in fish.

Bulletin of the Tokai regional fisheries

research laboratory .72± 67 - 81 (1973).

49. TAKANO Kazunori, KASUGA Seiichi, SATO Shigeru.

Jinko koshuki shita ni okeru medaka no seishoku

nichishuki.

Hokudai suisan iho 24 91 - 99 (1974).

K. Takano, S. Kasuea, S. Shigeru.

Diurnal• reproduction cycle in Oryzias latipes

under artificial control of the light cycle.

Bulletin of the Faculty of Fisheries,

Hokkaido University, 24 91 - 99 (1974).

• 58

4. Detailed characteristics of reproduction.

Sadaichi KATO.

(Freshwater fisheries research laboratory, Nikko).

ln order to produce fry efficiently in restricted

conditions, it is important to have a sufficient understanding

of the many characteristics peculiar to the reproduction of

the parent fish. By means of changing the methods and

conditions of rearing and the ways of breeding in accordance

with this understanding, it will be possible to make improvements

and to select parent fish which have desirable characteristics.

With this in mind, the Nikko division has investigated the

variability of many aspects of the growth of the rainbow trout

Salmo gairdnerli. Detailed results have been reported about

the relation of the age and the growth of the parent fish to **

the weight of the ripe eggs , the number of ripe eggs ' the ***

weight of an egg , and the season of spawning.

1. The ripe eggs.

The weight of the ripe eggs is determined by the

number of eggs and the weight of each egg, but since it was

found that the weight of the ripe eggs was the quantity which

among these three was most closely correlated to the body

* The gross weight of the completely mature eggs. ** The number of completely mature eggs. **** The average Weight of a completely mature egg.

P 31

•

59

weight of the fish at the time when the eggs were collected,

it was thought that the ratio of the body weight to the

weight of the gonads (which in this case is the weight of

the ripe eggs ) would be a most important reproductive index.

It is found that there is a linear relation between

the weight of ripe eggs and the body weight which is

independent of the age of the fish, and an increase of body

weight is accompanied by an increase in the weight of the

ripe eggs. The weight of ripe eggs shows the same trend when

the fish are large after being raised with a plentiful food

supply. Since the weight of one egg:hardly increases at all

as body weight increases, the increase in the weight of ripe

eggs must necessarily be due to an increase in the number of

eggs. The chance of survival of a single egg is therefore

not normally affected by rearing in conditions where there is

an extremely large food supply.

Since the gonad index is inversely related to the

body weight, its value is lowered in large fish.

2. The gonad index of mature individuals and the Pize

distribution of eggs in the ovary.

Many aspects of the degree of maturation can be

expressed in terms of the gonad index, but its value is low

in the early stages of maturation and it cannot be used to

In this paper the gonad index is _yr .ielt_g gonads x 100. Body weight

•• • 60

•

discriminate between individuals which will mature this year

and individuals which will not. These two classes can be

distinguished by the temporal change of the distribution of

egg diameters in the ovary. Figure 4.1 shows the relation

at various times between the gonad index and the diameter of

the eggs in the ovary .

As the spawning season approaches, two groups can be

distinguished. One group contains individuals in which the

average egg diameter and the gonad index is increasing, the

other contains individuals in which the average egg dnmeter

is less than one millimetre and the gonad index is still low.

As reported by Yamamoto et al 1 , it is supposed that the latter

group contains individuals which will not mature this year.

Since the two groups remain clearly separated after

July, it is believed that no individUals pass from the group

which will not mature into the group which will mature. Thus,

although it is not possible to distinguish positively between

the two groups in May, the individualsin which the egg diameter

exceeds one millimetre are believed to be those whicl-. will

mature this year.

The seasonal changes of the distribution of egg

diameters in the ovaries of individuals which will mature this

year are shown in Figure 4.2. In April and June, the gonad

indices are low, and it is difficult to determine whether an

* The average value for the group of eggs which is in an advanced stage of development.

p33

5

ro

ed 10

0 t..5 •

April GoNA0 INDEX (%).

0.7 .

. 1 . ,

: June . .

,r/\, . . 0.5

, August: , -

1.4 ..

. , September

. 5.2

_

. n October 6.1

,-...... i ....s. 4

10

0

e•—■

• o ,

P 0

0

p1 1

10

O

• 61

0

- May

•

d■ t I • 1

- Jùly

W. • . ,•• *, , , 'AuguSt

. te se, 1

• t

- :0Otober .: ••

>i • ,...... •-• ee

. s.. . - ..e

. .

te, , • , 0 2 4.

Mean diameter of ovarian eggs (mm).

Diameter of ovarian eggs (mm).

Figure 4.1.

Mean diameter of

ovarian es and

gonad index.

Figure 4,2,

Variation with time of

the distribution of diameters

of ovarian eggs in mature

individuals.

•

• 62

•

•

individual will or will not mature this year. However the

distribution of egg diameters in the ovaries shows two peaks,

one containing eggs which are in the course of maturation and are

advanced and one containing eggs which are not maturing and

it is to be supposed that the eggs in the former group, in

which the average egg diameter exceeds one millimetre, are

those which will mature this year. In August, the group in

which the egg diameter is less than one millimetre and the

group in which maturation is proceeding are completely

separated. In September and October the two groups are

similarly distinguishable, and as spawning approaches the

small-diameter eggs r.main unchanged and only the eggs of the

group which is maturing increase in diameter. It is

particularly remarkable that after the maturing and non-

maturing groups have separated, the maturing eggs are not

supplemented from the small egg group. This type of egg

diameter distribution is found to be quite normal in healthy

individuals, but in diseased individuals or in those which

have been kept in conditions of starvation eggs of size

intermediate between those of the maturing group and the

small egg group have been found. Even in these cases no

peaks other than the two already described have been found.

3. The ripe eggs.

It appears that after maturation has begun, none of

the eggs in the group which did not start to mature transfer

p34

• 63

1 0.743

0.838

0.707

0.745

0.582

0.740

0.617

0.686

2

0.587

0.785

0.634

0.635 4

Group No.

Group of fibh spawning for the first time,

Body weight in May and number of ripe eggs*.

Body weight in August and num-ber of ripe eggs*.

Body weight when mature and number of ripe eggs.

to the other group, and if this is so, the number of mature

es •will not exceed the number which were present when the

two peaks separated at the start of maturation. Those shown

in April . in Figure 4.2 can be said to determine an upper limit

to the number of eggs which can be spawned this year.

It is true that the size of the fish has a definite

influence on the number of ripe eggs, but it appears that the

number of ripe eggs will not be increased by delaying the

maturation season in order that the parent fish may be larger.

Table 4.1.

Comparison of correlation coefficients.

* Number of ripe eggs when fully mature.

• 64

•

•

Four groups of fish spawning for the first time were

investigated. The body weight of each individual was measured

in May, in August, and at the time of spawning in November to

January, in order to discover the season at which the body

weight was best correlated with the number of ripe eggs.

The results are shown in Table 4.1, and the highest degree of

correlation was that between the number of ripe eggs and the

body weight in August. As has already been stated, it is

believed that the number of eggs in an individual fish which

will mature during the current year does not increase after

May. Thus if there is any change at all in the number of

• eggs after May, this change can only be a decrease in the

number of eggs which may mature. Indeed, the fact that the ■

number of ripe eggs is highly correlated with the body weight

in August, shows that the number of ripe eggs has in many

individuals already been decided at some time in August. It

therefore appears that if it is desired to increase the

number of ripe eggs in the parent fish, the fish should be

chosen to be large by April at the latest, and from then

until August attention should be transferred to the nutrition,

especially the quantity and type of food, given to the

parent fish.

4. The weight of an egg.

There have been many studies of the relation between

the size of the fish and the weight of the egg, and it is

• 65

P35

•

•

reported that an increase in body weight is accompanied by an

increase in egg weight, but it is not yet known what relation

there may be between the weight of the egg and the size of

the fish in different year groups.

Figure 4.3 shows the relation between body weight and

egg weight according to age. It can be seen that on the

whole an increase of body weight is accompanied by an increase

in egg weight, but there is no evident relation between body

weight and egg weight when fish of the same age are compared.

In the case of the 3-year old fish shown in Figure 4.3, those

of body weight 1000g have eggs weighing 57mg and those of body

weight 2000g have eggs weighing 62mg, so that doubling the

body weight results only in an 8% increase of egg weight.

The relation between body weight and egg weight was also

investigated in the four groups of fish spawning for the

first time, and there was no correlation between the two

quantities in three of the groups. It appears from this that p36

factors other than the size of the fish contribute to the

size of the eggs.

An interesting question is whether individual fish

which have produced large eggs in one year will produce large

eggs in the next year, that is whether the size of the eggs

is a genetic characteristic. For this purpose individuals

were tagged and investigated,-and correlation coefficients

were obtained between the size of the eggs spawned in the

• 66

100

tx0

60 - • 4->

• • •

sa) 3 '

4.0 e) r

j4 t 6

cd

. .

• ..

• • a • .. -

• • • • •

o 0 0 .7

o 0 • •0 o•- • c o ° co! co

o oo. o o o • • • 6 o.

0 0 0 ' Jo. ... :

° • of ° . °

. • ° eeoe°

• • • : 0 0 . 0 .

• 000. _DO O. 0 " ° se.

e0 o •°C..e:e .° -. . •

0.00 .- -.0..0 . '.-.. ... • ....1!,...- a..

. 0 000 "6 . • 0 0 . • ••

• . ibe . 0 • • • %. .0 0 .0 ' O • 0 0

• . • • , .

° • • .. , •• ° : 0 • r.• •

-

•

1000 . 2P09 Body Weiiht (g).

Figure 4.3.

Relation between body wefght, age, and egg weigh.t.

2-year-Old. 0 3-year-old. 0 4-year-old.

3009

•

•

67

second year and the size of those spawned in the third year,

and also between those which spawned in the third and fourth

years. It was found that there was a great probability that

those individuals which produced large (or small) eggs in one

year would produce large (or small) eggs in the next year.

The distribution of weights of the eggs produced by

the fish spawning for the first time was investigated. The

individuals which produced large eggs could be distinguished

from those which produced small eggs, and it was found that

there was a clear difference between the two cases. Five

fish producing large eggs and five fish producing small eggs

were selected from thL group spawning for the first time,

and their individual egg weight distributions are shown in

Figure 4.4. The large eggs were relatively uniform in weight

with little difference, but the small eggs show relatively

large variations in weight. A similar tendency was found in

other first-time spawning fish. It is suggested that this

relation between the size of the eggs and the width of the

variations depends not only on the state of health of the

fish but also on genetic factors related to the accumulation

of yolk during development of the egg. This is thought to be

a very interesting quetion.

• 68

Egg weight (mg).

Figure 4.4.

The difference in e.g weiFht distributions in

individuals which.are producing large eggs (black)

and those producing small eggs (white).

•

• 69

•

•

5. The spawninu season.

The age at maturation and the date of spawning show

very large variations even between individuals of the same

group. In the rainbow trout, it is quite common to find

examples of differences of more than two years in the age of

first spawning, and even among individuals which spawn at the

same age the interval between an early spawning season and a

late spawning season is several months. These differences

lead to many difficulties in egg collection and in the

man -tgement of breeding. Fish which were believed to be

genetically similar were therefore investigated with the idea

of bringing their spawning dates closer together, and the

dates on which they first spawned were recorded. Figure 4.5

shows the length of the first spawning season of fish raised

from five female parents A - E. Shown for comparison is the

length of the first spawning season for fish raised from a

large number of parent fish from which eggs were collected

on the same day. The length of the spawning season was 86

days, which is not significantly different from the average

spawning season length of 82 days obtained from 54 years of

measurements made from 1911 to 1964 in the same place by the

former Nikko fish hatchery. However the length of the first

spawning seasons of the fish from selected parents were 39

to 60 days, so that these spawning seasons were considerably

shorter.

20

0 •

.--.. 20 •

0 •1-1 2

o 0

2

2

-- Oct Nov

• 70

•

Same day collection

2OF Fe,r0Up..

ne,-- 277

Oct NOV Dec Jan Feb

A n=118

Et it=62

ù .. 11=108

D n=49

. ,

. ,

Dec Jan Feb

Figure 4.5.

The length of the spawning season in the groups

hatched from eggs collected on the same day

(upper diagram) and in sibling fry (A - E).

* .The eggs were all collected on the same day, but from a large number of parent fish.

•

•

71

The variability of the day of the first spawning of

the fish from selected parents was small, whereas the

variability of this date in fish from eggs collected on the

same day was large. It may be inferred that eggs collected

onthe same day came from many parent fish whose spawning

periods were centered around different dates.

The next question was the variation of the date of

first spawning with age. This is shown in Figure 4.6 in

relation to the spawning date of the parents. It was found

that the date doe3 vary with the spawning age. The spawning

date of 3-year old fish was considerably earlier than the

spawning date of the parents and the date for 4-year old

fish was only slightly earlier. In 5-year old fish it was

much later. Thus from 3 years old onwards the date of first

spawning gets latser with increasing age. Individuals which

first spawn at two years do so later than the parental

spawning day, but thereafter the trend is the same as those

which first spawn at three years.

From these facts it would appear that if th::

attention were given to concentrating together those which

first spawn on the same date, and to the variation of the

spawning date with age, it might be possible in the management

of breeding not only to shorten the spawning season but also

deliberately to produce a series of spawning periods during

the spawning season.

• 72

-e -20

Days before parental spawnine date.

m 40. Days after parental spawning date.

-- -0 -

Figure 4.6.

Change of spawning season with age.

Changes of median spawning season of siblings.

Mean change with age.

• 73

The relation between size and spawning date in fish

which first spawned at three years of age was also investigated.

It was found that on the whole the large individuals tend to

spawn at an early period (Figure 4.7). However, considering

the results already mentioned that increase of age tends to

be accompanied by a later spawning date, it is thought the

growth is not the only factor that influences the spawning

season, and this question must be left for future clarification.

•

•

?OW

• • • •

• • • • • •

-P 1000 '

0.) ;e. •

•

• •

•

• •

•

• • • • • •

Nov Dec 1.1ari

• 74

Figure 4.7.

Relation between sibling size

and spawning season.

•

75

References.

1. YAMAMOTO Kiichiro, DOPA Isao, TAKANO Kazunori,

ISHIKAWA Tetsuji.

Nijimasu no siejuku ni kansuru kenkyu I.

Ichinen go no ranso no hattatsu ni tsuite.

Nichi sui shi 11 123-132 (1966).

K. Yamamoto, I. Oota, K. Takano, T. Ishikawa.

Studies of maturation of the rainbow trout I.

On the development of the ovary in first-year fish.

Bulletin of the Japanese Society of Scientific

Fisheries. 11 123-132 (1965).

2. KATO Sadaichi.

Nijimasu no seicho heni ni kansuru kenkyu III.

Mesu no sanranki no hendo oyobi sanranki kan

tanshutsu no tame no hitotsu no hobo.

Tan sui ken to 22 1, 41-51 (1973).

S. Kato.

Studies of changes during growth of rainbow trout III.

Variations of the period of spawning in the female and

a method of shortening the period of spawning.

Research reports of the Freshwater Fisheries

Research Laboratories. L2 1, 41-51 (1973).

• 76

•

5. Internal secretions, egg maturation and ovulation.

Fumio YAMAZAKI.

(Faculty of Fisheries, Hokkaido University).

The expression "egg maturation" may in principle be

taken to include both the growth of the egg and ovulation,

but since growth and ovulation differ considerably and there

is a clear endocrinological distinction, they should be

discussed separately. Reviews of knowledge about the

reproduction of fish have been published in Japan by Hibiya 1

and by Yamamoto2,3 but our present knowledge of the

endocrinology of maturation and ovulation remains fragmented.

At the present time t...ere is much which is difficult to under-

stand about the function of internal secretions in the

maturation and ovulation of eggs, and an outline will be given

in this paper of some new information about the secretions

from the pituitary and the ovary.

1. The maturation of the egg.

After finishing division in the ovary the oogonium

enters the growth phase as a primary oocyte (also called an

ovarian egg). In a discussion in outline of this growth

phase on the basis of maturation, it can be divided by the

accumulation of yolk into a primary and a secondary stage

of growth.

•

•• • 77

1.1 The primary stage of the growth phase of the oocyte.

This stage corresponds to the prophase of maturation

cleavage before vitellogenesis, and the nucleus having passed

through the stage of homologous pairs, the leptotene stage,

the zyeotene stage, the bouquet stage, and the pachytene

stage, enters the diplotene stage. The ovoplasm increases

and the oocyte enters the yolkless stage. The size of the

egg at this stage depends on the species and ranges from 13

to 250/).... The influence on the ovary of removal of the

pituitary has been investigated in many species, and it is

known4 that at this stage the egg does not disintegrate but

remains inside the ovary and grows slowly. As observation

of the recently discovered pituitary-deficient "cobalt" p42

rainbow trout has shown, the ovarian egg is in this stage

able to grow independently of the pituitary 5 .

It has been established with certainty that sex hormones

in particular female sex hormones, are secreted by the ovary,

but it is not known whether sex hormones are secreted from

the ovary in the immature stage in which only the yo7kless

primary stage has been reached. However 50 to 100 units of

estradio1-1 were found in ten extractions from the

pituitary of immature goldfish, and the oral administration

of 5?-g of the synthetic sex hormones diethylstilbestrol or

methyltestosterone in each uram of feed for three weeks

clearly caused an acceleration of growth of primary stage

•

•

• 78

ovarian eggs in trout, so that it cannot be denied that sex

hormones may participate in the primary growth stage. Twenty

days of oral administration of 100eg of methyltesterone per

gram of feed to Oryzias latines resulted in a very great

increase in the weight of the ovaries, and it is reported that

the ovaries became active 6 . It may therefore be possible that

ovarian activity may be significantly accelerated by means of

sex hormones, and this may be an important technique for the

control of maturation.

1.2 The secondary stage of the growth phase of the oocyte.

This stage is characterized by vitellogenesis, and is

the most important period during the maturation of the egg.

During this time the oocyte grows rapidly, and the diameter

of the egg, which in the avitelline stage was 100cto 200e,

increases to about 1mm in the carp and in some salmonids and

trout may reach more than 5mm.

1.2.1 Vitellogenesis and the pituitary.

When the fish pituitary is excised the eggs in the

secondary stage of growth disintegrate in a characteristic

way. The most easily affected eggs are those which are in

the process of accumulating yolk. In the goldfish they are

those which are surrounded by the most hypertrophic follicle

cells and, with diameters of 350/tuto 750?.., are in the first

or second stages of vitellogenesis. Those which have formed

yolk vesicles and yolk droplets and are in the third stage of

p4.3

pLA,

79

vitellogenesis remain in the ovary for a relatively long

time, but they are slow to develop and eventually disintegrate,

leaving yolkless eggs only in the ovary. The rapidity of

disintegration depends on the temperature. At 15o C it may

take several months, but above 20 °C it is extremely fast. For

example, at 25 ° C all the eggs containing yolk had disintegrated

in about two weeks4

This egg disintegration after hypophysectomy has been

found in the goby Gobius pagmel1us 7 , the plaice Pleuronectes

platessa8 , the catfish Heteropneustes fossilis 9 and several

other species. On the other hand, reimplantation of the

pituitary or injection of a fluid suspension or extract very

clearly accelerates the secondary stage of growth, and causes

vitellogenesis to be resumed. This fact alone shows that a

hormone which promotes vitellogenesis is present in the

piscine pituitary. This hormone is called gonadotropin (GTH)

and by promoting vitellogenesis and the secretion of sex

hormones it plays an important part in egg maturation.

1.2.2 Sex hormones and vitellogenesis.

GTH is not the only substance which affects

vitellogenesis. The sex hormones also participate in a

complex manner. The connection between the formation of

yolk materials and the female sex hormone has been clearly

shown in the recent work of Aida et al10,11 , and will be

outlined in the present symposium. The female sex hormones

80

••■■

■%

PREOPTIUUS

SUCLEUses\\

CÏÏERIOR\

PROTUBERANCE;

ECONUARY

SEX SYMPTOMS

Aer

.a

.0

o

w 0 m ic 0 r r a IC a a =

.....

Figure 5.1.

Endocrinoloeical processes related to maturation of fish.

• GROW-T-1.1

■ • THYROID. ■ # • GLAND

› •t-- ,

■%

. FEMALE sex NORMONES

1 4

YOL K MATERIAL' _ .

G - T

1H

\isî STADE 9* 2

e

. NDSTAGE

1\ )_ OROWTO ..-Yoik xceuek

WejmuLATIomeirAli

OVIDUCT

E G G

81

act on the liver, the yolk materials are secreted from the

liver and accumulated by the oocyte. The second stage of

growth has been shown to be stimulated in this way in many

species of fish12 .

There are many reports of studies of the sex hormones

secreted by teleost gonads. The female sex hormones most

often extracted from or detected in the ovaries of fish are

estradio1-1773 and estrone 13,14 . The male sex hormones

11-ketotestosterone and dehydroepiandrosterone have also been

isolated from the ovaries of Tilapia aureus and Mueil capito 15 ' 16 .

There are at present no reports in which these sex hormones

have been shown to sti -. -ulate maturity by direct action on the

ovary, but when Takano and Kasuga 6 administered 100/u.g/g of

methyltestosterone or ethinylestradrol orally to Oryzias

latipes for 20 days, they found an enormous increase of the

number of eggs in the yolk vesicle stage. The increase in the

ovarian index was much greater when chum salmon GTH and

diethylstilbestrol were simultaneously administered to rainbow

trout than when they were auministered separately. However

in neither of these experiments was there any great accumulation

of yolk, and the way in which vitellogenesis is related to the

action of sex hormones remains as an important subject for

future research.

•

82

1.2.3 The effects of administration of sex hormones.

The effects of administration of sex hormones depend

on the species. Even in the same species, they depend on the

condition of development of the gonads, the degree of

maturation, and the internal environment of the individual.

Even with the same internal environment, different results

are obtained with different types of hormones or with the use

of different concentrations. For example when 50),,tg/g of

methyltesterone was given to newly hatched rainbow trout for

five months, the development of the gonads was obstructed,

there was a complete absence of gametes and a sterile condition

was reached. However with 1 frg/g growth was stimulated, and

although the development of the ovary was inhibited in the

females, the males were clearly stimulated to mature. When

fry of the pink salmon Oncorhynchus gorbuscha were similarly

given 50 to 100 /kg/g in the feed the ovaries degenerated and

after two weeks the eggs which had been accumulating yolk had p45

completely disintegrated17

, but in Oryzias lijILing. the ovaries

were made to become active by a concentration of 100/g/g6

.

Kasuga18 suggests that at this concentration the cells in

Oryzias latipes which produce GTH were stimulated. Aida et al

injected 2 to 200 tAg/g per fish of estradio1-17p into the

abdominal cavities of Plecoglossus altivelis and found that

the amount of yolk material which appeared in the blood serum

increased in proportion to the amount of estradio1-17p

10

•

83

injected. However when more than an optimum concentration

of the female hormone was injected, maturation was inhibited.

This inhibition of maturation by a sex hormone can be

supposed to be the indirect result of the action of the

hormone in causing the sex control centre to inhibit secretion

of GTH from the pituitary. Such indirect influences on the

ovary will form subjects for future research.

The female sex hormones participate not only in

vitellogenesis but also in the thickening of the epithelium

of the oviduct. The male sex hormones participate in changes

in the pancreas and the digestive organs, and in salmonids

they also participate in changes of the skin thickness and

mucous secretion, in changes of body shape, in the relation

between muscle, blood serum and ovaries, and in the

carotenoid pigmentation of the skin. There are of course

queutions of the inherent natural balance between the two

types of hormones.

Hormones can be administered by injection or by being

included in the feed. Injection can easily damage the fish,

and is not easy to arrange when a large number of fish is to

be treated, so that if there is no need to determine the

exact dose administered to each fish, oral administration,

which is easy with large numbers, is much better than injection.

•

• 84

•

•

1.2.4 The sex control centre.

The sex control centre in fish is the diencephalic

hypothalamus. In particular the two important neurosecretional

nuclei are the nucleus preopticus in the anterior part of the

optic chiasma and the nucleus lateralis tuberis in the

protuberance of the hypothalamus close to the stalk of the

pituitary.

The GTH which is essential for the maturation of the

egg is secreted from the nucleous lateralis tuberis, and it

is believed to be under the control of a GTH releasing hormone

(GTH-RH) 19 . It is known that when the connections between the

pituitary and the hypo -:;halamus are severed, the secretion of

GTH is inhibited20

• 1.2.5 The thyroid hormone.

Changes in the thyroid gland parallel to the course

of maturation have been found in the goldfish and in other

fish. Thyroid activity is increased by the administration of

sex hormones, and the development of the gonads is inhibited

by excision of the thyroid and by the administration of anti-

thyroid materials. Maturation is reported21 to occur sooner

if thyroxin is administered, so it may be supposed that there

is a close connection between maturation and the thyroid

gland. The hormone which inhibits secretion of the thyroid

stimulating hormone (TSH-IH) and (GTH-Rh) are both secreted from

the nucleus lateralis tuberis 19 . They are similar in structure,

85

both TSH and GTH being glycoprotein hormones with molecular

weights of about 30,000, and though they differ in their

action on the control centre they are believed to have

common features, It is however doubtful whether the thyroid

hormone directly stimulates maturation by its action on the

ovarian tract, and it is thought that its action is a

secondary result of the stimulation of bodily growth. There

must be further investigation of the question.

2. Ovulation.

In ovulation the eggs which have completed

vitellogenesis are detached from the follicle and are

discharged into the ovarian cavity or into the body cavity.

Changes also occur in the egg itself, and with the maturation

division and the discarding of the polar bodies the diameter

of the egg is rapidly increased by the absorption of water22,23 .

In connection with ovulation the egg first becomes capable of

fertilization. Many studies in which the pituitary is

excised have shown that ovulation is caused by the secretion

from the pituitary of GTH, and that ovulation can be produced

in hypophysectomized fish by the administration of GTH. The

GTH producing cells in goldfish show extreme morphological

changes at the time of ovulation. For example, 10 hours

after goldfish which had matured in a pond at 13 ° to 14°C

86 •

•

were transferred to a tank at 20 ° C, the GTH producing cells

showed hypertrophy and vacuoles were formed in the cytoulasm,

and ovulation began to occur after 30 to 40 hours 24 . Although

there are few actual known facts about ovulation, it is

thought that ovulation follows the sudden secretion, about

20 hours earlier, of large amounts of GTH.

2.1 The amount of GTH in the blood at the time of ovulation.

It has recently become possible to measure the amount

of GTH in fish blood by means of radio-immuno-assay (RIA).

Breton et al25 used this method to measure the GTH in the

blood of goldfish and found the amount to be greatest during

the day, with 5.75 ng/ml at 8 a.m., 9.55 at 11 a.m. but

3.34 ng/ml during the night. They reported that the quantity

of GTH in the blood on the day of ovulation reached five times

that on an average day, and their results suggest that a large

amount of GTH is secreted by the pituitary at the time of

ovulation.

2.2 The mechanism of ovulation.

In considerations of the mechanism of ovulation it is

important to consider two sequences of events, those that

precede the secretion of GTH from the pituitary, and those

which accompany ovulation and are produced by the action of

the secreted GTH on the ovary.

External environmental factors which are of importance

to ovulation include the water temperature, the illumination,

the rainfall, the diurnal variation of illumination, and the

X/ERNAL

STIMULI. 7 Nuct.Eus _

reEOPTICUS..

HYF °THALAMUS

P.ROTUBER A.NCES

• • . • •• ADRENAL CORTEX

— —

• .. .

desoxycorticosterone

hydrocortisone •

•: .'0VAR IA N OR

ROOT CAVITY • •

:

% On- DUCT

Vital

WATER ADSORPTION AND

INCREASE OF DIAMETER

• 8 7

Figure 5.2.

DiaFram of the process of ovulation in fish.

•

• 88

•

weather conditions. It is believed that these stimuli affect

the sex control centre which by secreting GTH-RH causes the

secretion of a large amount of GTH from the pituitary.

Mammalian FSH releasing hormone (FSH-RH) and LH

releasing hormone (LH-RH) have been extracted from the

hypothalamus and purified. Their structural formulae have

been ascertained and it has become possible to synthesize

them26 . Studies by Hirose and Ishida27 of the effect on

ovulation of Plaecoglossus altivelis of mammalian LH-RH have

clearly shown the similarities between the mammalian and the

piscine sex control centres in their mode of action and in

the relation of LH to GTH p and this raises some very interesting

questions. It is important that the effectiveness of Li-RH

should be investigated in a number of species of fish. At the

same time GTH-RH should be extracted from these fish and p48

purified, so that synthesis can be used for comparative

chemical investigations.

