5.RCE[I-VE:"."3 .
•FISHERIES RESEARCH BOARD OF CANADA
•
Translation Series No. 1270
•
Biochemicarstudies on the lipids of cultured fishes
By Kazuo Ando •
•
Original title: Yoshoku gyorui no shishitsu ni kansuru
seikagaku teki kenkyu.
From:
Tàkyo Suisan Daigaku Tokubeksu Kenkyu Hokoku
(Journal of the Tokyo University of Fisheries),
54 (2): 61-98, 1968.
••■••■••■■■
Translated by the Translation Bureau(mK)
Foreign Languages Division
Department of the Secretary of State of Canada
Fisheries Research Board of Canada
Halifax Laboratory
Halifax, N.S.
1969
85 pages typescript
7 )3
)
SECRÉTARIAT D'ÉTAT
BUREAU DES TRADUCTIONS
DEPARTMENT OF THE SECRETARY OF STATE
TRANSLATION BUREAU
FOREIGN LANGUAGES
DIVISION
TRANSLATED FROM - TRADUCTION DE
•
Japanese
'
CANADA
DIVISION DES LANGUES
ÉTRANGÈRES
INTO - EN
English
AUTHOR - AUTEUR
Ando, Kazuo.
TITLE IN ENGLIÈFI - TITRE ANG LAI§
Biochemical studies on the lipids of cultured fishes.
Title in foreign language (transliterate foreign characters)!
Yoshoku gyorui no shishitsu ni kansuru seikagaku teki kenkyu
R5F5RENCE IN FOREIGN 1: ANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHA,RACTERS.
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Journal of the Tokyo University of Fisheries
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DATE OF PUBLICATION
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1968
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BIOCHEMICAL STUDIES ON THE LIPIDS OF CULTURED FISHES
By: Kazuo ANDO
UNEDITED DRAFT TRANSLATION
Only Ici
TRADLiCri.LCA NON REVISEE
Iniormation seulement
Received December 17, 1967
I.
(1)
Introduction
Purpose of this research
Japan's main cultured fresh water fish are carp,
eel, rainbow trout, smelt etc. These cultured fish were fed
pupae, marine fish etc., which were the main sources of
protein and fat in the past.
However, recently using these
feeds became difficult due to various reasons and therefore
a feed mixture having white meal which is made of low fat
content marine fish as a source of protein supplemented with
starch, fat, and vitamins became the main source of feed for
cultured fresh water fish.
Further, even brown meal which is
made of marine fish with a high fat content became available.
This material was believed to be useless as feed in the past,
due to the deterioration of the fat.
It is very important to know the effect of changes
on the growth of various species of fish due to the change in
•508-200... 10.- a
61
2.
feed system for parent fish.
Also it is necessary to know
the effect of changes in feed system on the growth of eggs
in a parent fish (carp, trout, rainbow trout, etc.) which
are artificially fertilized at an artificial spawning
•
ground. As far as the research is concerned on cultured
fish, there are many articles available on the growth of
cultured fish based on the effects of feed, but there are
not many articles concerning the effect of feed systems
on parent fish in. order to obtain superior seeds.
62
In order
to clarify the above problem, one must first , know the
'
chemical composition of the eggs, the metabolism of these
substances and the biochemical significances for the
development processes.
•
There are articles by Needham 1 / 2 , Brachet 3 and
others on the changes in egg composition due to the
.
development of eggs in the field of development chemistry.
Among these studies, there are many recent studies concerning
.the protein in the egg compositions but there are not many
studies done on the fat of eggs.
-
From the above observations, a . reasonable .
preparation of feed for parent fish was studied.
The problems .
.related to fat composition, as one of the basic studies,
was carried out using rainbow trout (Salmo irriseus GIBBONS)
egsincthywerasilobtne
and developed according
to standard growth.
The changes in egg composition of matured
unfertilized eggs and fertilized eggs which grew according to
3.
standard growth up to the swim-up-fry period and the
effects of added fat in feed on cultured fish were studied
using eels.
From the above studies, it became clear that the
nature of added fat in the feed had a large effect on the
nature and rise and fall of the neutral fat, phospholipids
and protein which existed in combination with lipids in
egg yolks. Furthermore it became clear.that the nature
of added fat in the feed produced, a large effect on the
fat of fish which was fed on a particular feed system.
These results are believed to be one of the
important basic materials for selecting the feed system or
improving the existing feed system. Before giving the
.
results of the experiment in each chapter hereafter, I
shall introduce already known biochemical studies on the
development of eggs and changes in forms.
(2)
Prior studies
There are many biochemical articles by Needham
concerning the developments of oviparous animals and
birds. These articles have become the basis for research
in this field. Needhamls topics of studies consisted of
'maturation of reproductive . organs, sperm, divisions of
the lung, formation of shapes and formation period, breathing y
of energy, hydrocarbons, protein, lipid, and
rise and fall of inorganic substances etc.
devlopmnt
4
There are many studies concerning the phenomena
associated with fertilization using the eu;s of sea urchins
among the recent studies on development chemistry.
Brachet
has done studies on protein using sea urchin eggs.
As far
as the studies of the development chemistry of eggs after
fertilization are concerned, there are studies by Lovtrup4
on Siredon mexicanum, Smith 5 on rainbow trout, and Hayes 6
on Salmo salar. A large number of these studies are on
large eggs of birds, amphibians and fishes.
These studies discuss metabolism from the changes
in amount (1.• hydrocarbons, protein and lipids during the
development of eggs but there is no investigation of the
content of components which make up each composition.
Suyamais studies on the egg yolk of rainbow trout are the
only information available on the subject of investigation
of the contents. His studies concern the changes in
aminoacid compositions in the egg yolk. There are complete
studies on lipid chemistry by Ishidag on the eggs of sea
urchins, but there are only studies by Murata-Takeshima 9
on the rise and fall of Vitamin A during the development
of eggs, in the larger fish eggs.
(3)
Common knowledF,e on changes of shapes and development
stages during development.
Yamamoto et all° have carried out studies on the
development of rainbow trout eggs in the body of parent
fish up to the period of spawning and the changes in shape
.
5.
from fetus to fry after fertilization in greater detail.
Egg yolk having glycogen, protein and lipid as its main
composition, is the required nutritious material for the
normal development of the fetus later and which is passed
along by the blood of parent fish to the eggs during the
maturing process of the ooyte.
Eggs which are fully developed in the parent fish
separate in regular sequence and develop to such forms
that they can be collected.
The changes in outside appearance
and stages of development after the start of development
in this research have been named as follows:
1) Unfertilized eggs.
Eggs which are collected by using an artificial
collection technique from the parent fish.
with a solution (example:
They are washed
10 litres of water, 2.4g of.KC1,
90.4 g of NaCl and 22.6 g of CaC1) to remove coelome fluid
and crushed eggs. Eggs have not yet taken in water. The
chorion is touching the vitelline membrane as shown in
Fig. 1-A, it has adhesiveness and is very soft