Sundaraj et al28 have made a series of studies of the

way in which the action of GTH on the ovary gives rise to

ovulation in the catfish Heteropneustes fossilis. They find

that GTH does not act directly on the ovary, but that GTH

acts on the adrenal to stimulate the secretion of cortisol

and deoxycorticosterone, and these adrenocortical hormones

act on the ovary to cause ovulation. They claim that the

action goes from the pituitary to the adrenal and thence to

• 8 9

•

the ovary. The experiments of Hirose 29 with Oryzias latipes

in vitro suggest that there is a transfer from pituitary to

adrenal to ovary similar to that in the catfish, in addition

to a direct transfer from pituitary to ovary. We must wait

for future research to establish whether or not the hormones

of the adrenocortex participate in the ovulation in all

species of fish.

Jalabert et al30 made in vitro experiments with

rainbow troUt and found that both progesterone and fish GTH

affected ovulation, but that adrenccortical hormones, male

sex hormones, and female sex hormones had no effect. They

considered that GTH indirectly induced ovulatica either through

progesterone or by stimulating the secretion in the ovary of

some unknown substance. Hirose 29 observed the damage to the

follicular structure during ovulation, and suggested that

some substance contained in the follicles was the primary

cause of ovulation. There is no present certainty about this

substance, but when one takes into account the gaps which

appear between the follicle cells during ovulation, The

fluids which appear in these gaps, the loosening of the

adhesion between the follicle cells and the egg, and the

absorption of water by the eggs, it is true that the damage

to the follicle cells is very large. It has recently been

•

•

90 ••

suggested 31 that the GTH acts on the genes in the egg itself,

and that functional maturity of the egg is brought about by

the development of new proteins through a typical procedure

from DNA to RNA to protein.

3. Piscine GTH.

3.1 Types of GTH.

As has been shown above, GTH secreted by the pituitary

is essential to the maturation and ovulation of the egg, but

it is not yet clear whether the GTH which participates in

maturation is the same substance as the GTH which participates

in ovulation. In other words it is not clear whether piscine

GTH exists in one or in two varieties.

The following facts can be used to support the

existence of two types of GTH.

1. The maturation of the egg (vitellogenesis) and ovulation

differ completely in detail.

2. Mammalian LH, pituitary extract, and chorionic

gonadotropin (HCG) are effective in causing ovulation

in many species of fish but they are not found to have

any noticeable effect on maturation .

3. It has been stated that two types of GTH producing

cells are present in the ee 1 32 .

1)49

•

91

4. Even though the GTH producing cells in the goldfish are

of only one type, two very different types of granules

are present in the cytoplasm, and they are observed to

have different behaviours during maturation and ovulation.

5. It is believed that gametogenesis and sex hormone

secretion in Xiphophorus maculatus are under the

control of different pituitary hormones 32 .

6. Maturation and ovulation in mammalian ovarian follicles

are under the control of two types of hormones, FSH and

LH. This may be thought to support the idea that there

should be two types of GTH in the piscine pituitary,

with individually separate functions in maturation and

ovulation.

The following facts can be used to support the contrary

view, that only one type of piscine GTH exists.

1. The GTH extracted from the pituitaries of carp and chum

salmon are biochemically similar and are of the same

type of glycoprotein34 .

2. Purified chum salmon GTH is effective in producing

maturation of the egg and ovulation in the female fish,

and in producing spermatogenesis and spermiation in

the male 35 .

3. There is only one type of GTH producing cell in the

pituitary of Gasterosteus aculeatus, goldfish, Oryzias

• latipes, Oncorhvnchus keta and Oncorhynchus nerka32,36

••

92

•

4, The changes seen in the GTH producing cells in the

goldfish after ovariotomy and the changes seen at the

time of ovulation have in common the appearance of

vacuoles due to the enlargement of the endoplasmic

reticulum, and both the large and the small types of

granules are observed to participate in the formation

of these vacuoles 36 . This appears to support the

idea that maturation of the egg and ovulation are

controlled by one type of GTH.

Burzawa-Gerard and Fontaine37 propose that there is

only one type of piscine GTH, but that the whole of the

reproductive phenomena are controlled by the existence of

two types of receptors in the gonads. Hyder 38 suggests that

there is only one type of GTH but that there may be two

active centres in the GTH molecule. Whatever the truth about

the types of GTH may be, it is important for the artificial

control of reproduction that it should be found out rapidly

by means of further studies.

3.2 The effectiveness of piscine GTH.

Up to the present time mammalian hormones have been

used to stimulate maturation and ovulation. Examples are

gonadotropin, synaholin, puberogen and gonadoplex. They

affect ovulation in goldfish4 , carp39 , Misgurnus

anguillicaudatus 40 , Plecoglossus altivelis 41 , Heteropneustes •

• 93

fossilis28 , Mylio macrocephalus42 , Mugil cephalus 22 and

Seriola quinqueradiata43 , and also affect maturation in

Mylio macrocephalus. However in salmonids, sturgeon,

Chenopharyncodon idellus, Hypophthalmichthys molitrix and

Anguilla japonica it is difficult to observe any particular

effect of these hormones on either maturation or ovulation.

Yamamoto et a144 have recently achieved maturation and

ovulation in the Japanese eel Anguilla japonica by using salmon

pituitary, and young eels were born from artificial

fertilization for the first time in history. It is supposed

that all types of pituitary hormones were present in the

pituitary fluid suspension used, but it must be considered

that there is at least a suggestion that piscine pituitary or

its extracts can be effective in the maturation and ovulation

of species which are difficult to breed. .

3.3 The purification and chemical properties of GTH.

GTH has recently been extracted and purified from

carp37 , chinook salmon, chum salmon, and Puntius .0 .onionotus46

an :. its chemical properties are gradually being found. The

extract ha s been fractionated with water or dilute ethyl

alcohol (40 - 57%) using Simplex G - 75 or G - 100 and

DEAE cellulose 34 ' 36 ' 37 ' 45

Carp GTH is a glycoprotein containing hexose 8.6%,

hexosamine 4.9% and sialic acid 0.35%, and this amount of

•

• 94

•

sialic acid differs from that contained in sheep FSH or LH34 .

The molecular weight in carp is 27,000 to 31,000, and in

chinook salmon is 28,5000 to 29,400 34 . It has been inferred

that carp or chinook salmon GTH, like mammalian GTH, is

formed from two units 34 . The purified GTH is reported to be

effective both in egg maturation and in ovulation.

The hormones known to be of greatest importance in

egg maturation and ovulation are the pituitary GTH and the P51

sex hormones secreted by the ovary and the adrenal cortex.

The individual and joint actions of these hormones are

essential in the artificlal control of maturation and

ovulation for the production of fry of useful species of fish.

Up to the present commercially available mammalian hormones

have principally been used to stimulate maturation and

ovulation in fish. However there are natural limits to the

results obtainable in fish in which the pattern of reproduction

is different from that in mammals. There is a requirement

for further studies of piscine GTH and for a coordinated

series of investigations of the effective individual actions

and joint actions of the male and female sex hormones, and for

simultaneous practical applications of the results.

•

• 95

References.

1)

SEE PAGE 99

SEL PAGE 99

3) Su PAGE 69

4) F. YAMAZAXI: Endocrinological stud-

ies on the reproduction of the fe

male goldfish. Carassius auratus L., with special reference to the func-tion of the pituitary gland. Mem.

Fac. Fish.Hokkaido Univ. 13, 1-44

(1965).

5)

SEE r AGE 100

SEE PAGE 100

chez les poissons sélaciens et

téléostéens. Bull. Biol. France et Belgique 75, 257--309 (1941).

8) W. A. BARR: The endocrine control of the sexual cycle in the plaice. Pleuronectes platessa (L),II. The

endocrine control of oogenesis. Gen. ComP. endocrinol. 3, 205-.215 (1963).

9) B. L SUNDARARAJ and S. V. Coswenr:

Effeet of short-and long-terrn hypo-physectomy on the ovary and inter-renal of catfish, Heteropneustes

jos•ilis (Bloch). J. Exp. . Zoo!., 168.

58-104 (1968).

10)

SE.E PAGE 100

II) SEE PAGE 1 00

7) J. H. VtviEN: Contribution a l'etude

de la physiologie hypophysaire dans

ses relations avec l'appareil genital, la thyroide et les corps suprarenaux

12) B. I. SUNDARARÀJ and T. C. ANA»:

Effects of piscine and mammalian

gonadotropins on gametogenesis in the catfish, Heteroneustes fossilis

(Bloch). Gen. Comp. Endocrinol.

Supple 3, 688--702 (1972).

13) • W. S. Hoes : Reproduction. in "Fish

Physiolgy" (W. S. Hou & D. J. RANDALL ed) Vol. III. 1--72, Acad. Press, New York (1969).

14) R. REINBOTR: Hormonal control of the teleost ovary. Amer. zool. 12, 307 --324 (1972). B. ECKSTEIN: Metabolic pathways of steroid biosynthesis in o‘arian tis-sue of a teleost, Tilapia aurea. Gen.

Comp. Endocrinol. 14, 303--312(1970).

16) U. Eviieru and B. ECKSTEIN : Isola-tion of 11-ketotestosterone and dehy-droepiandrosterone from ovaries of the common mullet, Mugil capito

Gen.. Comp. Endocrinol. 12, 58-62

(1969).

17) F. YANAZAKI: Effects of methyltes-tosterone on the skin and the gonad

of salmonids. Gen.ComP. Endocrinol.

Supple. 3, 741--750 (1972) 1

SEE PAGE I 01

19) R. E. «PETER: Hypothalamic control • of thyroid gland activity and

gonadal activity in the goldfish. Gen.

Comp. Endocrinol. 11, 334 (1970).

20) .1. N. BALL, M. OLIvEREAu, A. M.

SLICIIER and K. D. KALLMAN: Func- tional capacity of ectopic pituitary transplants in the teleost Poecilia

fornwsa with a comparative discus-sion on the transplanted pituitary,

Phil. Trans. Roy.Soc. Lond. Ser. B..

249, 69--99 (1965).

21) M. SAGE :The evolution of thyroidal

function in fishes. Amer. Zool. 13,

899-905 (1973).

22) Z. H. SUET:LADEN and K. S. NORRIS:

The grey mullet (11fugil ceplzalus L.): induced breeding and larval rearing. Oceanic Inst, Hawaii RePt. No. 01-

72-764, 1-402 (1972). K. HIROSE, T. HIRANO and R. Dunne: Effects of salmon gonadotropin on. ovulPtion in the ayu, Plecoglossur

altivelis, with special reference-to water balance. Comp. Biochem.

Physiol. 97A 283--289 (1973).

24) K.YAMAMOTO and F. Yitueza :Hor-monal control of ovulation and sper-miation in goldfish. Gunma Symposia

on Endocrinol, 4, 131---.145 (1967). B. BRETON, R. BILLARD, B. JALABERV

et G. 'KANN: Dosage radioirnmunolo-

gigue des gonadotropines plasmati-

qus chez Carassius auratus, au cours-

du nycthémère et pendant l'ovulati- on. Gen. Comp. Endocrinol. 18, 461

-.-468 (1972).

26) SEC PAGE 101

SEE PAGE 102

28) B. SlINDARARAJ • and S. Goswiiii:

Role of interrenal in luteinizing

hormone-induced ovulation and spa-wning in the catfish Heteropneuster

fossilis (Bloch). Gen. Comp. Endo-

crinol. Supple. 2, 374-384 (1969).

96

15)

18)

23)

25)

27)

• 97

29) .

SEE PAGE 102 30) B. JALABERT, B. Bavrou and C. BRY:

Maturation et ovulation in vitro

des oocytes de la Truite Arcen-Ciel Salmo gairdncrii. C. R. Hebd.

Seances Acad. Sci Ser. D.275, 1139—

1142 (1972).

31) Effect of actinomycin D, mitornycin,

C, puromycin, and cydoheximide on desoxycorticosterone-induced in vit-

ro maturation in oocytes of the catfish, Hetcropneustes fossilis (Bl-

och). J. Exp. Zoo!. 185, 327-332 (19

73). 1421. P. SCIIREIBMAN, J. F. LEATFIERL-

*ND, and B. A. Mc KEowm Functional

morphology of the teleost pitui,dry

gland.Amer. Zoo1.13, 719-742. (1973)

33) Y. Ne.c.inemA: Histo-physiological

studies on the pituitary gland of some teleost fishes, with special

reference to the classification of hormone-producing cells in the-

adenohypophysis. Mem. Fac. Fish.

Hokkaido Univ. 21, (1973).

34) E. M. DONALDSON: Reproductive'

endocrinology of 'fishes. Amer. Zool.

13, 903-927 (1973). F. ?AMAZAXI and E. M. DONALDSON

: The effects of partially purified

salmon pituitary gonadotropin on spermatogenesis, vitellogenesis, and ovulation in hypophysectomized goldfish (Carassius auratus). Gen.

Comp. Endocrinol. 11, 292-299 (1968).

38) SEE PAGE 102 •

37) E. HURZAWA-GERARD and Y. A. FON-

TAINE: The gonadotropins of lower

vertebrates. Gen. Corn p, Endocrinol. Supple. 3, 715-728 (1972).

38) M. HYDER: Endocrine regulation of reproduction in Tilapia. Gen. ComP.

Endocrinol. Supple. 3, 729 ,-740 (1972).

39)

SEE PAGE 103

40) SEE PAGE 103

SEE PAGE 103

42) SEE PAGE 104

43) SEE PAGE 104

-44) SEE PAGE 104

45) E. M. DONALDSON, F. ?AlIAZAXI, H.

M. Dis and W. W. Putwto: Preparation of gonadotropin from

salmon(Oncorhynchus tschauelscha)

pituitary glands. Gen. ComP.

Endocrinol. 18, 469-481 (1972). 46) B. I. SUNDARARAJ, T. C ANIND and

V. R. P. Smut: Effects of Carp

S.V.Gosweur and B. I. SDNDARARAJ:

32)

35)

41)

•

• 98

pituitary fractions on vitellogensis

ovarian maintenance, and ovulation in hypophysectomized catfish, liete-ropneustes fossilis (Bloch). J. En-door, 54,87-98 (1972).

47) B. I. SUNDARARAJ, T. C ANAND and E. M DONALDSON:. Effects of partia-

Ily purified salmon pituitary gonado-

tropin on ovarian maintenance, ovulation and vitellogenesis

in hypophysectomized catfish,

11 et eropn eust es fossilis (Bloch). Gen Comp. Endocrinol ; 18, 102-.114

(1972).

•

99

•

1. HIBIYA Takashi.

Horumon ni yoru gyorui no seijuku, sanran no kontororu.

Suisan zoshoku 12 239 - 259 (1965).

T.

Hormone control of maturation and spawning in fish.

Fish breeding 12 239 - 259 (1965).

2. YAMAMOTO Kiichiro.

Seishoku, gyorui sein.

KAWAMOTO Nobuyuki hen. 232 - 271.

Koseisha Koseikaku Tokyo 1970.

K. Yamamoto.

Reproduction and fish physiology. (Edited by N. Kawamoto) pp 232 - 271.

Published by Koseisha Koseikaku. Tokyo 1970.

3. YAMAMOTO Kiichiro.

Gyorui jinko zoshoku ni kanren suru naibunpitsu

gakuteki kenkyu, dai ikkai taheiyo no suisan

zoshoku ni kansuru Nisso godo shinpojiumu.

Ronbun shu 13 - 39 Tokai dal 1973.

K. Yamamoto.

Endocrinological studies related to artificial

fish breeding.

Collected papers from the first joint Japanese-

Soviet Pacific symposium on fish breeding.

13 - 39, Tokai University, 1973.

100

5. YAMAZAKI Fumio.

Nijimasu no kikeigyo no iwayuru "kobaruto" ni tsuite

Nichi sui shi ko 17 - 25 (1974).

F. Yamazaki.

On the so-called "cobalt" deformed type of rainbow trout.

Bulletin of the Japanese Society of Scientific

Fisheries, ko 17 - 25 (1974).

6. TAKANO Kazunori, KASUGA Seiichi.

Medaka no ranso ni oyobosu sel suteroido toyo no eikyo.

Do zatsu 82 263 (1973).

K. Takano, S. Kasuga.

The influence on the ovary of the administration of

sex steroids to Oryzias latipes.

Japanese Journal of Zoology, 82 263 (1973).

10. AIDA Katsumi, Fan ban gan, HIBIYA Takashi. • Gyorui no seishoku sen seijuku ni kansuru seini

gakuteki kenkyu I. Sei shokusen seijuku ni tomonau

ayu no kessho tanpaku sosei no shiyusa.

Nichi sui shi 1091 - 1106 (1973).

K. Aida, Phan Van NEan, T. Hibiya.

Physiological studies on gonadal maturation of fishes.

I. Sexual differences in composition of plasma protein

in relation to gonadal maturation.

Bulletin of the Japanese Society of Scientific

Fisheries 22 1091 - 1106 (1973).

•

•

•

101

11 , AIDA Katsumi, HIROSE Keiji, YOKOTE Motoyashi and

HIBIYA Takahashi.

Gyorui no seishoku sen seijuku ni kansuru seini gakuteki kenkyu II. Seijuku oyobi esutorojen shori

ni tomonau ayu kanzo no soshiki gakuteki henka.

Nichi sui shi 12 1107 - 1115 (1973).

K. Aida, K. Hirose, M. Yokota and T. Hibiya.

Physiological studies on the gonadal maturation of

fishes II. Histological changes in the liver cells

of Plecoglossus altivelis following gonadal maturation and estrogen administration.

Bulletin of the Japanese Society of Scientific

Fisheries ,3.2. 1107 - 1115 (1973).

18. KASUGA Seiichi.

Sei suteroido toyo ni yoru medaka no nokasuitai seishokusen shigeki horumon sansei saibo no henka.

Do satsu 82 263 (1973).

S. Kasuga.

Changes in the cells which produce the pituitary

gonadotropin in Oryzias latipes due to the

administration of sex steroids.

.Japanese Journal of Zoology 82 263 (1973).

26. IGARASHI Masao.

FRF to LRH.

Taisha 8 33 - 40 (1971). -

•M. Igarashi.

FRH and LRH.

Metabolism 8 33 - 40 (1971).

• 102

27. HIROSE Keiji, ISHIDA Rikizo,

LH - RH ni yoru ayu no hairan sokushin ni tsuite.

Nichi sui eakkai 49 nen koen hoshi shu (1974).

K. Hirose, R. Ishida.

On stimulation of ovulation in Oryzias . latipes by means

of LH - RH.

Japanese Society of Scientific Fisheries.

Collected Abstracts of Papers, 51 (1974).

29. HIROSE Keiji.

Gyorui no hairan no naibunpitsu shikai.

Tokai sui ken ho 21 67 - 81 (1973).

K. Hirose.

Endocrine control of ovulation in fish.

Bulletin of the Tokai Fisheries Research Laboratory,

21 67 - 81 (1973).

36. YAMAZAKI Fumio.

Gyorui no seishokusen shigeki horumon.

Nichi sui shi 695 - 709 (1969).

F. Yamazaki.

Gonadotropin hormone in fish.

Bulletin of the Japanese Society of Scientific

Fisheries, .25. 695-709 (1969).

•

•

103

39. KAWAJIRI Minoru ., SHIMADACHI Magoi, kOYAMA Haiime,

MIYAJIMA Chojiro.

Horumon ni yoru koi no sanran sokushin shiken (dal

ichi ho).

Nichi sui shi 14 13 - 16 (1948).

M. Kawajiri, M. Shimadachi, H. Koyama and C. Miyajima.

Experiments on the stimulation of spawning in carp

by means of hormones (Part 1).

Bulletin of the Japanese Society of Scientific

Fisheries 14 13 - 16 (1948).

40. MATSUURA Yasuasa.

Dojo no jinko saibyo shiken.

Sui san zo shoku 65 - 73 (1967).

Y. Matsuura.

Experimental production of young of Misgurnus

anguillicaudatus.

Fish breeding 65 - 73 (1967).

41. ISHIDA Rikizo.

Seishokusen shigeki horumon toyo ni yoru gyorui no

seijuku oyobi hairan no sokushin ni kansuru kenkyu II.

Ayu ni okeru taiju 1g atari no tekisei toyorà . o to

seijuku ni tomonau sokushin koka no henka.

Tansuiken ken ho 22 49 - 58 (1972).

R. Ishida.

Studies of the stimulation of maturation and ovulation

in fish by the administration of gonadotropic hormones.

IL Variation of the effectiveness of stimulation of

maturation with the quantity per gram of body weight

administered to Plecoglossus altivelis.

Research reports of the Freshwater Fisheries Research

Laboratory 22 49 - 58 (1972).

•• • 104

42, KASAHARA Shogoro, HIBIYA Takashi.

Kurodai no ryubyo seisan ni kansuru kisoteki kenkyu I. Seishoku sen shigeki horumon toyo ni yoru seijuku

oyobi sanran no sokushin ni tsuite.

Hirodai sui chiku sangaku bu kiyu 105 - 111 (1967).

S. Kasahara, T. Hibiya.

Basic research on the production of fry of Mylio

macrocephalus I. Stimulation of maturation and

spawning by means of administration of gonad

stimulating hormones.

Bulletin of the Department of Aquaculture, Hiroshima

University, 2. 105 - 111 (1967).

43. UMEDA Shin, HIROZAWA Kuniaki, OCHIAI Akira.

Kochi ken Komame gyojo ni raiyu suru buri sanran gun

to shinahorin ni yoru seijuku sokushin ni tsuite.

Nichi sui shi 15. 446 - 450 (1969).

S. Umeda, K. Hirozawa, A. Ochiai.

Stimulation of maturation by means of synaholin in the

yellow tail schools which migrate to Komane (Kochi

prefecture) to spawn.

Bulletin of the Japanese Society of Scientific

Fisheries, 3_5. 446 - 450 (1969).

44 • YAMAMOTO Kiichiro, YAMAUCHI Kohei, KASUGA Seiichi.

Unagi no shoki hassei ni tsuite.

Nichi sui gakkai 49 nen koen yoshi shu 102 (1974).

K. Yamamoto, K. Yamauchi, S. Kasuga.

On the early development of the eel.

Japanese Society of Scientific Fisheries, Collected

Abstracts of Papers, 102, 1974.

• 105

•

•

Environment, maturation, and spawning.

6. Freshwater fish.

Hiroshi YOSHIOKA.

(Hakodate Branch, Hokkaido University of Education).

The physical and chemical characteristics of the

environments in which freshwater fish and marine fish live

are different, and it is therefore to be expected that the

influence of environmental factors on maturation and spawning

will differ.

The principal factors affecting the maturation and

spawning of freshwater fish are illumination and temperature,

and this paper will discuss the relation of these two factors

to maturation and spawning.

1. Illumination.

When considering the relationships between an animal

and such factors of its environment as the illumination,

water quality and water temperature, it is important to

remember that they have . been established during a very long

period extending over many thousands of generations. For

this reason a given quality of illumination may exert a

great influence on one animal, no influence at all on another,

106

•

and even a contrary influence on a third. For example,

animals which normally live in bright daylight become

deficient in vitamin D if the illumination is insufficient,

but fish which live deep in the sea where the illumination

is normally insufficient contain large amounts of vitamin D.

Another important matter when deciding whether or not an

animal is being affected by the illumination, is that it is

often impossible to base a decision on the reaction during a

short period. Experiments concerned with the effect of

illumination on gonadal maturation in both birds and fish

must be continued for at least a month, and in some species

a period of not less than several months is necJIssary.

Studies of the effect of illumination on plants began

long ago, and many phenomena have been described. Basic

studies with vertebrates other than fish, such as birds and

mammals, have been completed and have reached the stage of

practical application. In contrast to this, apart from a few

early studies in Canada and America by Hazzard and Eddy1 and P56

by Hoover and Hubbard2 on the acceleration of spawning in

salmon and trout by artificial light cycles, regular research

on fish has only been carried on since the 1960's. An animal

experiences many variations of light during its life, from

those of a short period of one day to those of long period of

one year. Not all of these are necessarily influential, but

the length of the diurnal cycle of illumination is often

• 107

important. In the reproduction of the higher vertebrates,

this influence is exerted through the annual recurrence at a

fixed season of a diurnal cycle with a set length of

illumination. For this reason, the most frequent means by

which illumination influences maturation and spawning is

through changes in the diurnal cycle.

Garner and Allard divided plants according to the

relation between flowering and length of day into long-day

type plants and short-day type plants, and Yoshioka3 has

divided fish according to the relation between maturation and

length of day into long-day type fish and short-day type fish.

Fish which are categorzed as belonging to the first type are

those which spawn during the long-day season from spring to

summer, and include the medaka Orysiàs latipes, the honmoroko

Gnathopogon caerulescens, the shiner and the marsh killifish.

In these species maturation and spawning can be caused to

occur early by means of illuminated periods longer than those

which would normally occur at an early season (Yoshioka4-7 ,

Hibiya et al8,9 , Harrington10,11 ).

Species of the second type, principally salmonids,

which spawn in the autumn when the diurnal illumination is

short, behave in the opposite way, and can be made to spawn

earlier than in natural conditions by shorter periods of

illumination (Corson12 , Nomura13 , Henderson14 ).

• 108

For example Hazzard and Eddy1 took brook trout

Salvelinus fontinalis which had finished spawning in October

and November and exposed them from the middle of January to

the end of April to periods of illumination longer than

those which would occur in nature. Then the natural light

was blocked off and the period of illumination was made

shorter than in nature, and a species which spawns in November

was made to spawn in July. Shiraishi and Takeda have found

the same behaviour in ayu, Plecoglossus altivelis15,16

It has thus been shown that illumination influences

the maturation of fish, and there are two possibilites.

Either the total quantity of light or the increase or

decrease of the illuminated period may be effective.

Yoshioka4 therefore made observations on medaka Oryzias

latipes. He found that increase or decrease of the illuminated

period did not control maturation, but that there was a P57

critical length, between 12 and 13 hours, for the illuminated

period. Maturation was observed only when the illuminated

period was longer than this critical length. Similar

critical lengths have been observed with other species of

fish, and for Plecoglossus altivelis the period is required

to be less than 12 hour s. for maturation. In natural

conditions, of course, the period of illumination is not

normally constant, and there is some danger that the results

of increase or decrease of the length of the day may be

overlooked. This increase or decrease should be considered

1 09

to have a secondary role, supplementary to other influences.

An interesting observation with Oryzias latines is that if

the fish which were spawning were subjected to an illuminated

period less than the critical length, the large oocytes of

diameter more than 0.4mm completely disintegrated, but if

they were then returned to an illuminated period longer than

critical the oocytes resumed growth and spawning occurred.

Changes of illumination can thus stimulate, inhibit

or retard maturation, but there is a problem of when, during

the process of maturation, the influence is exerted.

Yoshioka4 found that apart from the extreme condition of

extremely long illumination and short dark period, the

length of the illuminated period had no effect on ovulation

or spawning of Oryzias latipes and its food gathering

activities were not affected, but he confirmed histologically

that there was a remarkable influence on the development of

the oocytes (Figure 6.1). Changes in diameter have been used

to observe the influence of illumination on the development

on #-he ovarian egg in the willow shiner Gnathopogon elongatus

caerulescens, the bridled shiner Notropis bifrenatus, the

rainbow trout Salmo gairdnerii, the brook trout Salvellinus

fontinalis and the ayu Plecoglossus altivelis.

Oryzias latipes was used in a further investigation

of the result of making the illumination cycle longer or

shorter than 24 hours, and the effect on the critical length,

•

• 1 10

Figure 6,1,

The influence of the photoperiod on the development

of the ovary in medaka (Oryzias latipes).

•

1. The medaka ovary during oogenesis when exposed to a short illuminated period, 14 days after the start of the experiment. Large oocytes are degenerating.

2. The ovary under a short illuminated period (at a time of no oogenesis). Large cells have almost disintegrated and been absorbed. The ovary is occupied by small oocytes of diameter less than 0.4mm.

3. The medaka ovary when oogenesis has been suppressed by a short illuminated period. 21 days after the start of the experiment.

4. The ovary of (3) after 21 days exposure to a long • illuminated period.

Figure 6.1 continued.

r ---- 7 ..ore.,-7.1M77--;:- . . • ...

,„........_ ,..„,...,...„......,...„,..,,...,,.....,... •

, ..‹,,..„.....,,,..„.,..„ ,, . ,

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01

111

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*-

4.

••• , (.1

bz.4 e\?1' '

.

-""Vr,,•p-,e?,1.-s . -- • .- - ;N-3' • • - --7e'e

,,à2.,•.?,'4, q---%_: -,... --'," ; i, 4 , .-.3.-. ?.....,C). ■ \I ej''''''Y e.‹. )• •••::. • i. t 1

t>e•Cic.e..:,•ei:,;.is2).,.Z.V:.,;.:,:i4.. ;keip:* „ .. «''''''-'0 et• ie ..,..!,..:y4 :.-='; -:,'-?-'".-)' 7 -

•

. . --"•-., - :•:,.t.f.m..-.-",.1,'-'"--- a

,--e...-1..,- ‘ e'. ...e% .K.f.:‘ pWr-,:b.:‘,4,,,,,te -,46.1c4::::,.'liu:'T.,.1

. . '",z7 ;15,ie-,...e.c,te,i, 1 .:-... 'f'■:e. `'. bi.:1-:-.-

..., ,r\:. ., . •. p .

. ‘ -,C:• ■ ,- . e.f,ocyi i:: Wi...

Ç. C::-.«:•"110:'1 'tic'. -.•-:-..--

I .. 1.,.:.-?' 'Zi..,-4 ■ *:ieft&o .'"?-1/j

5,6. The medaka ovary February, reared

in a period of no spawning in under a long illuminated period.

5. At 14 days.

6. At 21 days.

7,8. The medaka ovary in the period of no spawning in January, reared under natural photoperiod conditions.

5. At 14 days.

6. At 21 days.

112

••

when the cycle was lengthened so that the associated dark

period was even longer, maturation was not stimulated.

Conversely, when the dark period was longer than critical,

but the associated light period was even longer, maturation

was stimulated 7. Thus maturation can be stimulated or

retarded by changing the length of the cycle and by changing

the lit or dark periods. Hibiya et al8 have found

corresponding but inverse influences in Gnathopogon.

Furthermore Hibiya et al8,9 observed that there was

a range of periods in which the influence of illumination in

stimulating or inhibiting maturation was particularly strong.

This has not been observed in other species and is to be

awaited in further studies.

The next subject is the influence on maturation of

the strength of illumination. There exist only a few results

by Shiraishi with Plecoglossus 16 and by Yoshioka with Oryzia.

When Plecoglossus was illuminated by fluorescent light with a

16 hour illuminated period, maturation was inhibited in

females at 0.2 lux and in males at 0.11 lux. In Oryzia the

gonads were completely undeveloped with illumination less

than 5 lux, and with a low degree of illumination in the

range of 10 to 50 lux there was a slow and retarded

development of the gonads. The results were the same as in

natural illumination only with more than 160 lux, and there

•

• 113

was then no change in the rate of development of the gonads.

Up to the present there have been no observations of any

influence on maturation of illumination greater than the

critical amount, and the study of other species is awaited.

The effective critical illumination at which maturation is

inhibited may be thought to be rather low, but the sensitivity

to net changes, and the threshold value may be considerably

affected by variations of the environment in which the fish lives.

Light greatly influences spawning as well as maturation.

Oryzias normally spawns a little before dawn, and this shows

an influence of illumination on spawning. When day and night

were artificially internhanged, spawning occurred at the P59

beginning of the artificially lit period after a few days18 .

In order to find out the lowest illumination needed for

spawning, Yoshioka 19 caused Oryzias to stop spawning by

keeping them in darkness and then provided them with various

amounts of artificial light. He found that in illumination

greater than 5 lux all of the fish which were ovulating spawned,

but in illumination less than one lux some spawned and some

did not. Spawning was also induced by suddenly subjecting

individuals which were already well lit to a very strong light

(more than 5000 lux), and all ovulating individuals spawned.

This confirms that the occasional spawning of Oryzias during

the day or in the evening is the result of changes of illumination.

•

• 114

There are very few studies of the effect of wave-

length. Shiraishi 17 found that yellow and orange light was

more effective in controlling maturation than the short

wave-length blue and green, but these results were not

satisfactory because the filters used had very poor transmission

coefficients. Yoshioka19 used standardized liquid filters and

monochromatic filters, and tried the effect of selected wave-

lengths on the maturation of Oryzias. He found that wave-

lengths shorter than 5000 R had no effect on maturation, but

that visible light longer thal: 5000 R did have an effect. It

was interesting that visible light of wave-length 5800 R to

6700 R was particularly effective, and that nearby but shorter

wave-lengths had only a weak effect. The effects of infrared

and ultraviolet were tested with long periods of illumination,

but no effects were found. However the amount of light

passing the filters was said to be insufficient, and so the

experiments were not able to establish with certainty the

presence or absence of any effect. However, since the amount

of ultraviolet light at the çarth's surface is barely 1% of

the total, and since it is absorbed in water, it would be

very difficult for fish to utilize the ultraviolet in the

environment. In animals, other than fish, such the duck, the

development of the gonads is affected by wavelengths from

6640 R to 740 0 R. Conversely, it is known that the shorter

wavelengths in the blue and green are effective in insects.

115

It is supposed that these differences in the sensitivity of

animals are closely connected with the environment in which

they live. For example, the large amount of green and blue

light reflected from the leaves of the trees surrounding the

insects is in accord with the effect of these short wave-

lengths on their maturation. There are differences in fish

between the colour responses of the light sensitive cells in

freshwater fish and marine fish (the marine fish having

principally rhodopsin and the freshwater fish principally

polyrhodopsin), and it is known that the predominant

wavelength of the light depends on the depth in the water.

In deep places the light is generally blue or green,

(wavelength about 4700 R), in shallow places offshore there

is relatively a larger amount of long wave light. As a

result the deep sea fish are more sensitive to short

wavelengths than the shallow sea fish. Thus it is reasonable

to suppose that the pigmentation is appropriate to the light

conditions in the environment in which the fish live.

It is known that there are periods during which there

is no reaction to light, even though the illumination is

greater than the threshold for the species and is made up of

wavelengths which are believed to be effective. Harrington

found that in the bridled shiner Notropis bifrenatus the

spawning season could be made earlier by increasing the

length of the da y between mid-November and mid-,Tuly, but that

116

at the remaining season, from August to October, there was

no reaction to light. The period during which there is no

reaction is called the refractory period. Observation of

the refractory period in Oryzias showed that it is difficult

to get a' reaction to light for about one month after spawning.

However control of the length of the spawning season by means

of control of the illuminated period before the refractory

period begins makes it possible to some extent to shorten or

lengthen the refractory period. (Yoshioka 5 ). In both the

shiner and Oryzias the refractory period begins.immediately

after the spawning period. The metabolism of the fish is

very much reduced after a long spawning period of several

months, and it is thought that the refractory period is a

rest period of preparation for the following spawning period.

It is by no means clear how the external stimulus of

light can reach the gonads. It is supposed that the normal

channel for the effect will be from the light receptive organs

to the brain, from there to the hypothalamus, and then to the

pituitary and finally to the gonads, and it is believed that

it is the gonadotropin secreted by the pituitary which causes

maturation of the gonads. It is found that the gonads of

hypophysectomized Oryzias remain completely immature, even

if subjected to long days with long illuminated period, and

almost all individuals die within 30 days of the operation.

This shows that the pituitary has an important role and forms

part of the channel through which light affects the maturation

of the gonads.

•

•

1 17

2. Temperature.

Temperature is also an important factor which

influences the maturation and spawning of fish, and it has

been frequently studied. Since the living environment of

fish is in water, the environmental temperature range in which

life is possible is limited and is restricted to -2 ° to 35°C.

Since the range of temperature suitable for reproduction is

even narrower, temperature is frequently the factor which

sets a limit on reproduction. As an example, Oryzias latipes

will not mature at a temperature below 10 00 however fr2.vorable

the illumination conditions may be6 , and even in good

lighting conditions the minnow will not spawn below 7 °C

(Bullough20 ). The goldfish will not spawn at any season

unless the temperature is above 14 °C. In a detailed study of

the critical limits of temperature for Gnathopogon in which

the fish were cultured under a light cycle with an 18 hour

illuminated period and kept at various temperatures from 10 00

to 22 00 it was found that maturation occurred in all cases

except at 10 00. The greatest effect of temperature on

maturation occurred in the range from 16 00 to 1900 and

temperatures above or below this range resulted in a reduced

rate of maturation. In contrast, brook trout Salvelinus

fontinalis raised under natural illumination showed no

influence of temperature on maturation even with a temperature

difference from 8.5 00 to 16 00, and maturation was affected

• 118 ••

only when there were artificial changes between long-day and

short-day periods of illumination. Under short-day

conditions of illumination, maturation arrived sooner at 8.5 °C

than at 16 ° C, but the oocytes developed less rapidly than at

16 ° C. However, since growth continued longer than at 16°C

the eggs which had developed in the ovary were larger than

at 160 0. There are other examples of species in which the

effect of temperature changes when the light cycle is changed.

The minnow requires both a high temperature and a long

illuminated period for maturation, but when the light - cycle

is changed to a short day, the effect of a high temperature

is to inhibit development of the eggs. The sanie thing occurs

with the European bitterling and stickleback.

In the above examples both light and temperature are

limiting factors in maturation and *spawning, but there are

known to be species in which temperature alone is the main

factor in inducing maturation, anà illumination has no role

of importance. The development of the seminal glands in the

killifish Fundulus heteroclitus is not related to tl-„e light

cycle, and spermatogenesis proceeds at temperatures above 10 0 0

but is inhibited at 5.5 ° C. High temperatures from 16 ° C to

21 00 encourage proliferation of spermatogonia and spermiation

in the chub Couesius plumbeus, whereas low temperatures of 5 ° C

to 12 00 encourage the formation of spermatocytes and cause an

• 1 19

enlargement of the seminal glands 21 . It is very interesting

that such changes of temperature should have differing

effects on the process of spermatogenesis.

The relative importance of temperature and illumination

in regulating maturation and spawning of fish depends on the

species, and even in one species depends on the season of

reproduction. It may in general be said that there is a

narrow critical range for each species, and that circumstances

which make it easy to get outside this may be restrictive

factors. Temperature, as shown above, is the important factor

in the maturation of the goldfish, the killifish and the chub,

and illumination appea , 's to have almost no influence, but in

such cases it is to be thought that illumination is effective

over a wide range, and that it has vèry little opportunity to

operate as a limiting factor. For example Kazanskii22 found

that although illumination had been shown not to be important

to the killifish, the ovary did not develop under the most

extreme conditions of short illuminated period (one half hour

of light per day), even at the optimum temperature.

An important influence of temperature on maturation

and spawning is its contribution to the rate of oogenesis or

spermatogenesis. Yamazaki23 measured the changes of weight

of the ovaries of goldfish kept for three weeks from the end

of January into February in temperatures of 10 ° C, 15 °C, 20 ° C

and 25 °C. He found that the higher water temperatures within

• 120

•

•

his experimental range produced remarkable increases in ovary

weight. From a weight at the start of the experiment of

0.57g, the average increase at 10 °C was 0.4g, at 15 ° C 0.75g,

at 20 o C 12.0g and at 25 oC 13.5g . Yoshioka also found

greater increases in ovary weight in Oryzias with the higher

temperatures in the range of 10 °C to 16°C. Egami et al4

measured the rate of spermatogenesis in Oryzias by means of

thymidine labelled with H3 , and found that the interval

required for the cells synthesizing DNA (before meiosis) to

form sperm was about 20 days at 15° C and 12 days at 25° C,

showing that it was strongly influenced by temperature. In

general, oogenesis and spermatogenesis are accelerated by

high water temperature. However, according to Nomura, trout

which live in cold water show the inverse effect, and the

spawning season is earlier at low temperatures. However

according to Henderson's work on brook trout, the rate of

oogenesis is reduced by lowering the temperature, and since

the time taken for maturation becomes longer, cold water

water temperatures act agafaist maturation and spawning. This

must await further clarification in the future.

A further important action of temperature is its

effect on ovulation. K. Yamamoto et al25 found that

temperature plays an important part in the ovulation of the

* Sic. I have not seen the original paper, but could these be 1.20g and 1.35g? Translator.

121

goldfish, and that ovulation did not occur at temperatures

below 14 ° C however favorable the other conditions might be.

In fact, even though vitellogenesis proceeds to completion

in the oocytes in temperatures of 13 ° C to 14°C ovulation

does not occur, and even when males are present there is no

spawning. If these females are transferred to a tank at

about 20 ° C in the company of males, ovulation and spawning

occur in two days. According to Nagahama and Yamamoto26

great changes of the basophils in the adenohypophysis

precede ovulation, and secretory granules disappear. It is

clear from this that the influence of temperature on

ovulation is exerted through the pituitary, and results

from its effects on the secretion of the pituitary hormones.

References.

122

1) T. P. HAzzAan and P. E. EDDY:

Modification on the sexual cycle in,

brook trout, Salvelinus fontinalis by

control of light. Trans. Amer. Fish.

Soc., 80, 158-162 (1951). 2) E. E. Hoovait, and H. E. HUBBARD:

Modification of the sexual cycle in

trout by control of light. CoPeia, 4, 206-410 (1937)..

3) H. YOSHIOKA: On the effects of

environmental factors upon the

reproduction of fishes. 1. The effects

of day-length on the reproduction

of the Japanese killifish, Oryzias

latipes. Bull. Fac. Fish. Hokkaido

Univ., 13, 123-436 (1962). 4) H. YosinosA : Ditto. 2. Effects of

short and long day-lengths on Ory-

zias Wipes during spawnig season.

Ibid., 14, 137,-151 (1963).

5) H. Yostuose : Ditto. 3. The occurr-

ence and regulation of refractory

period in the photoperiodic response

of medaka, Oryzias latipes. J. Hok-

kaido Univ. Education, Ser. 2B 17,

23-43 (1966).

6) H. YosnioxA : Ditto. 4. Effects of

long photoperiod on the development

of ovaries of adult medaka, Oryzias

latiPes, at low temperatures. Ibid.,

21, 14- 20 (1970),

7) H. YOSHIOKA : Ditto. 5. The signifi-

cance of combinations of light and

dark periods in photoperiodic respo-

nse of the ovaries of medaka, Oryzias

latipes, in out-of-breeding seasons.

Seibutu Kyozai, 8, 76-82 (1971). 8)

SEE PAGE 124

9) SEE PAGE 124

10) R. W. HARRINGTON: SeXHal photo-

periodicity of the cyprinid fish, Noto-

pis bifrenatus(C,ope), in reation to

the phases of its annual reproductive

cycle. J. Exp. Zool., 135, 529-556 (19

57). 11) R. W. HARRINGTON : PreSeaSOnal

breeding by I he bridled shiner,

Notropis bifrenatus, induced uncle:

light-temperature control. Copeia, 304-311 (1950).

12) B. W. CORSON : Four year progress

in the use of artificially controled light to induce early spawning of

brook trout. Prog. Fish- Cult., 17, 99---102 (1955).

13)' SEE PAGE 125

14) N. E. HENDERSON. : Influence of light

•

25) 19) SEE PAGE 127 SEE PAGE 127

• 1 2 3

•

and temperature on the reproductive

cycle of the Eastern brook trout

Salvelinus fontinalis (Mitchill). J.

Fisheries Res. Bd. Can., 20, 859-897

_ (1963).

15), SEE P&GE 125

SEE PAGE 126

17)

SEE PAGE 126

18) N. EGAM1 : Effect of artificial photo-

periodicity on time of oviposition in

the fish, Oryzias • Wipes. Annot.

Zoo! , Jap., 27. 57-62 (1954).

21) S. N. ARSAN : Effects of temperature

and light on the cyclical changes

in the spermatogenetic activity of

the lake chub, Couesius plutnbeus

(Aoessiz). Can. J. Zool., 44, 161 ,-171

(1966).

22) B. N. 1{Kie.t;sxll : Experimental an-

alysis of the growth of oocytes in

fish. Dokl. Acad. Nauk. U. S. S. R.,

80, 277-280 (1951).

23) F. YAMAZAFI Endocrinolgical stud-

ies on the reproduction of the fe-

male goldfish, Carassius auratus L.,

with special reference to the funct-

ion of the pituitary gland. Mem. Fac-

Fish. Hokkaido Univ., 13, 1-64(1:45).

24)

SEE PAGE 127

16)

20) W. S. Bum.ouon: A study of the

reproductive cycle of thc minnow

in relation to the environment.

Proc. Zool. Soc. London, 109, 79-102

1939).

26) Y. NAGALTAMA and K. YAMAMOTO:

Basophils in the adenohypophysis

of the goldfish (Caraisius auratus).

Gunma Symposia on Endocrinol., 6,

39--59 (1969).

• 124

8 , HIBIYA Takashi ra.

Honmoroko no seijuku ni oyobosu suion narabi

ni hikari no eikyo.

Showa 47 nendo suisan gakkai shunki daikai koen happy°.

T. Hibiya et ai.

The influence of temperature and illumination on

the maturation of gnathougon elongatus caerulescens.

Papers presented to the Spring Meeting of the

Japanese Society of Scientific Fisheries (1972).

9. HIBIYA Takashi ra.

Honmoroko, Gnathopogon elongatus caerulescens (SAUVAGE).

no seijuku ni oyobosu suion narabi ni hikari

no eikyo III.

Koshu kanjusei no jikan ni yoru sai.

Showa 48 nen do nihon gakkai shunki daikai

koen happyo (1973).

T. Hibiya et al.

The influence of temperature and illumination

on the maturation of Gnathopogon elongatus

caerulescens (SAUVAGE).

Periodic changes in sensitivity to light.

Papers presented to the Spring meeting of the

Japanese Society of Scientific Fisheries (1973).

• 125

13. NOMURA Minoru.

Nijimasu no jinko sairan ni kansuru kiso kenkyu III.

Ko shiki no henka ni yoru sairan no sokika.

Nichi sui shi 28 1070 - 1076 (1962).

M. Nomura.

Basic research on the artificial collection of

eggs from Rainbow trout. III.

Changes of the illumination cycle and artificial

egg collection at an early 'period.

Bulletin of the Japanese. Society of Scientific

Fisheries. 28 1070 - 1076 (1962).

15. SHIRAISHI TAKEDA Tatsugo.

Ayu no seijuku ni oyobosu koshiki ni eikyo.

Tansui ku sui ken hohoku 11 69 - 81, (1961).

Y. Shiraishi, T. Takeda.

The influence of the illumination cycle on

maturation of ayu (Plecoglossus altivelis).

Reports of research of the Freshwater Fisheries

Research Laboratory. 11 69 - 81, (1961).

•

• 126

16. SHIRAISHI Yoshikazu.

Ayu no seijuku ni oyobosu koshiki no eikyo.

Dai 3 ho, shoshako no genkai shodo ni tsuite.

Tansui ku sui ken hohoku 11 69 - 76, (1965).

Y. Shiraishi.

The influence of the illumination cycle on

maturation of ayu (Plecoglossus altivelis).

Third report. On a critical quantity of illumination.

Reports of research of the Freshwater Fisheries

Research Laboratory 11 69 -76, (1965).

17. SHIRAISHI Yoshikazu.

Ayu no seijuku ni oyobosu koshiki no eikyo.

Dai 4 ho, shoshako no hacho to temmetsu shosha

no koka.

Tansui ku sui ken hohoku 11 69 - 76 (1965).

Y. Shiraishi.

The influence of the illumination cycle on

maturation of ayu (Plecoglossus altivelis).

Fourth Report. The effects of switching and of

wavelength of illumination.

Reports of research of the Freshwater Fisheries

Research Laboratory 11 69 - 76 (1965).

• 127

•

19 , YOSHIOKA Yasuko.

Kankyo to seijuku, sanran, tansuigyo.

Showa 49 nendo nihon suisan gakkai shunki dal kai.

Shinpojiumu koen happyo 1974.

Y. Yoshioka.

Environment, maturation and spawning. Freshwater fish.

Collected symposium papers, Spring Meeting of the

Japanese Society of Scientific Fisheries 1974.

24. EGAMI Nobuo, TAGUCHI Yasuko.

Medaka noseishi keisei sokudo ni oyobosu suion

no eikyo (yoho).

Shiken keitai gaku 21 500 (1968).

N. Egami, Y. Taguchi.

The predicted effect of temperature on

spermatogenesis in Oryzias latipes.

Experimental Morphology, 21 500, (1968).

25. YAMAMOTO Kiichiro, NAGAHAMA Yoshitaka, YAMAZAKI Fumio.

Kingyo no shunen sairan ni tsuite.

Nichi sui shi 12 977 - 983 (1966).

K. Yamamoto, Y. Nagahama, F. Yamazaki.

On a method for year-round collection of

goldfish eggs.

Bulletin of the Japanese Society of Scientific

Fisheries 32 977 - 983, (1966).

•

128

7. Marine Fish.

Tertio HARADA.

(Faculty of Agriculture, Kinki University).

It can be imagined that there is a close connection

between the maturation and spawning of marine fish and the

environment in which they live, but there is no detailed

account of the factors which are of importance for individual

species. Investigation of the environment is more difficult

than in the case of freshwater fish, and culture is not easy,

but the main reason for this lack of knowledge is that there

have been very few reports about maturation and spawning.

The intent of this paper is to describe experimental

work on maturation, spawning and egg collection which has

been undertaken by the author for more than ten years in the

Kinki University Fisheries Research Laboratory at Shirahama

in Wakayama prefecture. These studies have been based on the

black porgy (kurodai) Mylio macrocephalus, the porgy (kijinu)

latus, the red sea bream (madai) Chrysophrys major, the

porgy (hedai) Rhabdosargus sarba, the parrot bass (ishidai)

Oplegnathus fasciatus, the parrot bass (ishigakidai) Oplegnathus

punctatus, the yellow tail (buri) Seriola quinqueradiata, the

amberjack (kanpachi) Seriola purpurascens, the amberjack

(hiramasa) Seriola aureovattata, the jack (shimaaji) Caranx

delicatissimus, the sea perch (hirasuzuki) Lateolabrax latus,

and the Pacific fluke (hirame) Paralichthys olivaceus.

• 129

••

1. Water temperature, maturation, and spawning.

It is believed that temperature is the environmental

factor most closely connected to maturation. As is well

known, in natural conditions the gonads mature as the spawning

season approaches, and spawning soon begins. When the water

temperature during the spawning season is measured, it is

found that while there is vigorous activity the temperature is

well within the upper and lower limits of its range.

The natural spawning of yellowtail in the ocean has

been investigated by Uchida et al1 , by Mitani et a1 2 , by

experiments made by Harada et al3,4 on egg collection during

the 10 years from 1.960 to 1969 in the regions surrounding the

Orne islands and the Goto islands in Nagasaki prefecture, and

by Umeda et al5 on the spawning of yellowtail migrating to

the Komame fishing ground in Kochi prefecture. Most of the

spawning was from 17 ° C to 22 °C and the most suitable

temperatures appear to be from 18 °C to 20 0 0 . According to p68

Harada et al 6 , eggs can be collected from cultured yellowtail

by means of the injection of hormones when the water temperature

is 17 ° C to 21 0 0 . These authors cultured zero-year fish as

parent fish, rearing part of them in temperature-controlled

tanks, and part in netting fishpens offshore from Shirahama,

exposed to the natural temperature changes. Eggs were collected

by means of the administration of hormones and vitamin E, and •

samples of the 1970 experiments are shown in Table 7.1.

10 te t1

•

•

1 30

Table 7.1.

Records of the eggs obtained in 1970 by individual collection after the injection of hormones into yellowtail. 15 females and 5 males were used. When several fish spawned on the same day, 2 or 3 selected representative individuals are listed. The column headed "Number of floating egg..s" shows the number collected from a single parent yellowtail.

ATE. PLACE OF TEMPERATURE NUMBER OF RATE OF FER- NUMBER OF F ISH

REARING. AT TIME OF FLOATING TILIZATION FROM WHICH E3G

EGG COLLEC- EGGS. (%)* WERE TAKEN FOR

TIOW. FERTILIZATION. . -

LAND - ' - - -

W'12 17.3 193, 160 56.4 TANK 1

16.7 79,000 0 14 nm o

16.7 19,730 0 .

16 111.1

18.3 14,720 16.0 1

. NET FISH 18.0 199,260 9.3,

21 PEN.

18.0 345,600 -- 7.7 , 7

. 18.0 216,450 20.0 ,

Mu 18.1 93.100 54.2 22 11 .

18.1 40,000 25.0 . 18.0 147,400 81.5

M" 24 MA 201,040 76.7 .

18.0 26,200 84.7

tin 18.4 98,000 52.4 • 26 8

.18.4 155,000 35.2

,flt 18.4 10,920 84.4 28 8

' 18.4 144,000 . 66.7

tin

18.6 tWO 87.8 . 29 •

. 18.6 1,381 52.9 4

,10 1 19.0 39,900 28.3 ' ' 30

19.0 8,170 46.0 6

t”, 18.5 212,900 76.7 ,

V • 2 . • 18.5 209,000 55.3 4

• 18.5 187,350 36.0

18.3

18.3

20.3

20.3 wn

14 20.9

189,000

07,800

309,480

335,300

o

27.9

14.6 4

57.5

46.3

, 0 o

4

• 131

As can be seen from the Table, fertilized eggs were

not obtained below 17 ° C, and they were difficult to obtain

when the temperature was close to 21 00. Eggs could be

obtained from a large number of parent fish when the .water

temperature was between 18 ° C and 20 °C, and at these

temperatures the proportion fertilized was relatively large.

It was possible on rare occasions to obtain eggs from cultured

yellowtail even without giving injections of hormones or

vitamin E, but on such occasions the temperature was between

18 ° C and 20 °C. At temperatures below 16 ° C or above 22 00 no

eggs could be collected even with the use of hormones and

vitamins. Gathering together all these facts, it is evident

that temperatures suited to the spawning of yellowtail are

from 18 °C to 20 00. •

Experiments by Harada et al7,8with cultured parent

fish have shown that the temperature for active spawning of

Chrysophrys major, Mylio macrocephalus, and Rhabdosargus sarba

is almost exactly the same, from 17 °C to 21 °C. Accordinsx to

Fujita the puffer (torafugu) Spheroides rubripes spawns at

exactly the same time, and the author has found the same

temperature limits for the collection of eggs from cultured

karasu (gatora) . According to experiments with cultured parent

fish by Harada et al 10,11 , the temperatures required for active

* Karasu (gatora). I have not been able to identify this fish. "Karasu" is normally Rheinhardtius hippoglossoides, a coldwater fish. There is also the dogfish karasuzame, Etmopterus frontimaculatus, but some puffer species seems to be intended here, or perhaps a misprint for karasumibora, Mugil japonicus. Translator.

13 2

maturation and spawning of Oplegnathus fasciatus and . - Oplegnathus punctatus are 21 o C to 26 ° C, higher than those

for Chrysophrys and Spheroides.

Harada et al12,13 have also experimented with cultured

parent fish of Seriola purpurascens and Seriola aureovittata

and found that water temperatures for maturation and active

spawning were 20 °C to 25 °C.

Harada et al 14 collected eggs from fish in the open

ocean. Temperatures for the bonito (hagatsuo) Sarda orientalis

migrating to the Kii peninsula were 18 °C to 24°C. In further

experiments 15,16 the temperatures for the freight mackerels

(marusoda) Auxis than,rd and (marusoda)Auxis tapeinosoma were

18 °C to 26 00, and for the yellowfin tuna-(kihada) Neothunnus

macropterus the temperature was 26.2 0 0, so that the tuna was

by a little the warmest.

The spawning season for all these species is during

the warming up time from spring to summer, but some species

spawn during the cooling down period from autumn to winter.

The porgy (kijinu) Mylio latus spawns at just the same

temperatures as Mylio macrocephalus and Chrysophrys major, but

in the autumn. The jack(shimaaji) Caranx delicatissimus 18

matures at Shirahama in early winter from December to January,

and the Pacific fluke (hirame) Paralichthys olivaceus 19 spawns

from late winter to early spring in February and March. The

author's results from the spawning seasons and water

'temperatures of useful marine fish are listed in Table 7.2.

• 133

Parent fish culture.

Completely experimental.

tI

11 It

It

lu

11

It

It It

It

tl

Not completely.

Completely experimental.

It

ft

Table 7.2.

Water temperature and spawning seasons for useful marine fish.

Species

Paralichthys olivaceus

Chrysophrys major

Mylio macrocephalus

Rhabdosargus sarba

Seriola quinqueradiata

Sphreoides rubripes

Karasu

Oplegnathus fasciatus

Oplegnathus punctatus

Seriola purpurascens

Seriola aureovittata

Sarda orientalis

Auxis thazard

Auxis tapeinosoma

Neothunnus macropterus

Mylio latus

Caranx delicatissimus

Spawning season.

Feb -May

Mar-June

Mar-June

It fir

Apr -May

It It

May -July

11

May-June

II It

May-JuIy

May-Aug

11

June-Aug

Oct-Nov

Dec-Jan

Spawning temperature

1 4- 17 ° C

17-21 °C

17-21 ° C

▪ 0 0

• It 0

17-20 °C

ft

21-26°C

22 -25 °C

It It • It

18-24°C

18-26°C

II II II

24-28 °C

21-17° C

15-19°C

* See footnote, page 131. •

134

•

The temperatures in which marine fish are reared are

closely connected with their maturation. In the author's

experiments lasting over ten years on the rearing and maturing

of parent . fish of Seriola quiqueradiata, S. purpurascens,

S. aureovittatus and Chrysophrys major it has been found that

if there is a period of extremely cold temperature or extremely

warm temperature within several months before the expected

spawning season, and if the period during which spawning is

stimulated by change of temperature to the proper value is

short, it is difficult to obtain good quality eggs. In extreme

-cases, absolutely no eggs can be collected. This shows that

disturbance of the environment in which marine fish are reared,

may cause difficulty in maturation.

2. tj_ol i*Illumina- lmonandF_•_pa.ymi__na.

One environmental factor closely connected to the

maturation and spawning of marine fish is the illumination.

There are a good many reports about the influence of

photoperiodicity and day-length on the maturation and spawning

of freshwater fish, and marine fish will probably be similar.

The fact that Chrysophrys major and Mylio macrocephalus mature

and spawn in the spring whereas Mylio latus matures and spawns

at the same temperature in the autumn probably shows the

influence of illumination. In the spring the temperature is

rising and at the same time the illuminated part of the day is

lengthening, but in the autumn the temperature is falling and

the illuminated part of the day is becoming shorter.

• 135

Light is also closely connected to the time of

spawning during the day. Cultured Chrysophrys major, Mylio

macrocephalus, Rhadosargus sarba, Oplegnathus fasciatus,and

Oplegnathus punctatus almost always spawn between 3 pm and

10 pm. There is greatest activity around sunset. The eggs,

when discharged, float nearby on the surface of the sea and

directly after spawning they are crowded together, but then

are scattered by winds and waves and spread out. When eggs

are discharged in the light before sunset small fish such as

jack mackerel (maaji) which are in the vicinity gather

,together and they can be seen to be eating the eggs. As the

eggs are dispersed and become less crowded the amount lost

and eaten by small fish should be small. The frequent

spawning between sunset and nighttime may perhaps be an

instinctive use of the diminution orlight during the long

night for shelter against this loss by predation.

3. Other environmental factors, maturation and spawning.

Other environmental factors related to maturation and

spawning are the quantity of dissolved oxygen, the salinity,

noxious substances, and available space. If eggs of high

quality are to be obtained, the amount of dissolved oxygen

must not only be maintained, but an environment high in oxygen

saturation must be provided. There are no published reports

about the influence of low salinity on the maturation or

•• • 136

spawning of individual species, but in the case of the

yellowtail the buoyant eggs will probably sink if the

salinity is low and the density falls below 1.021, so that it

appears necessary to maintain a suitable salinity for spawning.

The environment will be degraded if dangerous substances,

accumulations of excrement or red tide are allowed to flow in,

so it can easily be supposed that maturation and spawning will

be adversely affected. It has also been demonstrated in

experiments on the culture of parent fish that normal

development is not possible if the rearing place is tifo

confined, and maturation and spawning will be hindered.

In short, a healthy environment must first be provided,

and then the conditions directly related to maturation and

spawning must also be provided. However further studies must

be awaited before a general account . can be given of the

relation of these individual conditions to maturation and spawning,

4. The culture of parent fish for eggcalleninE.

Eggs for the production of marine fish fry m,ly be

collected from wild or cultured parent fish. The disadvantages

of the use of wild fish include the uncertainty of the capture

of the parent fish, the relatively large expenditure needed

and the few opportunities for obtaining good quality eggs.

On the other hand, the use of cultured fish requires a long

time (often up to three years), and research on methods of

P7

• 137

•

maturation and egg collection is required, but it makes

possible a planned production and collection of reliable and

good quality eggs. Harada et al have experimented with the

culture of parent fish of Chrysophrys maior 7, Mylio macrocephalus 7

,

Rhabdosargus sarba8 , and Oplegnathus fasciatus 10 and have

found that eggs of better quality than those obtained from

wild parent fish could be reliably produced. With Seriola

guinqueradiata6, Paralichthys olivaceus 19 and Lateolabrax latus

there was no difference between the natural eggs and those

whose production had been pl,nned. Although it was not possible

to collect eggs from wild Seriola purpurascens 12 , Seriola

aureovittata13 , Caranx delicatissmus 18 , and Oplegnathus

Eunctatus 11 , it was possible to collect them from cultivated

Parent fish. It is considered that .future reliable production

of large quantities of fry will necessitate planning the

reliable supply of good quality eggs from cultured parents.

With the improved experimental netting fish pens which

Harada20,3 , has been using (in small sizes) since 1954 for

rearing quarters, the fish are reared in conditions close to

the natural conditions in the ocean. The culture density is

relatively high, the expense is low, they can easily be set up,

the fish can easily be harvested, and the fish can be moved

around in their normally submerged condition. These pens

have so many advantages that they are considered to be

138

appropriate places for rearing parent fish for egg collection.

Rearing places on land have the disadvantages of high building'

costs and high power costs, but they have advantages in the

gathering of naturally spawned eggs and in environmental control.

5. The stimulation and inhibition of maturation and spawning

by environmental control.

When parent fish are cultured, environmental control

can be used to stimulate or inhibit maturation so as to obtain

the desired quantity of eggs at the desired time. The period

during which fry are produced can be extended and their

production thereby prolonged. Harada et al21 reared Chrysophrms

major, Mylio macrocephalus, Seriola quinqueradiata, Oplegnathus

fasciatus and Oplegnathus punctatus in controlled environments

and found that eggs could be obtained earlier than the natural

season by raising the temperature. In these authors'

experiments on early egg collection from Chrysophrys major

from 1969 to 1974, naturally spawned eggs were obtained in the

middle of February, 1 to 1i months early. Figure 7.1 shows

an example of the relation between water temperature and

natural spawning of Chrysophys major reared in heated tanks at p72

the Shirahama Laboratory of Kinki University. It can be seen

in Figure 7.1 that when the water was gradually warmed,

natural spawning began when the neighbourhood of 17 ° C was

reached. When the temperature was lowered to 16° C spawning

139 •

• 1

■-•

a) e-1

4-)

(1)

a) 1 - 4-)

3 -

NATURAL

SPAWNING•

, NATURAL

SPAWN I NG.

NATURAL

. SPAWNING. %.

• NATuRAL

.4- SpAWNING •

NATuRAL ,"P•

SPAWNING;

NATURAL

SPAwNING,\Z,

,INATURAL7

SPAWNING.. •

Figure 7.1.

Culture water temperature and natural spawning of

Chrysophrys major in a heated tank.

10 15 20 15 20 25 1 - - 5

February March

Date.

140

stopped, and on rewarming to the neighbourhood of 17 ° C spawning

began again. Thus it is evident that spawning can be

controlled by control of the environmental water temperature.

Through the cooperation of the Owase City central Sanda steam

driven electricity generating plant from November 1967 to

April 1968, the authors obtained eggs from Seriola

quingueradiata by means of hormone administration on 4 April,

one half month early. Experiments were also made in the

Shirahama Laboratory heated tank, and an example of the

results is shown in the column for the tank on land in

Table 7.1. This shows that at the temperature of 16.7 °C no

fertilized eggs were obtained, but that thy were obtained

at 17.3 ° C and 18.3 ° C. From this it can be seen that Seriola

quinqueradiata can also be made to spawn early by controlling e 7.5

the environment to the optimum spawning temperature from 17 ° C

to 19 ° C. The authors have obtained similar results and

similar possibilities of early spawning with Mylio macrocephalus,

Oplegnathus fasciatus, and Oplegnathus punctatus.

The authors also found that a rapid increase of water

temperature was an effective method of stimulating spawning

in Chrysophrys major and Opleenathus fasciatus. The

temperature of water in which fish thought to be near spawning

were living was suddenly raised by 2 ° C to 3°C.

•

• 141

•

Tanks on land are considered to be appropriate as

rearing places for parent fish from which eggs are to be

obtained by means of control of the environment. Their

advantages include convenience of environmental factors such

as light, temperature, dissolved oxygen and salinity, the

possibility of preventing the entry of red tide and of

pollutants, and the easy collection of naturally spawned eggs

in nets stretched across the water outlets.

Other important points are:-

1. The exchange flow of sea-water requires power an6 is

expensive. This is particularly true with large tanks and

with those established in high locations.

2. The tanks must be suitable for the species used. In

particular, tanks must be deep and wide when large fish are

to be cultured, and this implies high construction costs.

3. Fish can be damaged as they are moved from the sea to

a tank on land. There is a need for improved ways of

capturing easily damaged species such as tuna, bonitos

and jacks.

4. Fish diseases can easily develop in land tanks. Epidemics

can easily cause great harm. Also, in many species, stress

may impede maturation.

5. Control of the rearing and environment by heating or

Cooling consumes a great amount of energy and is expensive.

• 142

6. Future prospects.

The planned production of large quantities of marine

fish fry requires the culture of parent fish to produce good

quality eggs which can be collected at the desired time. Up

to the present parent fish of more than ten species, such as

Chrysophrys maior, Seriola cuinaueradiata, Oplegnathus

fasciatus, Caranx delicatissimus and Paralichthys olivaceus

have been shown experimentally to be suitable for culture.

The individual environmental factors needed for maturation

and spawning are not known, and there are some species, such

as tuna and bonito, which have not been experimentally shown

to be suitable . In order that the technology needed for

economic culture of parent fish may be developed, more

detailed studies are needed of the relation between maturation

and spawning and the environmental and other factors concerned,

• 143

•

ReferenceS.

1. UCHIDA Keitaro, MICHIZU Yoshie, MITO Toshi, NAKAMURA Kantaro.

Buri no sanran oyobi shoki seikatsu shi.

Kyushu daigaku nogaku bu gakusei zasshi

16 (3) 336 - 337 (1958).

K. Uchida, Y. Michizu, T. Mito, K. Nakamura.

Spawning and early life history of the yellowtail,

Seriola ouinaueradiata.

Science Journal of tue Faculty of Agriculture,

Kyushu University, 16 (3) 336 - 337 (1958).

2. MITANI Fumio.

Buri no gyogyo seibutsu gakuteki kenkyu.

Kinki daigaku nogaku bu kiyo 1 211 , 212 (1960).

F. Mitani.

Biological study of the yellowtail (Seriola

quinqueradiata) fishery.

Bulletin of the Faculty of Agriculture, Kinki

University, 1 211 - 212 (1960).

3. HARADA Teruo.

Buri no zoshoku . ni kansuru kenkyu.

Kinki daigaku nogaku bu kiyo 2 40 - 54 (1965).

144

3. T. Harada.

Study of reproduction in yellowtail (Seriola

quinqueradiata).

Bulletin of the Faculty of Agriculture, Kinki

University. 1 40 - 54 (1965).

4. FUJITA Yaro, MICHIZU Yoshie, HARADA Teruo.

Horumon shigeki ni yoru buri no jinko sairan.

Nihon suisan gakkai showa 40 nendo shuki daikai

koen yoshj 15 (1965).

x._Eainp„, Y. Michizu, T. Harada.

Artificial collection of eggs from yellowtail

(Seriola quinqueradiata) by means of the

administration of hormones.

Abstracts of papers, autumn meeting (1965) of the

Japanese Society of Scientific Fisheries 15 (1965).

5. UMEDA Susumu, HIROZAWA Kuniaki, OCHIAI Akiru.

Kochi ken Komame gyojo ni raiyu suru buri sanran

gun to shinahorin ni yoru seijuku sokushin ni tsuite.

Nichi sui shi .m 446 - 449 (1969).

S. Umeda, K. Hirozame, A. Ochiai.

On the acceleration of maturation by means of synahorin

in the spawning shoals of yellowtail (Seriola

• quinqueradiata) which migrate to the Komame fishing

ground, Kochi prefecture.

Bulletin of the Japanese Society of Scientific Fisheries,

31 446 - 449 (1969).

1 4.5

6. HARADA Teruo, KUUI Hidemi, kIZUNO Kenpachiro,and

MURATA Osamu, NAKAMURA Motoji.

Yosei buri kara no sairan, jinko fuka ni tsuite.

Nihon suisan gakkai showa 42 nendo shuki daikai

koen yoshi 20 (1967).

T.Harada, H.Kumai, K.Mizuno, 0.Murata, M.Nakamura.

Collection and artificial hatching of eggs from

cultured yellowtail (Seriola quinqueradiata).

Abstracts of papers, autumn meeting (1967) of the

Japanese Society of Scientific Fisheries 20, (1967).

7. HARADA Teruo, KUMAI Hidemi, UMEDA Susumu.

Madai oyobi kurodai no shingyo no yosei ni tsuite.

Nihon suisan gakkai showa 39 nendo shuki daikai

koen yoshi 27 (1964). -

T. Harada, H. Kumai, S. Umeda.

Culture of parent fish of Chrysophrys major and

Mylio macrocephalus.

Abstracts of papers, autumn meeting (1964) of the

Japanese Society of Scientific Fisheries 27, (1964).

•

•

146

8. HARADA Teruo, MURATA Osamu, KUMAI Hidemi.

Hedai no shingyo yosei, jinkofuka oyobi shigyo

ikusei ni tsuite.

Nihon suisan gakkai showa 42 nendo shuki daikai

kOen hoshi 20 (1967).

T. Harada, O. Murata, H. Kumai.

On the culture of parent fish, the artificial hatching

and the rearing of fry of Rhabdosargus sarba.

Abstracts of papers, autumn meeting (1967) of the

Japanese Society of Scientific Fisheries 20 (1967).

9. FUJITA Yaro.

Yogyogaku kakuron. 554 - 562.

Koseisha Koseikaku (1967).

Y. Fujita.

Complete pisciculture. 554 - 562.

Published by Koseisha Koseikaku (1967).

10 KUMAI Hidemi, NAKAMURA Motoji, HARADA Teruo.

Ishidai no shingyo yosei, jinko fuka oyobi shigyo

ikusei ni tsuite.

Nihon suisan gakkai showa 42 nendo shuki daikai

koen yoshi 20 (1967).

•

• 1 47

10. H. Kumai, M. Nakamura, T. Harada.

On the culture of parent fish, the artificial hatching

and the rearing of fry of Oplegnathus fasciatus.

Abstracts of papers, autumn meeting (1967) of the

Japanese Society of Scientific Fisheries 20 (1967).

11. HARADA Teruo, KUMAI Hidemi, MIZUNO Kenpachiro,

NAKAMURA Moto.11, MIYASHITA Mori, FURUTANI Hideki.

Ishigakidai no jinko fuka to shigyo no shiiku.

Nihon suisan gakkai showa 45 nendo shuki daikai

koen yoshi 62 - 63 (1970).

T. Harada, H Kumai, K. Mizuno, M. Nakamura,

M. Miyashita and H. Furutani.

Artificial hatching and rearing of fry of

Oplegnathus punctatus.

Abstracts of papers, autumn meeting (1970) of the

Japanese Society of Scientific Fisheries 62-63 (1970).

12. HARADA Teruo, MURATA Osamu, MIZUNO Kenpachiro,

FURUTANI Hideki, KUMAI Hidemi, NAKAMURA Moto;li.

Kanpachi no shingyo yosei, jinko fuka shigyo shiiku.

Nihon suisan gakkai showa 42 nendo shuki daikai koen (1970).

T.Harada, 0.Murata, K.Mizuno, H.Furutani, H.Kumai, M.Nakamura.

Artificial hatching and rearing of fry of

Seriola purpurascens.

Papers of the autumn meeting (1967) of the Japanese

Society of Scientific Fisheries (1970).

•

•

148

13. HARADA Teruo, MURATA Osamu, MIYASHITA Mon.

Hiramasa no shingyo yosei, sairan, jinko fuka,

shigyo shiiku.

Nihon suisan gakkai showa 47 nen shuki daikai

koen yoshi chu 308 (1972).

T. Harada, O. Murata, M. Miyashita.

Culture of parent fish, egg collection, artificial

hatching and rearing of fry of Seriola aureovittata.

Collected abstracts of papers, autumn meeting (1972)

the Japanese Society of Scientific Fisheries 308 (1972).

14. HARADA Teruo, MURATA Osamu, MIYASHITA Mon.

Hagatsuo no jinko fuka to shichigyo no shiiku.

Kinki daigaku nogakubu kiyo 1 - 4 (1974).

T. Harada, O. Murata, M. Miyashita.

On the artificial hatching and rearing of fry of

Sarda orientalis.

Bulletin of the Faculty of Agriculture,

Kinki University, 2. 1 - 4 (1974).

15. HARADA Teruo, MURATA Osamu, MIYASHITA Mûri.

Hirasoda no jinko fuka to shichigyo no shiiku ni tsuite.

Kinki daigaku nogakubu kiyo 6 109 - 112 (1973).

T. Harada, O. Murata, M. Miyashita.

On the artificial hatching and rearing of fry

of Auxis thazard.

Bulletin of the Faculty of Agriculture,

Kinki University, 6 109 - 112 (1973).

• 1 49

16. HARADA Teruo, MURATA Osamu, MIYASHITA Mori.

Marusoda no jinko fuka to shigyo shiiku ni tsuite.

Kinki daigaku nogakubu kiyo 6 113 - 116 (1973).

T. Harada, O. Murata, M. Myashita.

On the artificial hatching and rearing of fry of

Auxis tapeinosoma.

Bulletin of the Faculty of Agriculture,

Kinki University, 6 113 - 116 (1973).

17. HARADA Teruo, MIZUNO Kenpachiro, MURATA Osamu,

MIYASHITA Mon, FURUTANI Hideki.

Kihada no jink‘ , fuka to shigyo shiiku ni tsuite.

Kinki daigaku nogakubu kiyo 4 145-151 (1971).

T. Harada, K. Mizuno, O. Myashita, H. Furutani.

On the artificial hatching and rearing of fry.

of Neothunnus macropterus.

Bulletin of the Faculty of Agriculture,

Kinki University, 4 145 - 151 (1971).

18. HARADA Teruo, MURATA Osamu, MIYASHITA

Shimaaji no shingyo yosei, sairan, fuka, shichigyo

no shiiku.

Nihon suisan gakkai showa 48 nendo shuki daikai

koen yoshi 65 (1973).

• 15 0

•

18. T. Harada, O. Murata, M. Miyashita.

The culture of parent fish, egg collection, hatching

and rearing of fry of Caranx delicatissimus.

Abstracts of papers, autumn meeting (1973) of the

Japanese Society of Scientific Fisheries 65 (1973).

19. HARADA Teruo, MURATA Osamu, MIZUNO Kenpachiro,

NAKAMURA Motoil, MIYASHITA Mon, FURUTANI Hideki.

Jinko fuka hirame no ikusei to seijuku ni tsuite.

Nihon suisan gakkai showa 44 nen shuki daikai

koen yoshi 1 (1964).

T. Harada, 0.Mur, -tta, K.Mizuno, M.Nakamura, M.Miyashita,

H. Furutani.

Rearing and maturation of artificially hatched

Paralichthys olivaceus.

Abstracts of papers, autumn meeting (1969) of the

Japanese Society of Scientific Fisheries (1964) 41*.

20. HARADA Teruo, KUMAI Hidemi.

Gosei seni gyomo shiyo ni yoru burl no ikesu

ami yosei ni tsuite.

Nihon suisan gakkai showa 34 nen shunki daikai

koen yoshi 44 (1959).

* Sic, though at least one of the dates must be in error.

•

•

151

20. T. Harada, H. Kumai.

On the culture of Seriola quinqueradiata in fish

pens made of artificial fibres.

Abstracts of papers, spring meeting (1959) of the

Japanese Society of Scientific Fisheries 44 (1959).

21. HARADA Teruo, KUMAI Hidemi, MIZUNO Kenpachiro,

NAKAMURA Motoji, MIYASHITA Mon, FURUTANI Hideki.

Buri, madai, ishidai, ishigakidai kara no kaon ni

yoru soki sairan.

Nihonsuisan gakkai showa 45 nen shyki daikai

koen hoshi 62 - 63 (1970).

T. Harada, H. Kumai, K. Mizuno, M. Nakamura, M.Mimashita

and H. Furutani.

On early egg collection from Seriola quinqueradiata,

Chrysophrys major, 02.1egnathus fasciatus and

Oplegnathus punctatus by means of warming.

Abstracts of papers, autumn meeting (1970) of the

Japanese Society of Scientific Fisheries

62 - 63, (1970).

•

•

152

IV. Maturation and Metabolism.

8. Maturation and fat metabolism.

Fumio TAKASHIMA.

(Tokyo University of Fisheries).

Many differing metabolic processes occur during the

development of the egg. Two which can be considered as

particularly important are the synthesis of the proteins

which comprise the embryonic tissue and those which generate

energy. In the eggs of the rainbow trout the amount of

acetone-soluble fat rapidly diminishes during development1 ' 2 ,

particularly at the time when movement becomes vigorous after

hatching2 . The fats are also diminished in the eggs of

Oryzias latipes just before hatching 3 , and in salmon eggs at

the time when the eyes deve1op4,5 . In all cases it appears

that fats are concerned in the generation of energy during

development. Fats are however not concerned solely in the

generation of energy, but are also concerned in the development

of the embryo. The phospholipids in the eggs of Oryzias

latipes diminish up to the moment of hatching, but it is

believed that after dissolution they are transferred to the

embryonic tissues 3. In these ways fats play important roles

in the course of development of the egg, and it is necessary

that the deposits of fat in the mother's body during this

development should be both qualitatively and quantitatively

• 153

sufficient. From the viewpoints of fat chemistry and of food

chemistry, much is already known about the fats in fish eggs,

but there is insufficient knowledge of the physiology of fat

accumulations and of the control mechanisms. The present

paper seeks to summarize the facts about fat metabolism in

relation to oogenesis from this physiological point of view.

1. The chemical properties of fish egg fats.

The overall analyses of the mature eggs of several

species of fish are shown in Table 8.1. The proportions of

the various components depend on the species but there is

more protein and less fat than in hen eggs. Each egg of the

Atlantic salmon Salmo salar contains 14.6mg of fatty tissue,

of which 67.9% is acetone-soluble fat, a large amount in

comparison to the 32.1% of acetone - insoluble fat . In chum p77

salmon, the egg contains 29.8mg of fat, of which as much as

75% is acetone-soluble. The acetone-soluble fats in the chum

salmon eggs are mostly glycerides, the acetone insoluble fats

which form 23% are compound fats in which the principal

components are phospholipids. The rainbow trout egg contains

10.7e fat, of which 6.4% is neutral fat and 4.3% is complex

fat. Thus fish eggs fats contain great amounts of glycerides

and complex fats, and the complex fats are mostly phospholipids,

the main comnonent being lecithin11 .

•• • 154

•

Table 8.1.

General analysis of fish eggs

Species Water Protein Fat Carbo- Ash Source hydrate (Reference)

Rainbow trout 59.0 28.8 11.8 --- 1.6 2

Rainbow trout 59.0 29.4 10.7 --- 1.7 6

Rainbow trout 66.2 20.2 7.4 0.2 1.3 7

Carp 66 28 2.5 --- 1.4 8

Chum salmon 54.5 30.1 10.4 --- 1.7 8

Cod 72.1 23.0 1.3 --- 2.1 8

Herring 69.2 26.3 4.2 --- 1)4 8

Sardine 70.7 21.0 7.0 0.3 2.1 9

Domestic hen 48.7 16.6 32.3 1.0 1.1 10

Ando investigated the fatty acid composition

of the fats in rainbow trout eggs2,12 , and found a high

proportion of unsaturated fatty acids, which could reach as

much as 82%. Of this 36% was higher fatty acids and 46% lower

fatty acids. 19.9% of the fatty acid constituents of the

acetone-soluble fats in chum salmon eggs13 were saturated, the

principal component being 016:0 , whereas 80.1% were unsaturated.

It is also reported14 that carp eggs were 28.3% saturated and

71.7% unsaturated. The unsaturated acids in rainbow trout

and chum salmon eggs contained more than 10% of highly

(%).

•

155

unsaturated C 22 acids but carp eggs only contained 1.5%14

It has been suggested 2 that the large amount of highly

unsaturated fatty acids contained in salmonid eggs is related

to their development at low temperatures around 10 0 at which

these fats are in a liquid condition.

The general analysis of the fats has been given above,

but in the living egg most of the fats are present either as

lipoproteins in the yolk or as fat droplets. This lipoprotein P78

is called lipovitellin, and reported fat contents are from

2 16. 22% to 25 16% in rainbow trout eggs and 9% in chum salmon

eggs. Half of the fat is phospholipids, mostly lecithin, and

in addition there are neutral fats, free fatty acids, and

cholesterol. The yolk components containing lipovitellin in

the young oocyte may be formed in tlie multithecal bodies

derived from the mitochondria, but in large oocytes they are

supplied from outside the egg by pinocytosis.

Fats are also present in the egg as fatty droplets.

These are entirely composed of neutral fats, and in rainbow

trout eggs 50% of the fatty acids were C18s 1 , C22:6 and

C16016 . They appear at first as small droplets surrounding

the nucleus, but often, depending on the species, they fuse

together into one or several drops.

•

• 156

2. Changes in body fat content during maturation of the ovary.

It is already known that the quantity of fat in the

body of many fishes shows seasonal changes. For example

Lovern19 reports that the fat in the body, though not in the

viscera, of rainbow trout increases from April to October, and

similar seasonal changes are known to Occur in mackere1 20 ,

sockeye salmon21 , and the Japanese sardine22 . It is often

suggested that these seasonal changes' of fat content are

related to water temperature and diet, but a response to the

reproductive cycle has also been suggested. Wada 22 found that

both the male and female gonads of the Japanese sardine enter

the phase of rapid development froM the end of January into

February, and that the fatty tissues decrease and the

proportion of fat in the muscle and the liver also become

lower (Table 8.2). .Suyama 6 found that the amounts of

phospholipids and aminolipids in the ovary of the rainbow

trout increase during maturation, but that at the same time

the amounts in the muscles decrease (Table 8.3). Thus the

changes of the fat content of the body are evidently connected

to the reproductive cycle, and it may be supposed that

oogenesis is accompanied by a "migration" of fat to the ovary

from all parts of the body.

P79

Table 8.2.

Seasonal variations of the fats in the body .organs

of female sardine ( 'o) .

Date caught Liver Fatty tissue Muscle

157

16 Mar 4.84

9 April 8.89

9 May 21.95 71.49

9 Dec 16.87 74.64

10 Jan 11.78 79.14

8 Feb 3.57 73.52

13 Mar 1.35

4.60

4.90

14.44

11.25

12.04

11.78

1.11

Table 8.3.

Changes of fat in the ovary of rainbow trout

during maturation (%) 22 .

Month Gonad Mean egg Extractable Phosphorus Nitrogen when index weight by ether. content of content of caught (%) mg. * (%) ** fat. ' fat.

(%) *** (%) ***

8 1.47 1.3 10.1 0.131 0.716 0.93 0.697 0.91

9 2.45 2.6 10.1 0.263 0.878 2.28 0.659 1.71

10 5.69 . 6.5 8.8 0.572. 1.220 7.39 0.747 4.86

11 9.06 8.2 7.1 0.582 1.300 10.66 0.794 6.51

12 17.2 16.6 6.4 1.04 1.580 26.3 0.930 15.44

* Assuming the average number of eggs per fish to be 2500. ** Converted to mg per egg. *** Converted to /ug per egg.

•

158

The quantity, the pathways and the conditions of this

fat migration contain many points of physiological interest,

23 but they are little known. Takashima et al made quantitative

measurements on 2-year old female trout obtained in December.

When they had been starved for the same period (85 days) the

amount of fat surrounding the digestive tract in immature fish

was 28.8%, but in mature fish it was 2.3%, and there was little

effect on the development of the ovaries. This showed that

the amount of depot fat diverted to the ovaries during

oogenesis is greater than the amount consumed for energy in

movement. Body fat in the female sockeye salmon also decreases

during migration from the river mouth to the spawning place,

but the 8% which disappears was found in an approximate

calculation to have migrated to the ovary24

Idler and Tsuyuki25 found that there was little

development of the ovary of the female sockeye salmon during

the spawning migration before the river mouth was reached, but

that there was an increase in the weight of the liver.

Nomura26 found that the beginning of the spawning season is

the period of greatest hypertrophy of the ovary, and that at

that time the weight of the liver is also high. The same

phenomena were found in land-locked sockeye salmon23 .

Takashima et al23 found hypertrophic livers of high fat

content in 2-year old rainbow trout caught in December.

During the spawning season the amount of fat in the liver of

•

•

159 ••

the Japanese sardine decreased22 , but in the directly

preceding period the liver was hypertrophied and had a high

fat content. Thus hypertrophy of the liver and an increase

of its fat content are found during the period of active

oogenesis. This shows that the liver is an active part of

the pathway through which fats migrate during oogenesis.

In order to investigate the way in which the depot

fat migrates, Takashima et al 28 measured by means of thin

layer chromatography the fat constituents around the digestive

tract, in the liver, and in the blood plasma of rainbow trout

in various stages of maturation. It was found that as

maturation proceeded the neutral fats and the free fatty acids

in the body fat around the digestive tract decreased. In

contrast, they were found to increase in the liver and in the

blood plasma (Figure 8.1). It is known that in birds and

amphibians 29-31 , the depot fats are transported in the blood

fluids as neutral fats and as free- fatty acids, and are

synthesized to lipovitellin on reaching the liver.

3. Endocrinological control of changes in fat metabolism

during maturation of the ovary.

Maturation of the ovary is principally controlled by

the diencephalon and the pituitary, and their internal

secretions are believed to take part in the changes of

metabolism during maturation. One of the ways in which this

16 0 ••

Figure 8.1.

Differences due to the state of ovary maturation of the

composition of the fats in the blood plasma, around the digestive

tract, and in the liver of two-year old female rainbow trout. In

mature fish there is little depot fat around the digestive tract,

but much in the blood plasma and the liver. There is very little

fat or free fatty acid in the depot fat, but much in the liver

and the blood plasma.

Cholesterol esters Neutra.1 fats Frp.e fa-tty acids: Free p.holesterol Non7-Dol ar fats

161

fat metabolism may be affected is through the action of the

sex hormones. It has been shown that the gonads of teleosts

secrete sex hormones under the influence of gonadotropic

hormones from the pituitary. Shreck32 found a concentration

of about 1.4 to 3.8 mg/mi of estrogen in the blood plasma of

rainbow trout, and Cedar et al 33 found that the blood plasma

of rire Atlantic salmon contained a high level of estrogen

which was reduced after the eggs had been discharged.

Eleftheriou et al 34 found maturation of the ovary of the

catfish to be accompanied by an increase of free estrogen in

the blood. The increase of estrogen during maturation is

thought to influence mctny other metabolic processes, such as

the metabolism of proteins, nucleic acid, and calcium, but fat

metabolism is also affected. Ho and Vanstone 3. 5 injected

estradiol monobenzoate into sockeye salmon, and reported an

increase of the total fats, the neutral fats and the total

cholesterol in the blood serum. Takashima et al 28 made four

injections, on alternate days, of 500t,g per fish of the

synthetic female hormone diethylstilbestrol into the abdominal

cavity of rainbow trout, and found an increase of the fats,

particularly the neutral fats and the free fatty acids, in

the blood plasma (Table 8., Figure 8.2). At the same time

there was an increase of lipoproteins in the blood plasma,

and hypertrophy accompanied by an increase of fat content of

the liver. These phenomena closely resemble the pattern of

p81

p82

• .

DES MT CS ACTE TP

• 162 Figure 8.2.

The influence of diethylstilbestrol (DES), methyltestosterone

adreno-cortico-tropic hormone (ACTH) and thyroid extract (TP)

on the fat in the blood plasma of two-year old female rainbow trout.

The blood plasma fats were increased by DES and reduced by TP. DES

also showed a tendency to increase the neutral fats and the free

fatty acids. CA and CS are controls.

Cholesterol . esters Neutral fats Free fatty aciO.s . Free ,cholesterol.; Mono - and di -glycer5.des Non-polar fats

Table 8.4.

The influence of diethylstilbesterol on the fat in the

plasma and the liver of the rainbow trout28 .

Amount injected (g) 0 5 500

Body weiuht (e) hepatosomatic index (%) Plasma fat (e/100m1) Liver fat (e0)

353 322 330

1.2 1.4 1.8

2.8 2.5 4.0

3.7 4.6 4.9 •

• 163

•

•

changes of fats already described in connection with

maturation. Thus the sex hormones take part in the

displacement of fats into the blood from the places in which

they accumulate. The sex hormones take part in the same way

as in birds36 and amphibians 37 in the displacement into the

blood of the accumulated fats, and it is to be supposed that

they are synthesized to lipoproteins in the liver. It has

been shown that thyroid hormones and adrenocortical hormones

take part in fat metabolism30 , and it is possible that they

may in this manner take part in the process of maturacion.

4. Fats in the diet of the parent fish.

As has already been discussed, the fats deposited in

the egg are derived from fats deposited in various parts of

the body, and these fat deposits are principally derived from

fats which have been taken by mouth. Therefore, if oogenesis

is to proceed satisfactorily, the parent fish must be supplied

with, and must accumulate, fats which are both qualitatively

and quantitatively sufficient. Ishida et al 38 explain the

so-called "shiniko" (literally "dead offspring") which is

found in herring roe as being composed of unripe eggs for

which the parent fish had insufficient nutrition before the

spawning season. In this example it is not only fats but

other nutritional elements whose insufficiently obstructed

the development of the reproductive elements. In salmonids,

• 164

the food intake is reduced during.00genesis, but it is

probably important that the conditions in which the parent

fish are reared before this period should provide for the

accumulation of sufficient fats in the body of the mother.

The eggs of rainbow trout contain many highly unsaturated

fatty acids, and it is supposed that they can be selectively

incorporated during oogenesis2 , but the diet given to parent

fish should be monitored with this in mind.

Lovern39 ' 4° has already shown that the fatty acid

components in the fats in the diet of fish have a considerable

influence on the fats in the body. Lasker et al40 and Ando 2

have demonstrated that the fats in the diet influence not only

the body fat but also the fat in the eggs. This is therefore

to be remembered when deciding the diet for the parent fish.

Furthermore, deficiency of essential fatty acids41 or of

choline42 leads to fatty degeneration of the liver, and the

administration of oxidized fats causes hepatoma43 , so that it

is known that nutritional deficiency can lead to abnormal fat

metabolism. However the investigations on this have all been

made with young fish, and there has been no study of parent

fish. It is therefore important that there should be further

studies of this matter in order to ensure satisfactory

development of the gonads.

•

•

165 ••

The eggs of salmonids exhibit a yellow-orange coloured

zone which is principally due to the presence of a carotenoid 4 pigment. Glover et al 1 observed that astaxanthin migrates

from the egg to the body of the embryo during the development •

of the salmon, and Deufel44 has.reported that an admixture of

canthaxanthin into the diet of rainbow trout improved the

quantity of eggs collected from the group and also the

proportion fertilized. This shows that the carotenoid

pigments take part in the formation of healthy embryos, and

suggests the possibility of using them as indicators of "egg

quality". There are other suggestions of the connection

between "egg quality" and pigments, but egg collection,

fertilization, hatching and fecundity ail vary according to

differences in the management and control of rearing. Very

careful and skilled investigation Of the value of pigments

as quality indicators is required. -

Grzenda et al45 reared goldfish on a diet containing

6.1 nmoles of 140 dieldrin, and found that after 32 days

47 picomoles/g had accumulated in the ovary, but that after

128 days there were 7209 picomoles/g, an increase of 150 times .

During this period increasing amounts accumulated in other

internal organs, but the rate of increase in the fatty tissue

and in the ovary was extremely high. Mayer and Sanders46

* Sic, but Grzenda gives 1836 picomoles in the ovary, and an increase from 649 to 7209 in the testes.

Translator. •

Guppy.

Fry per female 33 29

166

exposed Zebra danios (Brachydanio rerio) and guppies

(Poecilia reticulatus) for 90 days to diets containing 50

or 10g per gram of food, and found that the number of fry

decreased as the concentration increased, and that the

survival rate of the fry was reduced (Table 8.5). This may

be taken as a warning that water soluble or food soluble

substances may be concentrated in the ovary and hinder oogenesis.

Table 8.1.

The influence of di-2 -ethylhexyl Phthalat9 on

the reproduction of zebra danios and guppies.

Species. Dietary concentration Wg) 0 50 100

Zebra danio. Number of spawns 6 8 14 Eggs per spawn 20.3 15.2 10.1

'Percent of fry survival 51.1 31.7 11.5

•

•

16 7 • References.

•

1) M.Sureace and C. dorrio: Changes in

chemical composition during deve-

lopment of rainbow trout eggs.

'B. JAI.. Soc. SCI. FIsK. 23 2' 785 - 788 (1958)

: SEE P. 170

3) K.Y.sxso.tur: Phosphorus metabolism

in fish egg-I. Changes in the contents

of some phosphorus compounds

during early development of Oryzias

laliPes. Sci. Pap. Coll. Gen. Educ.,

Univ. Tokyo, 10, 99-408 (1960).

"4) M.GLovEs, R.A. MouTorr, and D.G..

. Rotors Astaxanthin, cholesterol and

lipins in developing salrnon egg.

Biochem. J., 50, 425-429 (1952).

t • 5) SEE P. $70

6) SEE P. 17$

7) S. Smut In "The physiology of

fishes" (ed. by M.E. Buowri), Vol.',

323-459.â, Academic Press, 1■'. Y.,

(1957).

8)

SEE P. 170

9) R. LAUER Efficiency and rate of

yolk utilization by developing em-

bryos and larvae of the Pacific

sardine, Sardinops caerulea. J. Fish-

Res. Bd. Canada, 19, 867-875(1962).

10)

SEE P. 171 11)

SEE P. 172

•

• 168

12) SEE P. 172

13) SE E P. 172

14) SEE P. 173

15) G.Youtio and L I. PITINNEY : On the

fractionation of the protein of egg

yolk. J. Biol. Chem., 193, 73-80 (19

51).

« 16) D. W. JARED and R. A .WAttAca :

' Comparative chromat,..r .aphy of the

yolk protein of teleosts. Comp.

Biochem. Physiol., 24, 437—.443 (19

68).

N. N. GUPTA and K. YAMAMOTO : Ele-

ctron microscope study on the fine

structural changes in the oocytes of

goldfish, Carassins auratus, during

yolk formation stage. Bull. Fac.

Fish. Hokkaido Univ., 22, 187---205

(1971).

18) $EE P. 173

17)

B. JAP. Soc. Sct. FISH

19 525-529 (1953)

21) D. R. IDLER and I. BITNERS : BiOCheITI•

ical studies on sockeye salmon • during spawning migration-II.

Cholestesol, fat, protein and water

in the flesh of standard fish. Can.

J. Biochem. Physiol., 36, 763-798

(1958).

SEE P. 173

23) F. TAEABHIXA, T. HIBIYA, T.

WATANABE, and T. HARA : Endocri-

nological studies on lipid metabolism

in rainbow trout-I. Differences in

lipid content of plasma, liver and

visceral adipose tissue between

sexually immature and mature

females. B. J. SOC Soi FISH _37 307-31 1 (1 9 7 1 )

24) D. R. InLan and L BITNE -RS: Biochem- i•

cal studies on sockeye salmon during

sp.awning migration-IX. Fat, protein

and water in the Major internal

organs and cholesterol in the liver

. and gonads of the standard fish. J.

Fish. Res. Bd. Canada. 17, 113-122

(1960).

25) D. R. IDLER and H. TSUYUKI : Bio-

chemical studies on sockeye salmon

during spawning migration-I. Physi-

- cal measurements, plasma chole-

- sterol, and electrolyte levels. Can. J. Biochem. Physiol., 36, 783-791

(1958).

Su P. 174

27) SEE P. 174

22)

19) 5. A. Lovaax Fat metabolism in

fishes-XII. Seasonal changes in the

composition of herring fat. Biochem. 26)

J.. 32, 676.-680 (1938).

20) E. NOGUCHI and M. BIT() : On the

seasonal variation of the liver weight

and oil content of the mackerel.

1 69

•

•

28) F. TAIIASIIINA, T. HIBITA, PI-tax-VAN Noss, and K. AIDA: Endocrinological

studies on lipid metabolism in

—rainbow trout-II. Effects of sex teroids, thyroid powder and adreno-

corticotropin on plasma lipid

•content. B. Jr. SOC. SC!. FISH. ' 38 43 - 49 (1 972 )

29) H. A. WALKER, M. W. Teroz,n, and

W. C. Rum.: The level and inter-

' relationship of laying hen. Pou!!.

• Sci., 30, 525530 (1951).

80) W.M. Mc Io: A lipophosphoprotein

• complex in hen plasma associated

• with yolk production. Biochem. J.,

72, 153-459 (1963).

31) P. J. HEALD and K. K. BOOKLEDGE :

Effect of gonadal hormones, gonado-

trophins and thyroxin on plasma free 38)

fatty acids in the dotnestic fowl.

J.Endocrinol., 30, 115--130(1964).

C. B. SCHRECK : Plasma oestrogen

leve,ls in rainbow trout. J. Fish.

Die!., 6, 227.--230 (1973). L. CEDARD, M. FONTAINE, and T.

Nouai: Sur la teneau en oestro-

genes du sang du saumon adulte

(Salmo solar L.), en eau douce. C. r.

•• •Seanc. Acd. Sc j., Paris, 252, 2656--

2657 (1961). 34) B. E. &minimal«, K. W. BOEIILICK,

and O. W. TIEMEIR : Free plasma

\ estrogens in the channel catfish.

• Proc. Soc. Exp. Biol. Med.. 121, 85

-48 (1966). 35) F.C. Ho and W.E. VANSTONE : Effects

of estradiol monobenzoate on some serum constituents of migratory

sockeye salmon (Oncorhynchus

nerka). J. Fish. Res. Bd. Canada,

32)

33)

39) J. A. Lovaaa : Fat metabolism in • fishes-XIII. Factors influencing the

composition of the depot fat of fishes. Biochem. J., 32, 1214-4224 (1938).

40) R. LASKER and G. H. THEILACKER :

The fatty acid composition of the lipids of some Pacific sardine tissues

in relation to ovarian maturation and diet. J. Lipid Res.,- 3, 60-44 (1962).

41) T. WsrANABE, F. TAKASIIIMA, and C.

Oottio : Effect of dietary methyl-

linolate on growth of rainbow trout.

B.JAP. Soc , SCI. FISH 40 181 - 188 (1974)

42) C. °onto, N. UKI, Z. IIDA, T. WATA-

NABE, and K. ANDO : 13 vitamin re-quirements of carp-IV. Requirement for cholin.

6. JAP. Soc. Sci. F :sn 36, 1140 -1146 (1970)

18, 859-.864 (1961). 36) D. A. Sciusins, M. WILKENS, R. G.

Mo LANDLESS, R. MUNN, M. PETERSON,

and E. CARSON : Liver synthesis, plasma transport and structural alterations accompanying passage of yolk protein. Am. Zoologist, 3, 167 ---184 (1963).

37) R. A. WALLACE and D. W. JARED t

Studies on amphibian yolk-V11. The estrogen-induced hepatic synthesis of a serum lipophosphoprotein and its selective uptake by the ovary and

transformation into yolk platelet protein in Xenopus laevis. Develop-

mental Biology, 19, 498-526 (1969).

SEE P. 175

43) D. G. ScAttraw : Ultrastructure and biochemical observations on trout bepatorna. U. S. DePt. Interior,

Bureau of Sport Fish. Wildlife, Res.'

70, 56-71 (1967). 44) J. Daum,: Pigmentierungsversuche

mit Canthaxanthin bel Regenbogen-forellen. Archiv. far Fish., 16. 125-- 132 (1965).

45) A. R. GRZENDA, W. J. TAYLOR, and

D. F. PARIS: The uptake and distri-bution of chlorinated residues by goldfish fed a "C•dieldrin contami-nated diet. Trans. Amer. Fish. Soc., No. 2, 215-223 (1971)•

46) F. L. MAYER JR. and H. O. SANDERS:

Toxicology of phthalic acid esters in aquatic organisms. Environm.

Health Perspectives, 4, 153-457 (19 -72).

170

2. ANDO Kazuo.

Yoshoku gyorui no shishitsu

gakuteki kenkyu.

J. Tokyo Univ. Fish.,

ni kansuru seika

ILI 61 - 98 (1968).

K. Ando.

Study of the fats in cultu'red fish.

Journal of the Tokyo University of Fisheries.

.5./1 61 - 98 (1968).

5. TAKAMA Kozo, ZAMA Koichi, IGARASHI Hisataka.

Sake tamago hassei katei chu ni okeru shishitsu

seibun no henka.

Hoku dai sui san iho 20 118 - 126 (1969).

K. Takama, K. Zama, H. Igarashi.

Changes in the composition of fats during the

development of the eggs

Fisheries Bulletin of the

20 118 - 126 (1969).

of the chum salmon.

University of Hokkaido.

17 1

6. SUYAMA Michizo.

Nijimasu tamago seijuku chu no ippan seibun no henka.

Nichi sui shi 24 656 - 659 (1958).

M. Suyama.

Changes of the normal composition of rainbow trout

fat during egg maturation.

Bulletin of the Japanese Society of Scientific

Fisheries 24 656 - 659 (1958).

8. TSUCHIYA Yasuhiko.

Suisan kagaku (kateibun). 243 peiji.

Koseisha Koseikaku, Tokyo 1965.

Y. Tsuchiya.

Fisheries Chemistry. (Revised edition) p. 243.

Published by Koseisba KoseikakU, Tokyo 1965.

10. FUJIMAKI Masanari.

Shin eiyogaku. 746 peiji.

Asakurà Shoten, Tokyo 1968.

M. Fujimaki.

New dietetics. p. 746.

Published by Asakura Shoten, Tokyo 1968.

•

17 2

11. ZAMA Koichi, KATADA Muneo, IGARASHI Hisataka.

Sake tamago no shishitsu - II. Fukugo shishitsu ni tsuite.

• Nichi sui shi 24 739 - 742 (1959).

K. Zama, M. Katada, H. Iuarashi.

Fats in the eggs of chum salmon II. On complex fats.

'Bulletin of the Japanese Society of Scientific Fisheries,

24 739 - 742 (1959).

12 , ANDO Kazuo.

Nijimasu tamago hassei chu no shishitsu no henka.

• Nichi sui shi 28 340 - 343 • (1962).

K. Ando.

Changes in the fat of rainbow trout during egg maturation.

Bulletin of the Society of Scientific Fisheries.

28 340 - 343 (1962).

13. ZAMA Koichi, KATADA Muneo, IGARASHI Hisataka.

Sake tamago no shishitsu - I. Yushi no seibun ni tsuite.

Nichi sui shi 24 569 - 572 (1958).

K. Zama, M. Katada, H. Igarashi.

Fats in the eggs of chum salmon - I.

On the components of the oils and fats.

Bulletin of the Japanese Society of Scientific

• Fisheries 24 569 - 572 (1958).

• 173 ••

14. IGARASHI Hisataka, ZA1U Koichi, KATADA Muneo.

Koi tamago no shishitsu - I. Yushi no seibun ni tsuite.

Nichi sui shi 26 326 - 329 (1960).

H. Igarashi, K. Zama, M. Katada.

Fats in the eggs of carp - I.

On the components of the oils and fats.

Bulletin of the Japanese Society of Scientific

Fisheries 26 326 - 329 (1960).

18. NAKAGAWA Heisuke, TSUCHIYA Yasuhiko.

Nijimasu tamago tanpaku no kenkyu - VI.

Hassei katei chu ni okeru shishitsu san sosei no henka.

Showa 44 nendo nihon suisan gakkai shunki daikai koen No. 466.

H. Nakagawa, Y. Tsuchiya.

Study of proteins in the eggs of rainbow trout - VI.

Changes in the fatty acid composition during development.

Paper No. 466, Spring meeting 1969 of the Japanese

Society of Scientific Fisheries.

22. WADA Shota.

Iwashi tai ni okeru shishitsu no seika gakuteki kenkyu.

No ka kai shi 22 339 - 475 (1953).

S. Wada.

Biochemical studies of body fats of the sardine

Sardincms melanosticta.

Journal of Agricultural Chemistry 22 339 - 475 (1953).

174 ••

26. NOMURA Minoru.

Nijimasu no jinko sairan ni kansuru kiso kDnkyu - V.

Seishokuso no hattatsu to shozangyo no okisa.

Nichi sui shi 22 976 - 984 (1963).

M. Nomura.

Basic studies of artificial egg collection from

rainbow trout - V. Development of the reproductive

organs and size of newborn fish.

Bulletin of the Japanese Society of Scientific Fisheries

z2 976 - 984 (1963).

27. IMURA Takatsugu, SAITO Tsuneyuki.

Gyorui soshiki kosei seibun no taisha kassei no

jiki teki henka I.

Nijimasu soshiki chu no kakusan ryo no henka.

Hoku dal suisan iho 20 202 - 210 (1969).

T. Imura, T. Saito.

Changes of the composition of fish tissues during

period of active metabolism - I.

Changes in the nucleic acid content of rainbow trout tissues.

Fisheries Bulletin, Hokkaido University,

20 202 - 210 (1969).

17 5

38. ISHIDA Rikikazu, SASAKI Takeo, ARITA Setsuko.

Nijimasu no ransoran ni kansuru so hiki gakuteki

kenkyu.

Iwayuru "shiniko" ni tsuite.

Hoku dai ken ken ho 24 171 - 176 (1962).

R. Ishida, T. Sasaki, S. Arita.

Histological studies of ovarian eggs in rainbow trout.

The so-called "dead egg".

Bulletin of the Hokkaidc, Regional Fisheries Research

Laboratory 24 171 - 176 (1962).

•

• 176

9, The accumulation of yolk protein.

KatsumLAIDA.

(Department of Agriculture, Tokyo University).

The ovaries of fish contain large numbers of oocytes, and

when yolk accumulates in the eggs as part of maturation, the

ovaries become greatly hypertrophie. Consequently the gonadosomal

index reaches 20% to 30% in many fish, and in the eel it may even

reach 60% to 75%. The composition of the mature ovary depends on

the species, but the values generally found are 55% to 75% water,

20% to 33% protein, 1% to 25% fat, and 0.7% to 2.2% ash1 . Thus

protein is the largest component other than water, and it

evidently has an important role in the process of morphogenesis

after fertilization. The protein can be divided into chorionic

protein and internal protein. In the mature egg the protein in

the yolk accounts for most of the internal protein of the egg,

and morphologically it is pricipally present as yolk globules and

yolk vesicles 2 . The protein of the fish egg is not a chemically

simple protein. Its main component is a lipoprotein corresponding

to the lipovitellin of the bird's egg, and it is found that there

are also phosphoproteins of the phosvitin type, and also livetin

proteins 3 8

•

110 1 77

There are three possible modes of formation and

accumulation of yolk proteins in the yolk.

Substances of low molecular weight such as amino

.acids and simple sugars are synthesized in the

oocyte itself.

2. Yolk proteins are synthesized in the follicle

cells and accumulated.

3. Substances synthesized in organs other than the

ovary are taken up and accumulated.

: Nakano et al have reported the occurrence of the

11, first Mechanism, the formation of protein in the Oocyte of

medaka Oryzias latipes during maturation 9 . However they did

not determine the type of protein which was produced. Almost

nothing is known as yet about the possibility of the second p89

type of production, the synthesis of protein in the follicle

cells. However, in the course of investigations of the

pl-ysiological changes in the body of the fish which accompany

maturation, Aida et al found results which lead to the third

possible process of yolk accumulation. These'results are

discussed below.

1.

•

• 178

•

1. The appearance and endocrine control of female

specific proteins during maturation.

It is already known that there are sexual differences

in the composition of the proteins in the blood plasma during

maturation of the gonads10-12 . Aida et al, used Ayu

(Plecoglossus altivelis) as the principal subject in a study

of the closeness of the relationship of these differences to

maturation of the ovary, and in particular, to the

accumalation of yolk.

Ayu from Lake . Biwa were reared under the natural

photoperiod. Yolk began to accumulate in early August and

the ovary began to hypertrophy rapidly. Ey late September

the gonadosomal index became 20% and spawning occurred from

late September to November. During spawning the gonadosomal

index was about 30%. The changes in the plasma proteins

during the process of maturation of the gonads were

investigated by means of starch gel electrophoresis, and

fractions 9 and 11 were found to increase rapidly after

vitellogenesis began in early August. After reaching a

maximum in early September they gradually decreased until

the time of death" (Figure 9.1, A). However no such evident

chancres were found in the males (Figure 9.1, B).

Next in order to investigate in detail the relation

between increases of these fractions in the females and the

state of maturation of the eggs, ayu which had been reared in

F. 9 F.11 0

•••- y • '

A , 'ez.e - • .%(

• • .1

7 .

179

1,

.. .

c

••••.; 071.,••• •

D ' , :.• e

•

• Figure 9.1. •

Starch gel electrophoresis (A - E) and agal" immunoelectrophoresis.

(F - J) of ayu plasma protein..

A, G. : Maturing female ayu.

B, F. : Maturing male ayu.

C, H. : Immature male ayu treated with 2 /mg of estradiol 17p .

D, I. : Immature male ayu treated with 20,ktg of estradiol 17C .

E, J. : Immature male ayu treated with 200/m.e of estradiol 17p ,

a : Maturing female ayu plasma and antibody.

Antibody of "a" absorbed by maturing male ayu plasma.

Origin,

180 ••

a suppressive photoperiod for maturation (16 hours of light

and eight hours of darkness, 16L-8D) were transferred to an

accelerative condition (8L-16D) and plasma samples were taken

on alternate days. The ninth fraction was observed to increase

on the 11th day, and at the same time egas appeared in the

ovary in the first stage of vitellogenesis with small

peripheral yolk droplets. The yolk droplets afterwards

increased with time. This result showed that the increase of

the fraction coincided in time with the appearance of yolk

globules around the eggs 13 .

It was next conformed by agar immunoelectrophoresis

that two types of protein (Fm-a, Fm-b) newly aueared in the

plasma of females which had begun to mature after early

August (Figure 9.1, F, G). The new protein Fm-a, present in

large amounts, was composed of the two fractions 9 and 11

found by starch gel electrophoresis. Aida et al named both

Fm-a (fractions 9 and 11) and Fm-b the "Female specific plasma

proteins" (FSPP). Our laboratory has since shown that the

same proteins appear in mature carp (Cyprinus carpioj, in

goldfish (Carassius suratus), in willow shiner (Gnathopogon

caerulescens), in makogarei (Limanda yokohamae), and in

konoshiro (Clupanodon punctatus).

The next question deals with the factors which

participate in the synthesis of FSPP. Urist and Schjeide14

reported an increase of a distinctive component of the blood

• 181

•

serum following injection of 10mg of estrone into the muscle

of the bass Paralabrax clathratus. Aida et al made

intraperitoneal injections of estradio1-17IS (0.2, 2, 20, 200?-g),

testeronè (10 /«g, lmg), hydrocortisone acetate (100tAg, 10mg)

and progesterone (10 .pg, lmg) into ayu in which maturation had

been suppressed. FSSP was found to develop only in the groups

in which 2g or more of estradiol 17/Shad been injected, and

the amount was shown to depend on the quantity of estrogen used 13 .

Figure 9.1, C, C - E, H - J). Estrogen also caused FSSP to

appear in immature female as well as matured ayu, and in

goldfish from which the seminal glands had been excised.

This suggests that estrogen stimulates the synthesis of FSPP

in some other organ than the gonads, and that it is discharged

from this organ into the blood.

2. The connection between FSPP and yolk protein.

Since it has been shown that the appearance of FSPP is

closely related to vitellogenesis, ovary extract from matured

ayu was used in antibody reaction with mature female blood

plasma. The precipitin bands, (Eg-a, b, c), were formed13

(Figure 9.2, A). Among these, Eg-a was of the same antigen

type as Fm-a (Figure 9.2, B, C) and, like Fm-a, was

fractionated by starch gel electrophoresis into the two

components found in the ovary extract. Fm-a and Eg-a combine

•

• 182

Figure 9.2

The relation between FSSP and yolk protein.

A, B Ovary extract from ayu. C. : Maturing female ayu plasma. D, E, F : Goldfish ovary extract.

a. Antibody and maturing female ayu plasma. 1 Antibody of "a" absorbed by maturing male ayu plasma.

Antibody and maturing female goldfish plasma.

d : Antibody and maturing male goldfish plasma.

Antibody of "a" absorbed by maturing male

goldfish plasma.

•

• 183

•

with fats, polysaccharides and calcium and the compounds were

found to have the same surface behaviour. Eg-b and Eg-c were

shown to be plasma proteins which are normally present in ayu

eggs. Yolk protein antigens to FSPP are also present in large

amounts in ovary extract from goldfish, and other serum

proteins are also normally present (Figure 9.2, D, E, F).

Krauel and Ridgway 15 showed by means of immunoelectrophoresis

the presence of a substance of an antigen type in the female

blood serum of red salmon in which the ovary weighed more than

10g, and reported the presence of a large quantity of the same

substance in the eggs. From these results it appears highly

probable that FSPP is synthesized under the stimulation of

estrogen, and is taken up by the egg from the blood in a

macromolecular condition as a precursor of the yolk protein.

3. The site of synthesis of FSPP.

The next question is the site in the body at which

FSPP is synthesized. Since it is known that this synthesis

occurs in the liver of other oviparous vertebrates, it is

supposed that FSPP will be synthesized in the liver of fish.

It is in fact known that the liver of the female is enlarged

during maturation, relative to that of the male, in the deep

water herring, (gisu) Pterothrissus gissu, the frog flounder

(meitagarei) Pleuroichthys cornutus, the half-beak (sayori)

•

• 184

•

Hemiramphus sajori 16 , the Pacific mackerel (saba) Scomber

japonicus 17 , the loach (dojo) Misgurnus anguillicaudatus 18 ,

the medaka Oryzias latipes 19 , and the three-spined stickleback

(togeuo) Gasterosteus aculeatus 20 . Aida et al made detailed

observations in ayu and in makogarei Limanda yokohamae. There

were sexual differences in the hepatosomatic index of ayu at

the time when the gonads begin to ripen, but after maturation

that of the female was 2 to 3 times that of the male.

Histological examination of the changes of the liver showed

that as tbe ovary commenced to mature the female liver cells,

the nuclei, and the nucleoli began to swell, the cytoplasm

became strongly basoellic because of an increase of RNA, and

fats and glycogens tended to diminish. These phenomena

continued until spawning, and sugget that the cells were

actively synthesizing protein21 . Maturation of males was

accompanied by atrViyof the liver and a lowering of its

activity. The hepatosomatic index of mature female Limanda

yokohamaemas three or more times that of the male, and the

cytoplasm was extremely basophilic. The water content was

high in females, but the fat content was found to be high

in males.

The cytological changes in the liver cells of female

which may accompany maturation are known to be reproduced

when estrogen is administered to males or to ovariotomized

females 18-20 . The administration of estradiol 173to immature

• 1 85

•

or mature male ayu induces changes similar to those in

maturing female ayu. The electron microscope shows that the

use of estrogen results in a considerable growth of the rough

surfaced, endoplasmic reticulum (rER) and of the Golgi

apparatus, and it is supposed that estrogen enhances the

ability of the male liver to synthesize protein.

The changes of the nucleic acid content of the liver

were investigated in order to corroborate the histological

results (Figure 9.3, A). The RNA density of the female liver

began to increase in the early part of August, reached a

maximum at the end of August and maintained until the spawning

season a value 1.5 to 2.5 times that normal to the male liver.

However there was practically no sexual difference in the DNA

density or in the protein content. The total DNA and the

total RNA gradually declined in the male, but in the female

they began to increase in early August and reached mardmum

values in mid September, when the RNA was about six times that

of the male, and the DNA about 3 times. They then,gradually

declined, but remained much greater than the normal male

amounts 23 . As shown in Figure 9.3, B, the relation between the

increase in weight of the ovary (at 10 day intervals) and the

integrated sexual difference in the RNA content of the liver

is linear from early August to mid September. After this time,

the relation breaks down, so it appears that some other

mechanism takes part in the increase of weight of the ovary as

-7-70 7/22 8/i

(Ix 100) •

A

Reln.tive quantity of nucleic acid inm the liver.

300

200

100

, FOU(derisity) _

• DNA(density)

11 30 31 21 10/10 9/10 • 20

• 186

Figure 9.3, A.

Changes in the nucleic acid content of the liver,

accompanying sexual maturation.

(The ratio of the weight of the female liver to

that of the male, which is taken as 100%).

•

• Increase in weight of the ovary.

(g).

• Figure 9.3, B.

The relation between the increase in weight of the ayu

ovary (in 10 days) and the total sexual difference in

the RNA content àf the liver..

1 87

1. 22 July to 1 Aug.

2. 1 Aug to 11 Aug.

3. 11 Aug to 21 Aug.

4 • 21 Aug to 31 Aug.

5. 31 Aug to 10 Sep.

6. 10 Sep to 20 Sep.

7. 20 Sep to 30 Sep.

8. 30 Sep to 10 Oct.

Total sexual difference in the

RNA content of the liver.

188 •

•

•

it becomes ripe. Creelman and Tomlinson have found that

female sockeye salmon at the time of going upstream have

larer amounts and densities of RNA-P in the liver than have

the males 23 , and this is in complete agreement with the

results in ayu.

Four types of steroid hormones were used for these

measurements, but, as in the investigation of FSPP, the

changes of the nucleic acid in ayu occurred only in those to

which 2p.g or more of estradiol had been administered, and

the amount of change depended on the quantity of estr-ugen

used (Figure 9.3, C).

Furthermore, when mature female ayu are reared for

one month in conditions which suppress maturation (161, - 8D),

not only does the development of the ovary cease, •but FSPP

disappears, the liver atrophies, its RNA density, RNA quantity,

and DNA quantity greatly decline, and the gonadotrophic

PAS-positive substances diminish.

Ovariotomized immature females were reared for one

month in conditions which accelerate maturation. A ritrong

development of gonadotroph was observed in both the ovariotomized

and the sham operated groups, but FSPP was not observed in the p94

ovariotomized group. The hepatosomatic index, the RNA density,

the quantity of RNA and the quantity of DNA increased very

greatly in the sham-operated group, but no changes were

observed in the ovariotomized group.

k,nm17n,17.171,,vem.5?!mrym M,".137VF's,.

RNA (toU')

400

Cie)

Relative quantity of nucleic acid in the ! liver.

(Treated group 1 X 00 ) (Control group )i

100

HSI

(total) (total)

RNA (density)

DNA (density)

0.2 2 2'0 200n EsuacIMA7/1

• 189

•

Figure 9.3, C.

The effect of estradiol 17C on the quantity of

nucleic acid in the liver.

(The quantity in the treated group relative to that

in the control group which is taken as 100%).

•

• 190

•

In order to observe the action of estrogen in detail,

300 it,1/4g of estradiol 17(3 was administered to immature ayu, and

the times needed for the appearance of FSPP and of chances in

the composition of the nver were measured. FSPP was found

in the plasma after 48 hours, and the increase of FSPP was

accompanied by an increase of the total blood protein to 2.7

times that in the controls. The hepatosomatic index also

began to increase after 48 hours, and reached a value twice

that of the controls. The water content of the liver also

suddenly increased by about 5% after. 24 hours, but after

120 hours it showed no. difference from the controls. The

density of RNA waJ found to increase after 48 hnurs, and remained

high for 240 hours, being 1.6 times that of the controls. The

DNA density showed a tendency to decline after 72 hours, and

this is thought to be connected witli the decline in water P 9

content. The total quantity of RNA increased after 48 hours,

and after 240 hours was 4 times that of the controls. The

increase of total DNA was at least 24 hours later than the

increase of RNA, showing that RNA synthesis precedes DNA

synthesis. All these results are now considered to conform

with the ideas .that FSPP is synthesized in the liver because

of the increase of the quantity of estrogen which accompanies

maturation, and that it is discharged into the blood. Furthermore,

this estrogen produces development through its action on the

metabolism of the water content, the nucleic acids, the sugars,

the fats, and the proteins.

• 191

•

4. The introduction of FSPP into the maturing egre.

The facts so far described suggest that FSPP is taken

up by the egg as a macromolecular substance, and the following

experiments were made in order to test this hypothesis.

Firstly, male gold fish in which FSPP synthesis had

been stimulated by the use of estradiol 17(3 were treated with

100 .eCi of L-leucine 4.5 - H3 , and plasma from blood taken

48 hours later was fractionated in a Sephadex G-200 column.

An extremely high radioactivity was found in the FSPP fraction,

which confirmed the active s-,:mthesis of new protein25 .

This newly synthesized FSPP was administered to

maturing goldfish and its process of migration was investigated.

As shown in Figure 9.4, A, the protein fraction in the plasma

reached a maximum after 10 hours and then rapidly declined in

the females, whereas a relatively high level of radioactivity

was maintained in the males25 . The radioactivity in the ovary

protein fraction gradually increased, as shown in Figure 9.4, B,

and after 120 hours it was about 10 times that in the blood

plasma fraction. However, the radioactivity of the seminal

glands was only one third that of the blood plasma. Moreover,

more than 50% of the quantity administered accumulated in the

ovary, whereas scarcely 2% was present in the seminal glands.

Male and female plasma and ovary extract were fractionated in

Sephadex G-200 96 hours after treatment, and the site of the

radioactivity was measured. In all three, the peak was found

192

Figure 9.4.

410 24 48 .. .72 96 120 Houftl:

1420 24 - 48 • 72 06 Hourts

__ • •

à 0.6 ro csJ

—OA

D 0.2

4 n

2

10 20 30 40 60

Ffte.C1104._

60 70 80

•

• A, B. Changes with time after the administration of

labelled FSPP of the plasma protein (A) and of

the gonad protein (B).

o Male, o Female, Dpm, Disintegrations per minute.

C. The location, in Sephadex G - 200, of the radioactivity

in the ovary extract 96 hours after administration.

Cpm, Counts per minute.

•

• 193

to be in the same place as FSPP. In particular, no radio-

activity was found to have been transferred to the other

protein fractions or to any low molecular weizht fractions in

the ovary extract, and this strongly suggests that the

labelled FSPP was transported to the egg as a macromolecular

substance 25 . In addition, since micropinocytotic vesicles

have been observed during the process of maturation in some

eggs26,27

the egg by micropinocytosis. Aida et al, have shown by means

of the fluorescent antibody method that FSPP is present in

the yolk globules, and that the normal plasma protein

components are contained in the yolk vesic 1es 25, it is concluded from these results that FSPP is

synthesized by the liver when stimulated by estrogen, and

after being discharged into the blood it is taken into the egg

in the macromolecular condition and becomes a structural

component of the yolk globules. (Figure 9.5).

To sum up, there are three possible mechanisms for

the formation and accumulation of yolk protein, but he

author concludes from the results given here that the third

mechanism must now be considered to be the principal mechanism.

However since the other two mechanisms may operate at the

same time, further detailed studies will be required in order

that the full mechanism may be made plain.

it appears reasonable to suppose that FSPP enters

SE CO4DAi; , SYMPTOMS. ;

LIVER. 00,RY.

I ,POLLICLE

CELL LAYER IVOLK

vES ICLE

GLOBULE

• ••

Figure 9.5.

The mechanism of the formation and accumulation of

the yolk rrotein in.fish.

' MrPoTHiLAmus.

194,

PITuiTARY.

•GONADOTROPH IC HORMONE. •

CELLCaÀVAGE Oftt INUCLEATE CELLS ; • •

Estrogen

RNA

I 1 FRESH PROTE IN , k

. SYNTHESIS.

1

ROUGH-SURFACE AM I .NO . RET ICULUM.

DNA

PLASMA

', ROTE IN,

a

r. ACIDS.

4 Boor

TISSUE.

•

References.

•

•

1) SEE P. 197

2) SEE P. 197

3) E. C. YouNo and J. T. Pruxusr: On

the fraction of the proteins of egg

yolk. J. Biol. Chem., 193, 73-80 (19

: 51).

4) T.. E. BARMAN, NOUYEN-KIM BAI

and Nnurstz-Veli THOAI: Studies on

a herring-egg phosphoprotein. Blac-

kens. J., 90, 555-558(1964).

z,) K. ANDO : Ultracentrifugal analysis

of yolk proteins in rainbov trout egg

and their changes during develop-

• ment. Canadian J. Biochem., 43, 373

.--379(1965). •

Y. ITO, T. FUJI', M. NAKAMORI, T.

! HAT-rout and R. YOSHIOKA: Phosvitin

\ of the trout roe. J. Biochem., 60, 726

- —.428(1966).

7) Y: MANO and F. LIPMAN: Characte-

! ristics of phosphoproteins(phosvitin)

from variety of fish roes. J. Biol.

- ChWit., 241, 3822—.3833(1966).

8) D. W. JARED and R. A. WALLACE :

Comparative chromatography of the

yolk proteins of teleosts. ComP•

Biochem. Physiol., 24, 437-443(19

68).

9) E. NAKANO and M. ISHIDA-YAMAMOTO:

Uptake and incorporation of labeled

amino acid in fish oocytes. Acta.

Embryo'. Morphol. ExPeri., 10, 109

"416(1968).

10) W. E. VANSTONE and Cm:run-Wm

Ho: Plasma proteins of coho-salmon.

Oncorhynchus kisutch, as separated

•by zone electro phoresis J. Fish.

Res. Bd. Canada, 18, 393-399 (1961).

11) A. Datum and J. •U. Pius :

Dimorphisme sexual dans les pro

serique de Salmo salar: Etud-éte

electrophoretique. Compt. Rend.

Soc. Biol. , 157, 11, 1897—.1900(1963)

12) R. V. TnousroN: Electrophoretie

pattern of blood serum proteins

from rainbow trout (Salmo gaird

nerii). J. Fish. Res. Bd: Canada;

24, 10, 2169-2188(1967).

13) K. AIDA Pusx-VAN-Nomr and T1

• Phy.sioloïical studies on

gonadal maturation of fishes-I.

' Sexual difference in composition o

•

Bd.

• nucleic acid. J. Fish. Re:.

' Canada, 16, 421-426(1959).

24) SEE P. 199

SEE P. 197

17) SE ( P. 198

• 196

References. continued.

plasma protein of Ayu in relation

to gonadal maturation. Bull. Japan.

Soc. Sci Fish., 39, 1091-1106(1973).

14) M. R. URIST and A. O. SCHJEIDE :

The partition of calcium and protein

In the blood of oviparous vertebra-

tes during estrus. J. Gen. Physiol.

44, 743-456(1961).

15) K. K. KRAUEL and G. J. RIDGWAY :

Immunoectrophoretic studies of red

' salmon serum. Int. Arch. Allergy,

23, 246-253(1963). .

16)

Histological changes in the liver cells of Ayu following gonadal maturation

and *estrogen administration. Bull.

Japan. Soc. Sci. Fish., 39, 1107-- 1115(1973).

22) SU P. 198

23) V. M. CREELKAN and N. TOMLINSON

Biochemical ' studies on sockeye salmon during spawning migration

VI. Ribonucleic acid and deoxyribo-

18) H. KOBAYASHI Effects of eStrogen . upon the structure, weight and fat

• content.

of the liver in the fish,

• Misgurnus anguillicaudatus. Annot.

Zoo!. Japon., 26, 213-416(1953).

19) N. Diem: Effect of estrogen and

androgen on the weight and struc-

ture of the fish, Oryzias latipes.

. ibid., 28, 79-435(1955).

20) C. 00IIRO: Some observations on the • . effect of estrogen upon the liver oi the three spined stickleback, Gasterosteus aculeatus aculeatus

• L., ibid., 29, 19-23(1956). 21) K. AIDA, K. HIROSE, • M. YOROTE

and T. HIBITA: Physiological studies

' on gonadal maturation of .fishes-II:

25) SEE P. 199

26) K. Y,o/AuciTo and I. OOTA :.* An

electron microscope study of the

formation of the *yolk globule in the

• oocytes of zebra-fish, Brachydanio

rerio. Bull. Fat. Fish. Hokkaido

Gniv., 17, 16i-.:174(1967). 7) N. N. GUPTA and K. YAMAMOTO:

Electron microscope study on the fine structural changes in the oocytes

"of goldfish, Carassius auratus,

'during yolk iormation stage. ibid.,

22, 187-205(1971).

• 197

1. TSUCHIYA Yasuhiko.

Suisan kagaku p234 - 251.

Koseisha Koseikaku (1962).

Tsuchiya.

Fisheries chemistry. p234 - 251.

Published by Koseisha Koseikaku, (1962).

2. YAMAMOTO Kiichiri.

Gyorui sein i (sel Shoku). p233 - 271.

Koseisha Koseikaku (1970).

K. Yamamoto.

Fish physiology (reproduction). p233 271.

Published by Koseisha KoSeikaku, (1970).

16. AMEMiYA Ikusaku, TAMURA Tamotsu.

Gyorui kanzo juryo shiyusa ni tsuite.

Sui san gaku kaiho 10 10 - 13 (1948).

I. Amemiya, T. Tamura.

Sexual differences in the weights of the

iivers of fish.

Reports of the Society of Scientific Fisheries,

10, 10 - 13, (1948).

• 1 98

17. NOGUCHI Elzaburo, BITO Masamichi.

Saba kanzo no juryo oyobi shiboryu no

kisetsuteki henka.

'Mehl sui shi 12 525 - 529, (1953).

E. Noeuchi, M. Bito.

On the seasonal variations of the liver weieht

and oil content of the mackerel.

Bulletin of the Japanese Society of Scientific

Fisheries 12 525 - 529, (1953).

22. AIDA Katsumi, HIBIYA Takashi.

Gyorui no seijuku ni kansuru seirigaku teki kenkyu - II.

Shitoku isei kessho tanpaku to rano tanpaku

oyobi kanzo kino to kankei.

Showa 45 nendo nihon suisangakkai nenkai happyo.

K. Aida, T. Hibiya.

Studies of the physiology of maturation in fish - II.

The connections between female specific plasma

protein, the yolk protein, and the liver processes.

Collected papers of the 1970 annual meeting of the

Japanese Society of Scientific Fisheries.

•

1 99

24. AIDA Katsumi, HIBIYA Takashi.

Gyorui no seijuku ni kansuru seirigaku teki kenkyu IV.

Estrogen no kessho tanpaku sosei oyobi kanzo seibun

ni okeru eikyo.

Showa 46 nendo nihon suisan gakkai nenkai happyo.

K. Aido, T. Hibiya.

Studies of the physiology of maturation in fish - IV.

The influence of estrogen on the protein component

of the blood plasma and on the composition of

the liver.

Collected papers of the 1971 annual meeting of the

Japanese Society of Scientific Fisi:eries.

25. AIDA Katsumi, HIBIYA Takashi.

Gyorui no seijuku ni kansuru seirigaku teki kenkyu - V.

Shitoku isei kessho tanpaku no tamago e no torikomi.

Showa 48 nendo nihon suisan gakkai nenkai happyo.

K. Aida, T. Hibiva.

Studies of the physiology of maturation in fish - V.

The take-up of female specific plasma protein into

the egg.

Collected papers of the 1973 annual meeting of the

Japanese Society of Scientific Fisheries.

•

•

•

200

••

V. The evaluation of ep-,g quality in spawn.

10. Freshwater fish.

Kiyoshi SAKAI.

(Tokyo University of Fisheries).

The management of the hatching and rearing of fry, and

the steady artificial production of fingerlings would be

greatly facilitated if it were possible to develop methods by

which the quality of the eggs could be appraised immediately

after collection, and those eggs which were suitable for

artificial fertilization could be selected.

It is at present believed that the quality of the eggs

is closely related to the age of the parent fish, to their

nutrition, ancestry*, conditions of rearing and spawning

season, but studies of the relationships between these factors

and the proportions of the eggs which are fertilized or which

develop have not yet given concordant results1-4

The colour, transparency and elasticity of the eggs

of freshwater fish are used as criteria for the appraisal of

egg quality, but the relations between these criteria and egg

quality are by no means clear 7-9, 14

* For example, it is said that there are differences between marine, lake and artificially reared Ayu-(Plecoglossus altivelis).

201

Experimental work was therefore undertaken with the

objectives of appraising the satifactoriness of the quality

of the spawn, and of defining this quality in terms of

•

•

satisfactory fertilization and hatching of the eggs when

collected. The results of these experiments are discussed

in this paper.

The experiments were made in the following manner.

The fish used as parents were Rainbow trout (Salmo gairdneri),

Ayu (Plecoglossus altivelis), Sogyo (Ctenopharyncodon idellus)

and Hakuren(Hypophthalmichthys molitrix). These were reared

in field ponds at natural temperatures and in laboratory

aquatrons with the temperature controlled in three steps.

Small quantities of eggs were soueezed out at intervals from

fish which had ovulated naturally or . had been caused to ovulate

by hormone treatment, and these eggs were examined by eye and

by microscope. The external examination of the eggs included

the colour, the transparency and the elasticity, and also the

size and distribution of the fatty droplets. An internal

histological examination of the morphological characteristics

wàs also made. At the same time, measurements were made of

the proportion of the eggs collected which were fertilized

and which developed, and these measurements were compared

with the morphological observations. The pH of the eggs and

the refractive index of the yolk obtained by centrifuging

were also measured.

p101

202

1. The Quality of the spawn and its internal and external

morphological characteristics.

Eggs which,remain in the body after ovulation pass

successively through the four following morphological stages

In rainbow trout and ayu, eggs which have just been

ovulated are semitransparent and small fatty droplets are

uniformly distributed throughout the body of the eggs (Stage 1)

(Figure 10.1, -1, 11).

Next, some of the fatty droplets distributed throughout

the egg increase in size and begin to accumulate at a

particular location near the animal pole (Stage II),

(Figure 10.1, -2, 12). Soon after, all the fatty droplets

gather together, and in rainbow trout they form a single

cluster while the remainder of the egg becoMe's transparent or

cloudy white and translucent. In ayu, several large drops

are formed, and the remainder of the egg continues to be

translucent (Stage III), (Figure 10.1, -3, 13).

In rainbow trout, the egg next contracts and loses

its globular shape and its elasticity. In ayu, the fatty

droplets and the protoplasm are collected into one place, and

the rest becomes completely transparent (Stage IV), Figure 10.1

-13). Rainbow trout eggs in Stage III and ayu eggs in Stage IV

are generally called overripe eggs.

* An introduction is presented in this paper to the four stages which have been used as normal divisions of the process of maturation by Nomura et al. in rainbow trout15, 16, by Sakig e al. in Sogyo and Hakuren 1 7 and by Sakai et al. in Ayu1°,19,

• 203

•

From the histological point of view eggs in Stage II

are distinguished from those in Stage I by the increases in

size and the gathering together of the fatty droplets, and by

the loss . of some of the cortical alveoli from the cortical

cytoplasm, but the yolk and the fatty droplets have not yet

penetrated into the cortical cytoplasm (Figure 10.1 - 4, 5, 7,

8, 15, 16). At the time of ovulation, the yolk of the rainbow

trout appears to be homogeneous, but in the ayu it consists of

innumerable small droplets which coalesce and form large drops

during passage through this stage. In the Stage III rainbow

trout egg, the cortical protoplasm which has hitherto surrounded

the periphery of the egg atrophies and contracts, and the yolk

percolates in between the contracted cortical protoplasm and

the chorion. The fatty droplets and the yolk penetrate the

atrophied cortical protoplasm (Figure 10.1-6, 9). In the ayu

egg, the fatty droplets and the yolk globules become large and

penetrate the contracted cortical protoplasm and the chorion

(Figure 10.1 - 17). In Stage IV the content of the rainbow p106

trout egg becomes disordered (Figure 10.1 - 10). In the ayu

egg, the yolk percolates between the atrophied cortical

protoplasm and the chorion (Figure 10.1 - 18).

The eggs of sogyo and hakuren contain no fatty droplets

immediately after ovulation. They are elastic and translucent

(Stage I) and the yolk consists of innumerable small globules

(Figure 10.1 - 19). After ovulation the elasticity is

204

maintained as time passes and the transparency is increased

(Stage II). In this stage the yolk globules coalesce and

increase in size but do not penetrate the cortical protoplasm

(Figure 10.1 - 21). Next, the egg loses its elasticity

(Stage III), the coalesced yolk globules join together in

large clusters, and some of them penetrate the cortical

protoplasm (Figure 10.1 - 21). Next the protoplasm gathers

together in one place and the rest becomes completely

transparent. The egg swells up again and becomes cloudy

(Stage IV). The yolk percolates between the contracted

cortical protoplasm and the chorion in these eggs in the same

way as in overripe eggs of rainbow trout and ayu (Figure 10.1-22).

When the eggs are divided among stages in this way, it

is found that the quality of rainbow trout and ayu eggs is

related to the stage. About 70% to 80% of Stage I eggs develop,

40% to 50% of Stage II, and none at all of Stage III. In sogyo

and hakuren the proportion of live eggs (defined as the

percentage of the total number of eggs which are alive one day

after being exposed to fertilisation) in Stages I and II

shows great variability from 0% to 97 2 with an average of 36%,

but in and after Stage III it is zero.

This shows that the quality of the eggs decreases

gradually after ovulation.

•

urrit.•- ••••

• • ./..•7!--.- r.! • re ,

j*, Jt!.. • , 7 ,

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Figure 10.1.

Part 1. Rainbow trout. p102

1. Stage I. Small fatty droplets distributed through the body of the transparent egg (Live egg).

2. Stage II. Fatty droplets beginning to increase in size and coalesce (Live egg).

3. Stage III. Fatty droplets and protoplasm assemble in one location and the remainder of the egg becomes transparent (Fixed egg).

4. Stage I. There are 1 or 2 rows of small fatty droplets immediately inside the cortical protoplasm, and the yolk is forming apparently ho:aogeneous clusters in the centre of the egg. (Chorion removed).

5. .Stage II. rrhe fatty droplets are increasing in size and beginning to coalesce (Chorion removed).

6. Stage III. The surface protoplasm surrounding the egg has contracted and the yolk has percolated 'between the chorion and the cortical protoplasm.

The vicinity of the animal pole of the egg in Stage I. There is a small thickness of fat in the cortical protoplasm, and many

• cortical alveoli are visible.

8. The vicinity of the animal pole of the egg in Stage II. Some of the cortical alveoli have disappeared, but the fatty droplets and yolk globules have not yet penetrated.

9. The atrophied cortical protoplasm of the egg . in Stage III. The fatty droplets and yolk

globules are seen to be penetrating the contracted cortical protoplasm.

10. The profile becomes oval and the interior contents are disordered.

7.

Figure 10.1..

206

p103 Part 2. Ayu.

11. Stage I. The ege transparency is low, and small fatty droplets are distributed throughout the egg (Live egg).

12. Stage II. The ego: transparency and the size of the fatty droplets have slightly increased, and the fatty droplets are beginning to collect at the animal pole (Live egg).

13. Stage III. The egg transparency is increasing and several large fatty droplets can be seen (Live egg).

14. Stage IV. The protoplasm and the fatty droplets are gathering into one location and the remainder becomes completely transparent.

15. Stage I. The cortical protoplasm rs seen inside the chorion and the adhesive membrane. Small fatty droplets are distributed throughout the egg, and innumerable small yolk globules are present.

16. Stage II. The yolk globules and the'fatty droplets slightly increase in size, and the fatty droplets move toward the vegetative pcle. The yolk and the fatty droplets have not . penetrated the cortical protoplasm.

17. Stage III. The fatty droplets have greatly increased in size, and the cortical protoplasm, being penetrated by the enlarged yolk globules, becomes thin.

18. Stage IV. The cortical protoplasm having atrophied, the yolk percolates between the chorion and the cortical protoplasm.

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Part 4. Sogvo and hakuren ovarian eggs. •

24. Ovarian egg

Stage II.

25. Ovarian egg

Stage III.

26. Ovarian egg

Stage IV.

20 7

11, Figure 10.1. .

p104 Part 3. Sogyo and'hakuren eggs.

19. Stage I. There is a small thickness of fat near the vegetative pole. The yolk is formed of 'numerous small globules, and no fatty droplets are present.

20. Stage The yolk globules are coalescing and increasing in size, but not penetrating the cortical protoplasm.

21. Stage III. The coalescence of the yolk globules has greatly progressed, and the large clusters of yolk globules are penetrating the cortical protoplasm.

22. Stage IV. The cortical protoplasm is atrophying and gathering • into one place, and the yoU.c is penetrating between the.chorion and the cortical protoplasm.

23. Ovarian egg The yolk globules are small, the Stage I. germinal vescicle (nucleus) is situated

in the centre of the egg.

The germinal vesicle is beginning to migrate to one pole of the egg. No change of the yolk globules is seen.

The germinal vesicle has migrated nearly to the periphery of the egg. A few of the yolk globules surrounding the germinal vesicle are beginning to increase in size.

The germinal vesicle has migrated to the periphery of the egg. The protoplasm is collecting around the germinal vesicle, and an increasing number of the yolk globules are increasing in size.

When the egg reaches Stage V it is ovulated (Stage of the spawned egg, Photograph 19). In this case Stage III and Stage IV of the spawned egg become Stage VI and Stage VII (Photographs 21 and 22).

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•

• 208

2. Change of egg quality with time.

The proportion of eggs which live or develop changed

with time after ovulation. The initial rate of development

in rainbow trout reared at 10 ° C to 12 oC was 75%. After 3 to

6 days it rose to 86% and then sank during the course of time

to 0% after 27 to 30 days. The initial rate in ayu at 15° C

to 16° C was 70% but was zero after 4 to 5 days, and in sogyo

and hakuren (at 20 °C to 22 ° C) an initial proportion of live

eggo of 34% became zero after 6 hours. .

Thus the quality of unspawned eggs decreases after

ovulation, and this decrease is more rapid in fish which

spawn at higher temperatures. For this reason the proper

moment for the collection of eggs must be precisely grasped

if eggs of good quality are to be obtained from fish which

spawn at high temperatures.

3. Hormone administration and the process of maturation

of the ovarian egg.

In some species spawning does not occur unless

hormone treatment is used. Since the quality of unspawned

eggs decreases after ovulation it is particularly necessary

with these species to grasp the moment of ovulation after

hormone treatment in order that eggs of good quality may

be procured.

p107

•

•• • 2 09

•

The process of maturation of the ovarian eggs of

sogyo and hakuren has been divided into 5 stages according to

the location of the nucleus in the egg and the size of the

yolk globules (Stages I to V) (Figure 10.1 -23, 2 )4V , 25, 26, 19).

After the hormone had been administered, the state of maturation

was measured by the use of a narrow vinyl tube on the nozzle of

a hypodermic syringe which was periodically inserted into the

genital opening in order to extract some of the ovarian eggs.

The hormone chosen was the sogyo or hakuren pituitary, and it

was administered twice, with an interval of 6 hours, to each

fish. The results are shown in Figure 10.2.

The fish (No. 3) treated at stage I and kept in water

at 20 °C to 22 °C reached the ripe stage V in 21 hours, and the

fish (No. 23) which started between stages II and III reached

stage V in 15 hours. At 25° C to 26°C, the fish (No. 24) which

started in Stage II reached stage V in 12 hours. The fish

(No. 28) which started in stage II but was treated with

pituitary only once.had not reached stage IV after 24 hours,

but' after being again treated with pituitary reached stage V

in about 12 hours.

From these preliminary experimental results, it is

considered that the moment of ovulation after hormone

treatment can to some extent be predicted from the stage of

maturation of the parent fish, the temperature of the water

P108

Degree of ; maturation ..tr (stage). •

n _u. Hours.

42 30. 36 48

210

Figure 10.2.

Stage of maturation and time elapsed since

hormone treatment of sogyo and hakuren.

The arrow shows.the time of hormone treatment.

.For stages I to VII see the present paper

and Figure 10.1 21 to 26.

• 211

•

and the quantity of hormone. It also considered:that a

catheter of the type described could be used for the

extraction of some of the ovarian eggs from large fish in

order to determine the degree of maturation of the ovary at

the time of treatment.

4. Assessment of the quality of . eges - spawned near the

It has been stated10-12 that the quality of eggs spawned

by rainbow trout and by ayu near the upper limit of spawning

temperature is unsatisfactory. An investigation was made to

find out whether the a -opraisal of the quality of eggs spawned

by rainbow trout and ayu reared at three different controlled

water temperatures could be expressed in terms of the

morphological stages described above.

. Rainbow trout were reared for 30 days from 3 November

to the following 21 January at temperatures of 10 ° C, 15°C and

18 00. Eggs in stages from I to II were extracted from all

f,ish in the 10 °C group. At 18 ° C eggs almost in stage III

were obtained after 20 or more days from the start, and the

group at 15° C was intermediate between the other two. The

mean proportion of development of the spawned eggs was 45% at

10 ° C, 1.6% at 1500 , and zero at 18 ° C, so that the high

temperature lowered the quality of the spawn.

•

• 212

Next ayu fry from Lake Biwa were reared to full growth

for 115 days from 28 June to 20 October at 15o C, 20 0 C and 25 o C,

and the relationship between the proportion of development and

the temperature was measured in eggs ovulated naturally or by

means of hormone treatment. In spawn ovulated naturally at

15° C and at 20 0 0 there was not much difference in the frequency

of arrival at a particular stage or in the proportion which

developed, but in eggs which were spawned after hormone

treatment there were more in stage III at 20 ° C than at 15o C,

and the average rate of development at 15° C was 25% whereas

at 20 o C it was 2.5%. At 25o C there was some vitellogenesis,

but there was also much degeneration, and there was neither

natural nor hormone induced spawning.

It is thus found that the classification by stages

which is applicable to eggs incubated but not spawned after

ovulation is also applicable near the upper temperature limit

for spawning, and that these stages can in general be used as

criteria on which to base appraisal of egg quality.

5. Egg quality and egg fractionation by centrifuging.

When eggs were homogenized and fractionated by

centrifuging, (3000 rpm for 30 minutes) the contents of the

eggs are separated into several layers in the centrifuge.

The number of layers and the volume of each layer in proportion

p109

• 213

to the total volume in rainbow trout eggs was found to remain

unchanged for 36 hours after ovulation. However sogyo and

hakuren eggs showed the following fivefold pattern of

change (Figure 10.3).

Type 1. Four layers are formed. In order from the centre

they are

A layer of protoplasm

A fluid layer (transparent)

A yolk layer

A layer containing cortex and chorion.

Type 2. A part of the protoplasm sinks into the transparent

fluid layer.

Type 3. The sinking of the protoplasm is pronounced, and it

is difficult to distinguish.it from the fluid layer.

Type 4. The protoplasm and fluid layers can be distinguished

but the fluid layer is turbid.

Type 5. The protoplasm layer sinks through the fluid layer

to the centrifugal side, and the four layers in

order from the centre are:

The fluid layer (turbid).

The protoplasm layer.

The yolk layer.

The layer containing cortex and chorion.

The fluid layer widens and the protoplasm and yolk

layers shrink.

1 1

A

D

A

D

0

2 Time elapsed since 3 ovulation. (Minutes)* 6

•

:11

22

25

Figure 10.1.

The centrifue.al fractionation patterns Of soeyo and

hakuren . eqes, and the change with elapsed time of the

percentaq- e of parent fish which show each pattern.

type

214

16.7 16.7 8.3 33.3

memme Kmemi ...........« .... N..1 EMM \s 'N

100 . \\\.

100, \\

50.0 50.0

. - 100.

100 \\

\ 11 \ \

-.‘ \ 100

\ • . • 100

■ 100 \

The arrow shows the direction of centrifugal force.

A. Protoplasm layer. B. Fluid layer. C. Yolk layer. D. Cortex and chorion layer.

111› * Sic, but the text says hours. Translator.

215 • Directly after ovulation all stages from 1 to 5

were displayed by some parent fish, but as time passed many

parent fish changed type, and after 22 hours all parent

fish showed type 5. There was a connection between the type

of stratification of the spawn and the average proportion of

development. In type 1 it was 51% and decreased as the type

changed, being zero in type 5.

It thus appears that the quality of the eggs of

sogyo and hakuren can be to a certain extent determined by

cer..Jrifuging a portion of the spawn. p110

6. Egg quality and other criteria.

Criteria other than those already described which

were investigated were the colour and pH of the'eggs and

the refractive index of the yolk. However no changes were • p111

found and there was a large individual scatter of the values,

so it is considered that these criteria are not suitable

for the appraisal of egg quality.

Experiments were made with the objective of developing

methods for the appraisal of egg quality. If eggs of good

' quality with satisfactory fertilization and development ratios

can be obtained, the yield of fry could be increased and

stabilized.

• 216

•

•

It was found that the quality of the eggs of rainbow

trout, of ayu (Plecoglossus altivelis), of sogyo (Ctenopharyncodon

idellus), and of hakuren (Hypophthalmichthys molitrix), could

to a certain extent be determined from the transparency of the

spawn, from the size and distribution of fatty droplets and

from the morphological features shown in histological sections

of the cortical protoplasm, the yolk globules and the fatty

droplets. It was also shown that it is possible to determine

to some extent the quality of sogyo and hakuren spawn from the

stratification pattern obtained when a portion of the spawn

was centrifuged.

Since these criteria can be applied not only to eggs

which have not been spawned after ovulation, but also to those

formed at the upper limit of spawning temperatures and to the

quality of abnormal eggs directly after ovulation, it is

possible to use them as general criteria for the appraisal

of egg quality.

Eggs formed near the upper limit of spawning temperatre

were of extremely low quality, and the quality of eggs not

spawned after ovulation also decreased with the passage of

time. Since this decrease of quality was particularly rapid

in species in which the temperature suitable for spawning is

' high, the period for collecting eggs from such species is

short. Consequently it is to be supposed that the moment of

ovulation must be precisely ascertained if eggs of good quality

are to be collected.

• 2 17

References. .

1. ITO Takashi, IWAI Hisao, FURUICHI Tatsuya.-

Ayu shubyo seisan ni kansuru kenkyu - XXXIV.

Kaku shu chiayu no yosei shingyo no jinko juseiran

to fuka shigyo no tokusei.

Kiso mikawa kako shigen chosa hohoku No. 4 521 -542 (1967).

T. Ito, H. Iwai, T. Furuichi.

Studies relating to the production of ayu fry - XXXIV.

Artificial fertilization of culture fish as parents

of various types of young ayu, and the characteristics

of the young,fish.

Reports of studies of the resources of the Kiso Mikawa

estuary, No. 4, 521 - 542 (1967).

2. Hiroshima ken tansuigyo shido jo.

Kurogoi shingyo yosei jiryo to ranshitsu, shigyo

ni hikaku (2).

Showa 47 nendo jigyo jisseki 12 (1972).

Hiroshima prefectural freshwater fish promotional centre.

Comparisons of the diet of cultured black carp with -

quality and fry (2).

Annual reports of progress 12 (1972).

• 218

3 , KONDO Kei.

Kingoi no shubyo seisan ni kansuru kenkyu I.

Sairan, saibyo ni kansuru kiso chosa.

Hiroshima ken tansuigyo shidojo chosa kenkyu hohoku No.12.

Kingoi tokushu 1 - 13 (1973).

K. Kondo.

Studies of the production of brocade carp fry - I.

Basic studies of the collection of eggs and of fry.

Research reports of the Hiroshima prefectural

freshwater fish promotional centre, No. 12.

Brocade carp issue, 1 - 13 (1973).

4 • TACHIKAWA Wataru.

Nijimasu shingyo kairyo shiken - I.

Shingyo jiryo no tekisei tanpaku ryo o motomeru

•

shiken narabi ni botai to ranshitsu ni kansuru kento.

Gifu sui shi ho No. 12, 64 - 88 (1965).

W. Tachikawa.

Experiments on the improvement of parent rainbow

trout - I.

Experiments on the quantity of protein required in

parent fish diet and an investigation of the

relation between egg quality and the maternai body,

Research reports of the Gifu fisheries research

laboratory, No. 12 64 - 88 (1965).

• 2 19

•

S. KOGISO Takuo, ISHII Shigeo.

Ayu no shingyo yosei jiryo narabi ni seijuku

togyo ni kansuru shiken.

Gifu sui shi ho No. 13, 19 - 25 (1966).

T. Togiso, S. Ishii.

The diet of cultured parent ayu and experiments

on the control of maturation.

Research reports of the Gifu fisheries research

laboratory No. 1? 19 - 25 (1966).

6. KONDO Kei, FUSHIMI Toru.

Ayu shigyo yo:,ei to sairan shiken (jiryo no sa ni

yoru sairan oyobi fuka kekka).

Hiroshima ken tansuigyo shidojo chosa kenkyu hohoku

No. 10, 73 - 96 (1971).

K. Kondo, T. Fushimi.

Experiments on diet and egg collection with ayu

parent fish. (Results on the variations produced

by various diets on egg collection and hatching).

Research reports of the Hiroshima prefectural

freshwater fish promotion centre .

No. 10 73 - 96 (1971).

p112

•

220

7. ISEDA Hiroshi, HIRATA Mitsuru, ITASAKI Kiyoshi.

Ayu shingyo yosei shiken - * I.

Kumamoto sui shi jigyo hohoku showa 46 nendo 336-363 (1971).

H. Iseda, M. Hirata, K. Itasaki.

Experiments on the rearing of parent ayu - I.

1971 progress report of the Kumamoto fisheries research

laboratory 336-362 (1971).

8. GOTO Katsuaki, KOGISO Takuo, HONJO Tetsuo.

Yosei ayu no ranshitsuni kansurtukenkyu - I.

Gifu sui dai 9 kai ayu bukai shiryo I (1973).

• K. Goto, T. Togisr, T. Honjo.

Studies of the quality of cultured ayu eggs - I.

The appraisal of egg quality ..

Materials from the ninth meeting of the ayu section,

Gifu fisheries research laboratory, I. (1973).

9. ZHONG Lin, LI You Guang, ZHANG Song Tau, LIU Jia Zhao,

CHEN Fen Chang.

Jia yu di sheng wu he ren _gong fan ji.

Ko xue qu ban she (1965).

CHUNG* Lin, LI Yu Kuang, CHANG Sung T'ao, LIU Chia Chao,

CH'EN Fen Ch'ang.

The artificial breeding of domestic fish.

Science publishers (Peking) 1965.

* This reference is in Chinese. The authors' naines have been transliterated in the modern (pin-yin) romanization, but the translation gives the names in the Wade-Giles romanization more commonly used in English language publications. Translator.

•

221

10. OWATARI Satoshi.

Shingyo yoshoku ni yoru sairan shiken.

Saitama sui shi ho 18 86 - 90 (1963).

S. Owatari.

Experiments on egg collection from cultured parents.

Reports of the Saitama fisheries research laboratory,

18 86 - 90 (1963)

11. OWATARI Satoshi.

Shingyo yoshoku shiken.

Saitama sui shi ho 20 56 - . 69 . (1964).

S. Owatari.

Experiments on cultured parent fish.

Reports of the Saitama fisheries research laboratory,

20 56 - 69 (1964). .

12. KOGISO Takuo, FUNASAKA Yoshiro, ISHII Shigeo.

, Ayu no shingyo jiryo narabi ni seijuku togyo ni

kansuru shiken.

Gifu sui shi ho, No. 11 74 -83 (1965).

T. Kogiso; Y. Funasaka, S. IShii.

The diet of cultured parent ayu, and experiments on

the control of maturation.

Research reports of Gifu fisheries research laboratory.

No. 11 74 - 83 (1965).

•

• 222

•

13. OWATARI Satoshi.

Nijimasu shingyo shiiku ni .okeru suion sza sairan

seiseki ni oyobosu eikyo ni tsuite.

Saitama sui shi ho 28 58 - 78 (1969).

S. Owatari.

On the influence of the culture temperature on the results

of egg collection from parent rainbow trout.

Reports of the Saitama fisheries research laboratoty,

28 58 - 78 (1969).

14. ITO Takashi.

Ayu shubyo no jinko seisan ni kansuru kenkyu LXX.

Ayu no jinko saibyo gijutsu no genjo to mondaiten.

Ayu no jinko hoshoku kenkyu.. 1 1 - 7.

Mie ken tate dai suisan gaku bu tansurzoshoku

gaku kenkyushitsu.

•• T. Ito.

Studies of artificial production of ayu fry. LXX.

The present state and problems of the technique of

artificially obtaining ayu fry.

Studies of artificial ayu culture 1 1 - 7.

Freshwater propagation laboratory, Faculty of

Fisheries, Mie prefectural University.

•

• 223

•

15. NOMURA Minoru, TAKASHIMA Fumio, OWATARI Satoshi,

UEMATSU Zenjiro.

Nijimasu no kajukuran keisei katei ni tsuite - I.

Kajuku katei ni okeru juseiritsu sono ta no hendo.

Showa 41 nendo suisan gakkai shunki daikai, koen (1966).

M. Nomura, F. Takashima, S. Owatari, Z. Uematsu.

On the process of oogenesis during maturation of

rainbow trout - I.

On fluctuations of the rate of fertilization during

the process of maturation.

Papers of the 1966 spring meeting of the Japanese

Society of Scientific Fisheries (1966).

16. NOMURA Minoru, SAKAI Kiyoshi, OWATARI Satoshi.

Nijimasu no kajuku rankeisei katei ni tsuite - IV.

Hakken ritsu to tamago kussetsu ritsu to no kankei.

• Showa 42 nendo suisan gakkai shuki daikai koen (1967).

M. Nomura, K. Sakai, S. Owatari.

On the process of oogenesis during maturation of

rainbow trout - IV.

The relation between the proportion developing and

the refractive index of the eggs.

Papers of the 1967 autumn meeting of the Japanese

Society of Scientific Fisheries (1967).

•

224

17. SAKAI Kiyoshi, TSUCHIYA Minoru INABA Denzaburo, NOMURA Minoru

Sogyo no horumon shori go no ransoran no jotai to

sairan seiseki.

Showa 42 nendo suisan gakkai shuki daikai koen (1967).

K. Sakai, M. Tsuchiya, D. Inaba, N. Nomura.

The condition of ovarian eggs in sogyo after hormone

treatment, and the results of egg collection.

Papers of the 1967 autumn meeting of the Japanese

Society of Scientific Fisheries (1967).

18. SAKAI Kiyoshi, TACHIKAWA Wataru, NOMURA Minoru.

Ayu tamago no kajuku katei ni tsuite.

Showa 48 nendo suisan gakkai shunki daikai koen (1973).

K. Sakai, W. Tachikawa, N. Minoru.

On the process of maturation of eggs in ayu.

Papers of the 1973 spring meeting of the Japanese

Society of Scientific Fisheries (1973).

19. SAKAI Kiyoshi, NOMURA Minoru, KOGISO Takuo, GOTO Katsuaki.

Ayu tamago no kajuku katei ni tsuite

Yosei shingyo to tennen shingyo ni okeru hikaku.

Showa 49 nendo suisan gakkai shunki daikai koen (1974).

K. Sakai, M. Nomura, T. Kogiso, K. Goto.

On the process of maturation of eggs in ayu II.

Comparison of cultured and natural parent fish.

Papers of the 1974 spring meeting of the Japanese

Society of Scientific Fisheries (1974). •

• 225 ••

11. Marine Fish.

Michiyasu KIYONO.

(Faculty of Agriculture,. Tokyo University)..

Experience in rearing has suggested that the survival

of fish larvae is greatly influenced not only by the

environmental conditions but also by the quality of the eggs1

.

Nevertheless, this has been little studied. It has been

clearly shown2 ' 3 that the fertilization and hatching of the

eggs are greatly influenced by the ripeness of the eggs (the

length of time after ovulation to exposure to the sperm), by

the treatment of the parent fish just before sp , wning, and by

the timing of the hormone administration. Further studies

are needed to determine whether the problems in the rearing

of fry which are due to egg quality arise simply from the

methods used in the collection of the eggs or whether they

arise from earlier factors such as the state of nutrition of

the parents4 , the rearing environment, or the state of

development of the methods used for culturing the parent fish

and of collecting eggs.

The proauction of fry would be helped if ways not yet

established could be found of appraising the quality of the

eggs in the spawn. In this paper good quality eggs are

provisionally defined, on the basis of the rearinp; of larvae,

•

•

226

as eggs from which satisfactory fry are hatched after they

have survived incubation. A number of investigations have

been made in order to test methods of determining the

proportion of good quality eggs in the spawn, and an outline

introduction to these investigations follows.

1. The characteristics used in determining egg oualitv.

Many of the marine fish of importance in the production

of fry produce floating eggs. The fish used in these

investigations all lay floating eggs and include the black

porgy (kurodai) Mvlio macrocephaluF1 BASILEWSKY, the sand borer

(kisu) Sillago sihama FORSKRL, and the stone flounder (ishigarei)

Kareius bicoloratus BASILEWSKY. Eggs naturaily spawned after

hormone treatment were obtained 6 times from 6 parent porgy in

May and June 1971, and 12 times from 7 parent sand borers in

August and September 1973. Eggs were collected by stripping

25 times after hormone treatment of 13 flounder in December

1973 and January 1974.

With the idea of estimating the proportion of good

quality eggs in the spawn, the characteristics and the

relations between the characteristics of the spawn were

investigated. Careful attention was given to changes of the

feed in the rearing environment, which greatly influence the

satisfactoriness of the survival of the larvae. It was

decided to measure the proportion of good quality eggs,

p114

2 27

the number of eggs in the spawn which did not hatch, the

number which died before the yolk was absorbed, and the

number of fry which hatched but were considered to be abnormal

(in the following called the abnormal egg rate*). The

results are shown in Figures 11.1 to 11.3. The individual

properties will be described below.

1.1 Egg density.

The density was determined by observing whether the

eggs floated or sank in seawater of known density kept at the

proper temperature . This temperature was 20 °C to 23 00 for

porgy, 23.5° C to 24.5°C for sand borer, and 11 °C to 13 ° C for

floun der.

Eggs with opaque portions when discharged were all

unfertilized and sank to the bottom of high density seawater

(C1 240/00). Only those which were transparent were therefore

measured. The eggs in a single group were all very similar

in density, the differences being 0.0002 to 0.0005. Groups

of flounder eggs containing a low proportion of abnormal eggs

were found to have a tendency to gather together, but no such

tendency was found within the limits of the experiments in

sand borer eggs. It was also observed that unfertilized eggs

and eggs with abnormal development often tended to sink before

incubation.

* For convenience, this is the ratio to the total number of floating eggs,

• 228

•

Figure 11.1.

Properties of porgy eggs and larvae.

LARVAL PROPORTION

'ARENT IATCHING SURVIVAL OF EGGS EGG FATTY

;II O. RATE RATE (YOLK WITH MULT.. DIAMETER** DROPLET DENSITY

ADSORBTION tPLE FATTY DIAMETER**

STAGE)* DROPLETS*

.----... -

% 0/

.2,, % mm

1. 99 95 2 0.83±E0n92 — 1.0233 • 2 100 90 7 0.91±0.089 0.24±0.050 —

3 • 99 62 9 0.87±0.093 0.23±0.036 —

4 98 50 21 0.85±0.068 0.23±0.037 1.0233

.5 99 98 2 0.85±0.088 . 0.24±0.048 1.0244

92 40 — 0.87±0.075 —

The abnormal egg rate is (100 - percentage of fry

surviving to the yolk absorption stage) %. The egg

diameters are given as mean standard deviation.

Many eggs from No. 3 and No. 6 were in the cloudy

white morula stage when gathered.

Percentage of the floating . eggs gathered.

Observed in the morula stage.

FI ••

* *

•

• 229

•

Figure 11,2.

Properties of sand borer eges and larvae.

FRY

PARENT SURVI • HATCHED LARVAE , ."0

F ISH No. HATCH VAL EN

•—• INC RATE. EGG FATTY • ITY LENGTH YOLK YOLK VOLUME/

COLLECTION SATE (YOLK DIAMETLR DROPLET • * VOLUME LENGTH

No. (,:l ABSORB *4 DIAMETER

TION 4* STAGE)

4

. _ „, " —

'A 0.,

,0 mm mm mm mm 1- 1 99 78 0.67±0.013 — 1.0214 1.46±0.044 — —

1- 2 88 16 0.68±0.007 0.15 ±0.0041.0207 — — —

1- 3 100 70 0.67±0.014 0.13±0.0061.0215 1.45±0.050 0.18±0.0200.124±0.028

2—Ï 95 90 0.65±0.021 — 1.0185 1.43±0.029 —

2-2 99 80 0.69±0.009 — 1.0192 1.52±0.063 0.18±0.0220.118±0.014

3-1 97 90 0.69±0.017 0.16±0.0051.0217 1.501-0.066 0.19±0.025C.127±0.020 .

4-1 91 84 0.68±0.015 0.16±0.0061.0206 1.50±0.047 0.17±0.0250.113±0.014

4-2 88 58 0.70±0.006 0.15±0.0041.0197 1.52±0.053 0.19±0.0320.125±0.022

5- 1 0 — — — 1.0232 — — —

5- 2 98 74 0.66±0.012 0.16±0.0051.0230 1.39±0.038 0.15±0.0170.108±0.010

6- 1 96 37 0.70±0.010 0.15±0.061.0195 1.52±0.054 0.22±0.0040.145±0.033

7- 1 86 7 0.65±0.010 0.15±0.0031.0235 1.40±0.051 0.19±0.0300.136±0.025

Values for egg diameter are + standard deviation.

In 1-2, 5-1, 5-2 and 7-1, many opaque eggs in the morula stage were found at the time of .collection..

Relative to the number floating when collected.

Observed in the morula stage.

***• Yolk volume =axbx c.

* 41.

•

FI

COL

•

Figure 11,3:

Properties of rock flounder eggs and larvae.

PER.. AVAL ARENT CENT- SH No. AGE OF IVAL EGG DEN- COLOUR PH WATER --, ' FLOAT... NORMAL HATCH RATE DIAMETER SITY. TONE. CONTENT.

LEcTioN iNG Ly bey 1 NG (You( 4... .*** ...I, 1.4.1. •4, 41.

No, RATE. ELOPED RATE. ADSORB • EGGS '1 ** TION

•* STAGE) ***

■-,-.., % % % % mm I DARK g.

1-1 98 0 0 - 0.97±0.021 10217 - 92.36

2-1 51 49 • 49 34 1.02:10.015 1.0210 L IGHT 5. 5 _

2-2 83 4 4 0 0.99±0.020 1.0223 L IGHT 5.6 -

2-3 .83 15 12 4 1.01±0.019 1.0236 MED I um 5.7 -

2-4 - 27 24 19 1.07±0.020 1.0210 I L iGHT _ 52.56

2-5 59 71 69 13 1.01±0.014 1.0223 DA IIK 5.6 -

3-1 50 26 18 5 1.07±0.017 1.0198 - - 92.88.

3-2 95 15 13 • 0 • 1.03±0.020 1.0198 LIGNT - 91.92

3-3 62 32 32 19 1.06±0.022 1.0192 L 8 GHT 6.0 92.96.

4-1 58 43 43 29 1.04±0.014 1.0204 --- 5.8 92.87

4-2 -. 2 2 0 - 1.0210 --- 5.6 -

4-3 47 31 16 7 1.06±0.012 1.0192 L I GHT 6.0 93.02

5-1 - 28 18 16 1.09 ± 0.023 1.0204 --- - -

5-2 - 3 0 - 1.04±0.019 1.0192 MED i um 5.9 92.90.

5-3 74 25 25 20 1.05 ± 0.015 1.0204 LIGHT - 92.62

6-1 0 73 56 53 1.04±0.014 1.0210 ele "" 5.9 -

6-2 13 68 68 27 1.04±0.014 1.0198 MEDIUM 5.9 -

7-1 - 43 24 21 1.03±0.015 1.0217 --":" - -

7-2 9 37 35 18 0.99±0.017 1.0223 MED tun • - 92.41.

8-1 - 44 42 32 - 1.0210 - - -

9-1 - 84 83 75 1.05±0.023 1.0210 L IGHT- 92.46. ---

10-1 9 70 59 45.1.04±0.017 1.0204 5.9 92.11

11-1 53 87 87 78 1.04±0.019 1.0204 ---.. - 92.72

12-1 35 51 25 18 1.04±0.016 1.0204 L i c 87 ' - 92.92

13-1 87 58 21 5 1.05±0.014 1.0204 MED I UM 5.8 92.98

Egg diameter: Mean + standard deviation.

PH : MR and BOP as indicators.

: The percentage of all eggs discharged.

: At the morula stage, as a percentage of the floating eggs exposed to fertilization.

Percentage of floating eggs exposed to fertilization.

23 0

**

***

** * * Measured at the 2 to 8 cell stage.

23 1

1.2 Egg colour tone.

Some of the floating eggs were transparent when

gathered, but many were yellow. Three shades of yellow could

be distinguished in the flounder eggs. The sample was small,

but within the experimental limits, groups of a dark yellow

colour tended to have a high proportion of abnormal eggs.

p115

1.3 Egg diameter and fatty droplet diameter.

The values for groups of flounder eggs containing

small percentages of abnormal eggs had a slight tendency to

be concentrated. There was also a tendency towards a small

number of abnormal eggs in groups with a large degree of

scatter (as shown by the standard deviation). These tendencies

were especially noticeable in the sand borers.

1.4 Length and yolk volume of newly hatched larvae.

An investigation was made of the sand borer larvae.

Groups in which the average value of the specific yolk volume • pt16

(yolk volume / length), and the standard deviation of the

specific yolk volume were both low, tended to be those in

which the percentage of larvae which died before reaching the

yolk absorption stage was also low.

1.5 The percentage of abnormal eggs.

• The observations made includes-

When the percentage of sinking eggs (the ratio to the

total number of eggs discharged) is high, the percentage which

hatches (the ratio to the number which float) is in general low.

23 2

In the groups in which the proportion which hatch is

low many of the larvae die before reaching the yolk

absorption stage.

If opaque eggs are found in large numbers on the

bottom of the tank when eggs are collected after natural

spawning, many will die before reaching the yolk absorption

stage.

Black porgy eggs normally contain a single fatty

droplet, but if the percentage of eggs which contain multiple

droplets is high, many of the larvae die before the yolk

absorption stage.

Many of the eggs which progress to or beyond the

morula stage will hatch.

The pH of flounder eggs was measured after homogenization.

It was found to be 5.8 to 5.9 in grOups with few abnormal eggs,

and 5.6 to 5.7 in groups in which the percentage of abnormal

eggs was high.

2. Discussion of methods of appraisal of egg quality.

It appears from these results that groups of eggs in

which abnormal eggs were numerous before hatching produced

abnormal larvae. It seems that the proportion of good quality

eggs in the spawn can to some extent be estimated from the

proportion of abnormal eggs at the time of discharge or in the

early stages of development. It also appears that further

criteria, such as the density and the colour, could be obtained

from future trials with separate species.

t

• 233

•

•

The values of pH found for rock flounder are rather

lower than those of Fundulus and medaka (Oryzias latipes) 6 ,

but it can be deduced that the normal and the abnormal eggs

differ in physical composition.

When groups in which the percentage of larvae which

die before reaching the yolk absorption stage is low are

compared with those in which the percentage is high, it is

found that there is relatively little variation in the yolk

volume and that such groups can be considered homogenous.

There was to be founa some relation between the

proportion of abnormal eggs and the degree of scatter of the

egg diameters and of the fatty droplet diameters, However

it was found that in individual sand borers and flounders

which discharge eggs several times, the values, and the

scatter of the values, of the egg and fatty droplet diameters

may vary even between discharges from the same fish. As

silown in Table 11.4 it appeared that there was some connection

between these variations and the administration of the

hormones. In order to investigate the connection between

these values and the quality of the eggs, it will be necessary

to clarify the mechanism which through its action on the eggs,

determines the egg diameter.

Larvae can survive for a long period when reared

without food but they will strongly degenerate unless there

is sufficient food in the surroundings. Some investigations

p118

Parent fish No. -- Collection

Days since EFe. hormone diameter*. treatment

mrn- 2 0.65±0.021

0.69±0.009 2 0.68±0.015 5 0.70±0.006 2 0.67±0.013 5 0.68±0.007 2 (2fiD T Imt) 0.67±0.014

2-1 2-2 4-1 4-2. 1-1 1-2 1-3

• 234

Figure 11.4.

Changes in egF diameter and +he number of days

elapsed after hormone treatment (sand borer eggs).

* Mean + standard deviation.

•

•

• 235

•

have been made to estimate the length of the survival Period.

It is known that there is in herring a normal relationship

between the dry weight of the eggs and the number of days of

survival of the larvae without food ' . In the present

experiments we measured the number of days of survival

(defined as the number of days in which the number of

survivors was reduced to 50%)of each of the groups which had

survived to the yolk absorption stage. This was compared

with the many other characteristics which depend on the

quabtity of yolk* such as the egg diameter, the volume of

yolk in the larvae, and the dry weight of the eggs**, and

also with the density of the eggs and the abnormalities, but

no definite tendencies appeared.

Future investigation of the quality of the eggs must

first of all clarify the problems concerning fry production

which have been discussed in this paper. It will also be

necessary to include the . wayè.ihWhich'quality participates

in the survival and growth of the larvae after the yolk

aborption stage.

The values including the abnormal eggs and abnormal larvae.

** Values calculated from the egg diameter, dehsity, and water content.

•

• 236

•

•

References.

1. KURATA Hiroshi.

Nishin chigyo no'shiiku ni tsuite.

Hoku sui ken ken ho 20 117 - 138 (1959).

H. Kurata.

On the rearing of herring fry.

Bulletin of the Hokkaido Regional Fisheries Research

Laboratory, 20 117 - 138 (1959).

2. HIBIYA Takashi.

Horumon ni yoru gyorui no seijuku, sanran no kontororu.

Sui san zo shoku 12 (4) 239 - 259 (1965).

T. Hibiya.

Control of maturation and spawning 6f fish by

means of hormones.

Fish breeding 12 (4) 239 - 259 (1965).

3. HIRANO Reijiro.

Kurodai no chigyo

Nichi sui shi 11 (6) 567 - 569 (1969).

R. Hirano.

Rearing of black porgy fry.

Bulletin of the Japanese Society of Scientific

Fisheries 31 (6) 567 - 569 (1969).

• 237

•

4. ITO Takahashi, ta.

Ayu shubyo no jinko seisan ni kansuru kenkyu VIII.

Yoshoku ayu no jinko juseiran oyobi fuka shigyo

no tokusei .

Kiso mikawa kako shigen chosa hohoku (2) 825 -381 (1965).

Ito et al.

Studies relating to the artificial production

of ayu fry VIII.

Characteristics of fry hatched from artificially

fertilized eggs of cultured ayu.

Reports of studies of the resources of the Kiso Mikawa

estuary (2) 825 - 881 (1965).

T.

5) R. Cnàbissus :Intracellular hydrogen-

. Ion concentration studies.-V The pHpf

the protoplasm of the Fundulus egg.

J. Cal.. Comp. Physiol., 1. 65-40

(1932).

6) T. Yamaxoyo: Studies on the rhyth-

mical movements of the e3.rly embryo

. of Oryzias -V1 Anaerobic

• movements and oxidation-reduction

potential of the egg limiting the

rhythmical movements. J. Fac. Sci.

Tokyo. Imp. Univ. sec. IV (Zool.), 4.

233--247 (1936).

7) J. l-1. S. &Aetna and G. HE UPEL : The

influence of egg size on herring larvae

(Clupea harengus L.). J. du Cons.. 28, 211-440 (1963).

• 238

QUESTIONS.

I. Present positions and uncertainties.

Chairman: Tsuyoshi HACHIZUKA.

(Kochi University).

Parent fish for eg.9 collection.

Chairman: Kiichiro YAMAMOTO.

(Hokkaido University, Faculty of Fisheries).

For Mr. Takano.

Tamura. (Nagoya University, Agriculture).

To what extent do secretions rather than the villi of

the ovary wall govern or participate in the transport of ova?

Takano. (Hokkaido University, Fisheries).

I believe that the turbulent motion of the secreted

fluids around the egg contributes to local motion in the

reproductive cavity.

Hibiya. (Tokyo University, Agriculture).

Fish begin to accumulate yolk when they reach the

appropriate age. What do you think the trigger mechanism to be?

Takano.

The direct cause of the start of yolk accumulation is

the activation of the productive cells after the gonadotrophic

hormone (GTH) is produced by the pituitary, but the factors

required for this are not known.

•

239

For Mr. Kato.

Hibiya.

How is the weight of the ripe eggs measured? What

is the teMperature used for rearing at the Nikko branch of

the Freshwater Fisheries Research Laboratory?

Kato. (Nikko branch, Freshwater Fisheries Research Laboratory).

The ripe egg weight includes the eggs which have been

squeezed out and the total weight of the eggs which remain in

the body cavity, (partly emptied), but it does not include

the ovary, the ovary fluids or the unripe eggs. The

temperature was 9.5 + 1 00.

For Mr. Yamazaki.

Hibiya.

The cells which produce GTH were stimulated to high

activity by control of the temperature>of the goldfish, but

what happened to the cells which produce thyroid stimulating

hormone (TSH)?

Yamazaki. (Hokkaido University, Fisheries).

Insufficient observation was made of the TSH

producing cells.

•

• 240

Hibiya.

Were any forerunner changes observed in the GTH

producing cells when the temperature was controlled?

Yamazaki:

Some changes are to be expected in the hypothalamus,

but they have not yet been investigated.

111.1 121-Ya.

What do you suppose to be the site which is stimulated

by temperature?

Yamazaki.

The whole body is affected by changes of temperature,

but it is thought that the ovary is made more sensitive by

hormones. Recently there has been discussion of the action

within certain temperature limits of an isohormone.

Hibiya.

Temperature stimulation invigorates the TSH producing

cells, and promotes secretion from the thyroid. Do you think

it possible that this is connected with the secretion of LH

from the pituitary?

Yamazaki.

It has long been known that the thyroid hormone promotes

growth but it is difficult to think of any direct connection

with maturation. Of course one may conjecture that its influence

on the metabolic level could result in promoting maturation.

• 241

Oguri. (Nagoya University, Agriculture).

It is possible that methyltestosterone promotes

maturation of the eggs, but how are we to explain its

physiological action?

Yamazaki.

At the time of maturation the skin of both male and

female salmonids thickens. This has been shown experimentally

to be caused by both male and female hormones. It has recently

been shown that the females also secrete male hormone from the

ovary, and it is supposed that this may have something to do

with the maturation of the eggs, especially in the early stages.

Hirose. (Tokai Regional Fisheries Research Laboratory).

The action of GTH on the interrenal causes the

secretion of corticoids, and these ipromote ovulation, but the

amount of corticoids needed to cause ovulation experimentally

iS large. If instead, corticoids were secreted from the ovary,

could one suppose that ovulation occurs when the local

concentration is high?

Yamazaki.

Corticoids are known to be effective in the ovulation

of catfish and medaka, but I have heard that they are not

effective in salmonids or mullet. Evidently there are

differences between species. Of course it is undeniable

that a condition of strong local concentration would increase

their effectiveness.

p121

242

••

Environment maturation and spawning.

Chairman: Tamotsu TAMURA. (Nagoya University, Agriculture).

For Mr. Yoshioka.

Shiraishi. (Tokyo University, Oceanology).

When the length of the day is limited, what is the

effect of unnatural day lengths?

Yoshioka. (Hokkaido University of Education).

When very short or very long photo periods, such as

8 hours, 48 hours, or 64 hours, which cannot be obtained in

natural conditions, are used, maturation is not so well

encouraged as with a 24 hour period,

For Mr. Harada.

Murakami. (Hiroshima University, Agriculture).

What is the definition of "the proportion which

develop?

Harada. (Kinki University, Agriculture).

This is the percentage of live eggs which progresses

into development, and here it is the percentage which, about

8 hours after fertilization, have reached the 8 to 16 cell stage.

243

Yamato. (Hokkaido University, Fisheries).

If cleavage does not occur after exposure to sperm,

one cannot speak of fertilization. Can one not therefore

say that the proportion which develop is the same as the

proportion fertilized?

Harada.

I would say that that is so immediately after exposure

to sperm. However, in time, development may stop and death

of the egg may be produced, so that the proportion developing

is not in general the sa -, e as the proportion fertilized.

IV. Maturation and metabolism.

Chairman: Michizo SUYAMA. (Tokyo University of Fisheries).

For Mr. Takashima.

Yamamoto.

Has it been reported that changes in the diet produce

quatitative and qualitative changes in the triglycerides in

the fatty droplets?

Takashima. (Tokyo University of Fisheries).

There are very few examples of investigation of such

phenomena in fish eggs, and I know of no such study.

• 244

Tamara.

Has there been any study cif a bad influence on

oogenésis of unnatural circumstances such as those produced

by free fatty acids?

Takashima.

don't believe that this has yet been studied in

fish. However I have heard that growth, survivability, and

oogenesis in rainbow trout were not affected by rearing with

hydrolyzed oils containing C 17 .

For Mr. Aida.

11, Oeuri.

Estradiol 17p was chosen as an estrogen, but were

other estrogens tested?

Aida. (Tokyo University, Agriculture).

Diethylstilbestrol was also used, and produced the

same results.

Oeuri.

Was estradiol 17(3 chosen because it is a fish

estrogen?

Aida.

That is correct.

p122

•

• 245

•

V. The a -Opraisal of egg quality.

Chairman: Hiroshi TSUKAHARA. (Kyushu University, Agriculture).

For Mr. Sakai.

Yamamoto.

How were observations made immediately after

ovulation in rainbow trout? How many specimens were used?

Sakai. (Tokyo University of Fisheries).

Individuals from which eggs could not be collected

on the previous day, but from which they could be collected

the next day were taken to have just ovulated. The number

of specimens was 30.

•

• 246

General Discussion.

p123

Chairmen: Takashi HIBIYA (Tokyo University, Agriculture).

' Minoru NOMURA (Tokyo University of Fisheries).

Reijiro HIRANO (Tokyo University, Agriculture).

Hibiya.

Let us start the discussion with the question of

maturation and external environmental factors.

Oguri.

111, would like to enquire about pathways for the

influence of light in stimulating development of .the gonads.

Yoshiaka.

Up to now it has been supposed that the stimulation

of gonad maturation by light was effected through the

diencephalon pituitary, but there has been no experimental

search throughout the body for pathways for this stimulation.

Medaka mature in long days, but if the pituitary is extirpated

the ovary degenerates. This shows that the pituitary has an

important role in maturation under long daylight. Comparison

of medaka reared under short and long days shows that those

reared under long days contain more aldehyde-fuchsin staining

neurosecretions and more cells containing these secretions in

the diencephalon hypothalamus. This shows that the diencephalon

is concerned in stimulation by light.

24 7

However when the eyeballs are removed from the medaka,

the optic nerves cut, and the pineal body removed at the same

time, the frequency of spawning is reduced, but it is recovered

after 5 weeks. This shows that there is a direct path for

the action of light on the hypothalamus. Since the pigment

in the scales absorbs light whose wavelength is less than

400m/..., it is to be supposed that the light which is effective

has a wavelength longer than 400mp-, It has been experimentally

shown that medaka with the upper part of the eyes removed and

reared in light of wavelength less than 400m/.., do not mature,

but that in longer wavelength light they do mature. Thus there

must be a direct pathway for light to act on th-: hypothalamus

other than that from the eyes to the nerves and thence to the

hypothalamus and the pineal body. However since there is

little pigment which will absorb light-in.:the vicinity of the

hypothalamus, further investigation of the mode of direct

action of light on the hypothalamus is required.

Tamura.

It must be assumed that there is a path for light

stimulation which does not involve the eyes, and this will

probably be a path through the pineal body. The receptor

for the day length is the pineal body, in which melatonin

is produced during dark periods. The melatonin acts on the

pituitary and inhibits the secretion of gonadotrophic hormone.

248

During periods of light, less of this inhibiting substance

is uroduced, and consecuently it is to be supposed that the

secretion of gonadotrophic hormone by the pituitary is

increased. It is also said that melatonin acts directly on

the gonads of the higher animals and inhibits their

development. I feel that this is a field in which there

should be further experimental studies of fish. The fish

pineal emits pulses durilw dark periods which are suppressed

and not emitted during light periods. For this reason

removal of the pineal body is, so far as concerns the pulses,

the same as being in an illuminated place. I think that the

pineal body should be experimentally removed with this point

in mind. The absorption of the pigments suggests that the

wavelength of the most effective light should be 520 to 530y-

for the pineal body and 610mr for the eye. This does not

disagree with the effectiveness of light of wavelength longer

than Lkoome, which has been described by Mr. Yoshioka. However

the shorter wavelengths which are not wholly ineffective must

be included, and the intensity of the light must also be

remembered.

Yamagishi. (Teikyo University).

In natural conditions, fish live in an environment

with seasonally changing temperatures. What is the effect

on spawninn- of rearing them from the fry stage at a fixed

temperature?

p124

• 249

•

•

Yamamoto.

Many fish will form yolk even when reared at temperatures

lower than that required for spawning, but the rate of

vitellogenesis is slow. A definite temperature is needed

for ovulation and spawning. Nevertheless vitelloeenesis often

occurs at temperatures at which spawning is not possible.

Yamagishi.

There is a connection between this and the problem of

rearing parent fish for egg collection. If the fish are

reared at the appropriate temperature for growth and the

temperature is also changed only at the maturation and

spawning season, what is the effect on the maturation and

spawning of these fiSh?

Nomura. (Tokyo University of Fisheries).

In order to obtain good eggs with a development

percentage of not less than 80% from rainbow trout the parent

fish should be raised in temperatures not above 12 °C to 13 °C

and not below 3 °C to 4° C. For maturation and spawning the

needed temperature changes from 13°C to about 18 °C. With ayu

a temperature of 20 °C or higher is good for growth, but for

spawning the temperature is 17 °C to 18 ° C or lower. Mr. Sakai

observed experimentally that oogenesis occurred above 20 0 0,

but that the eggs became abnormal and remained unovulated.

In the culture of rainbow trout, they are kept at a high

• 250

ve

•

in order to increase the number of egg collections, to

obtain eggs from two-year-old fish, and to promote rapid

large growth, and the fish are made to spawn about 2 months

early by being moved into water at the spawning temperature.

In the Kumagaya trout culture experimental ponds of the

Saitama fisheries experimental station, the temperature one

month before the active spawning season is 14.6° C or lower,

and the temperature is then lowered about 1 0 0 every 10 days,

with good results in egg collection .

Hirano. (Tokyo University, Agriculture).

At the Owase station porgy are reared with the water

kept at the high temperature of 20 ° C, and eggs are successfully

obtained in mid-February, 1.5 months earlier than in natural

conditions. Would it be possible to cause them to spawn even

earlier with still higher temperatures? I would like to hear

Mr. Harada's opinion.

Harada.

If porgy spawn 1.5 to 2 months earlier when cultured

than in natural conditions, the fry can be successfully

reared. For early egg collection, porgy are reared at the

spawning temperature or at a slightly lower temperature, and

when the eggs are ripe the temperature is raised by 200 to 3 ° C

* Sic, though it would appear from earlier in this statement that the temperature should be raised. Translator. •

• 251

from just below the spawninP: temperature to just above it.

Ovulation is then efficient. The fish will grow well in

temperatures higher than the spawning temperature, but I do

not think that they will be caused to spawn.

Fushimi. (Hiroshima University Fisheries Research).

The spawning season for cultured red sea bream (madai)

Chrysophrys major in the Inland Sea is from May to July, but

in order to obtain eggs early the fish were reared from the

end of November and through the winter in water in which the

lowest temperature reached was 8 00, 13 00 or 15 0 0. Those for

which the lowest temperature was 15 00 produced mature eggs,

but up to 10 May they had not spawned, and eggs were not

obtained early. Those for which the lowest temperature was

• 13 ° C spawned on 14 April. •

In natural conditions of low winter temperatures they

do not feed and the body weight drops by about 15%, but when

the temperature is increased they start to feed and the

weight increases. The nutritional condition of parent fish

reared at the higher temperatures' remained satisfactory and

it was found experimentally that the amount of spawn and the

proportion hatched were both good.

Hirano.

When porgy are reared in warm water, do you think

that spawning is influenced by the len7th of time before

. spawning at which they are shifted?

• 252

•

Harada.

If there are no large changes in environmental factors

other than temperature, the change of temperature when

shifting will have some effect, but if the change of

temperature is small, shifting the fish will not cause much

of a problem. If the living space after shifting is not

restricted, they will spawn if shifted two months earlier.

I believe that rapid changes of temperature before or after

shifting can cause problems.

Hirano.

I think there are some temperature changes, but at

the time when we move the cultured parent fish in our

experiments into the spawning pond, most of the parent fish

which are treated with hormone and given sufficient food will

spawn. If more than one week elapses between the moving and

the hormone treatment, the ovarian eggs all degenerate and

there is no spawn.

Hibiya.

In general the influence of stress is greater in

marine fish than in freshwater fish. Handling of the fish

may cause polyuria, and there should be a study of the

dehydrated condition. I suppose that this question is

related to the physiology of the interrenal glands, but I

p125

II› would like to hear Mr. Oguri's opinion.

• 2 53

•

•

Oguri.

1 think this is related to the secretion by the

interrenal glands of cortical hormones in order to regulate

the osmotic pressure. For examule, if the interrenal glands

are removed from the eel it becomes unable to live in sea

water, but when cortisol is injected it is found experimentally

to be able to live in sea water. However since the eel is

the only fish from which the interrenal glands can be removed,

and since the method of removal is difficult, there are very

few experiments available, and further inveutigation of methods

of removal is needed. The interrenal glands may thus be

essential to the life of the fish. The data connecting the

interrenal glands with the problems of dehydration are at

present in an unsatisfactory condition.

Yamazaki.

Cortical hormones are secreted in large amounts in

response to stress. According to Fagrlund's experiments with

sockeye salmon the females are more sensitive to stress than

the males. For example, when put into a scoop net in a tank

for 30 minutes, the cortisol in the blood increased five-fold

in the males but ten-fold in the females. Also when chinook

salmon were caught in the wild and reared for one to ten days

in a tank, the cortisol increased by 50 times, and they then

died. Furthermore, when cortisol was injected in Robertson's

experiments, the ovary and other organs deg,enerated. This

254

shows that stress is extremely deleterious to reproduction.

Nevertheless a great deal of cortisol is secreted by salmon

and trout while goinp: up river, but there are few actual

measurements. It appears from the experimental results of

Donald et al that the injection of sex steroids causes a

large discharge of cortisol from the body. Thus, when hormone

is used to cause ovulation in fish which are greatly

influenced by streSs, and sex hormones are administered at

the same time, it is possible that cortisol may bé rapidly

discharged.

Nomura.

In Mr. Kato's paper it was stated that the largest

number of spawnings of rainbow trout were in March to May,

and that the actual number of spawnings was mot closely

correlated with the weight of the parent fish in August. In

other words, eggs which had not yet grown in August were

among those which appeared in March to May. Since it is

important, in the production of fry, to obtain a large number

of eggs spawned from each parent fish, I would like to ask

how this fact can be explained on the 'basis of the

fundamental endocrinology, of the nutritional environment or

of the metabolic process, so as to extract the maximum

possible amount of spawn.

•

• 254a

•

Yama s ak i.

It is the feeding of the body that determines the number of eggs

laid. When the parent fish is poorly fed, few eggs mature and are

released. When it is not fed, regressive eggs grow in number and few

eggs are laid. When the fish is not healthy, the hormone treatment

is ineffective.

Yamamoto

The relationship between growth and multiplication may be

considered as follows. The total amount of energy in the fish is given. If

much energy goes to reproduction it does not go to growth. The opposite

holds too. Generally among cold blood organisms, at a certain time energy

is stored within the body for reproduction and at a certain time it is

used for reproduction. At another time, it is used for growth. I think

that this tendency is the general rule and that fish are no exception.

As we treat scientifically in a concrete fashion of the relationship

between growth and multiplication, I would like to hear the opinion

of a specialist in metabolism.

255 ••

Hibiva.

I would like tc, kncw the opinion rf Mr. Takeuchi.

Takeuchi. (Tokai regional Fisheries Research Laboratory).

As you have suggested, the components of the diet are

applied towards growth and reproduction, and the proportions

allotted to the two functions vary greatly from one stage to

another. The number and quantity of spawnings are important,

and one must also consider the satisfactoriness of the spawn

and the way in which the components of the diet are assembled

into the eggs. It has been found in tuna that some of the

materials taken in during maturation are accumulated in

quantity in the eggs, and some are not. For example, in ayu,

when the fat level in the feed is increased (to the extent of

3% to 20%), the fat content of the body increases, but the fat

content of the ovary does not change. If a large amount of

vitamin E is supplied, much is accumulated in the ovary, but

if only a small amount is supplied there is no spawning even

though the GSI does not change. These migrations of the diet

vary greatly, but may perhaps supply some hint on how to

obtain good eggs.

Hirano.

When one is concerned with the actual production of

fry, egg quality is the most important question. Mr. Kiyono

made his experiments on the assumption that good quality eggs •

• 256

were those with a high larval survivability rate, but I

suggest that egg Quality depends on the process of

maturation and the method of collecting the eggs. What,

from the fundamental point of view, is Mr. Yamamoto's

opinion about this?

Yamamoto.

I originally supported Mr. Sakai's definition of the

satisfactoriness of egg quality in terms of high fertilization

and hatching percentages of the eggs. However I think that

apart from the reduction of fertilization percentage with

lapse of time after ovulation, the essential causes of

reduction of the fertilization and hatching qualities of the

eggs are changes in the metabolism of the parent fish during

the process of oogenesis. For example, when the eggs hatch,

the important factors which influence the metabolism of the

embryo are not only the accumulation of nutrients such as

yolk and fats, but also the substances such as vitamin E

which are present in very small amounts. It is important

that this should receive further study.

Hirose.

I have found that the water content and the ions

which enter the eggs at the time of ovulation are important

to egg quality.

• 257

•

Ogino. (Tokyo University of Fisheries).

The phosphorus content of‘rainbow trout eggs is un-

usually large, and the calcium content unusually low. The

Phosphorus is present in the egg as phospholipids and

phosphoproteins. The phosphorus in the egg cornes from

materials in the mother's body, and probably is derived from

the bones. The relative proportions of phosphorus and

calcium in the mother's body will be very different during

maturation from those at other times. Consequently, simple

reasoning suggests that the supply of phosphates to the

parent fish during maturation of the eggs will be important.

Yamazaki.

It is believed that the yolk c'ontains substances

which promote the development of the embryo. It is not known

when these substances are accumulated, but is it not true

that if they are not properly accumulated the eggs will not

be of good quality even if sufficient yolk has been accumulated?

Hibiya.

There are differences between different species of

fish. For example, even among the same family such as the p127

Salmonidae, the salmon.die after spawning once, but the trout

spawn repeatedly.To add to Mr. Ogino's remarks about phosphorus,

if a fundamental investigation were made of the differences

258

in the state of these species after ovulation, it might be

possible to get some ideas for a technique for their

regeneration. I would like to as Mr. Suyama for his views

on this.

Suyama (Tokyo University of Fisheries).

The composition of the phosphoproteins in fish differs

from that in domestic fowl. For example phosvitin, a protein

containing a large amount of phosphorus, can be isolated from

the fowl egg, but 10 times as much protein of the phosvitin

type is found in rainbow trout eggs. This phosphorus is

present as laro;e quantities of serine esters and small

quantities of threonine esters, but serine is not classed as

a very important amino-acid in fish. If these are decomposed

during development, the phosphorus obtained may be expected

to have an important metabolic action in the formation of

high energy types of ATP and of creatine-phosphoric acid.

For this reason I believe that the phosphoproteins will be

sources of interest in future research.

Murakami.

Supposing that egg quality is so good that the

fertilization and hatching percentages are 100%, is it possible

that 100% fry could be obtained by a perfected technology of

rearing,.? I would like to ask Mr. Hibiya and Dir. Yamamoto

about this.

•

259

Hibiva, Yamamoto. .

Given your conditions, it is possible.

Hibiya.

The information obtained by basic research is detailed,

that obtained by experience is generalized. The two don't

mesh together very well, and this distresses those of us who

participate technically or scientifically in production.

Today we may feel some relief from our distress. Much remains

for the future, such as the most suitable regulation of the

environmental conditions, the development of standard feed

for the parent fish, and the application of genetics to the

breeding of superior parent fish of both sexes. I feel that

this symposium has been successful in fostering debate and

bringing out concrete proposals for future research, and I

may perhaps say that it was been more successful even than

was hoped. All those who were charged with the responsibility

of planning are happy that it was possible to debate so many

important questions during this successful meeting, and I

wish to express our cordial thanks to all the participants

for their cooperation.

Shuzo EGUSA Minoru OKADA Shunji KAWABATA hitoshi KONDO Shoichi TANAKA

Junsaku NONAKA Yoshiro HASHIMOTO Takashi HIBIYA Yoshihiro MACHIDA Motonobu YOKOZEKI

Publishing Committee.

260

Fisheries Science Series (6). 0362-150060-2244.

The Maturation and Spawning of Fish.

Fundamentals and Application.

Price 1100 Yen ,

Published 15 October 1974.

Editors: The Japanese Society of Scientific Fisheries (Incorporated).

c/o Tokyo University of Fisheries Minato minami 4 - 5 - 7 Minato ku Tokyo Japan 108.

Place of publication: Koseisha Koseikaku Ltd.

Saneicho Shinjuku ku .Tokyo.

Gyro .Tokyo 59600

Telephone (359) 7371 - 5.

Copyright: Japanese Society of Scientific *Fisheries, 1974.

Printed by Daishin. Bound by Nakajo.

261

Food Science for Pisciculture.

(Yogyo shiryo gaku)

Edited by Yoshiro HASHIHOTO.

A5, 282 pages, 1600 Yen.

The latest developments in nutritional science are

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An introduction to the Science of Pisciculture.

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A5, 720 pages, Lp000 Yen.

Concentrating of the fundamentals of pisciculture,

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the outset of the ae of cultured fisheries, this book of

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p130

a 262

Complete Pisciculture.

(Yogyo gaku kakuron).

Edited by Noboyuki KAWAMOTO.

A5, 840 pages, 5000 Yen.

This large volume gives detailed explanations of

all important matters in methods of rearing, the nutrition,

the prevention of disease, the shipping and care of fry of

all the principal species which are cultured in Jauan,

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trout,yellow tail and porgy to oysters, abalone, laver,

pearls and prawns.

"_Thorough Culture" in shallow seaR.

(Senkai kanzen yoshoku).

Joint Editors:

Takeo IMAI Shun INO Munehisa KUROKI Moritaro YAMAMOTO. Motosaku FUJINAGA

B5, 464 pages, 3500 Yen.

The completely artificial culture of all stages from

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Forthcoming! Diseases of cultured fish. by Shuzo MUSA.

KOSEISHA KOSEIKAKU.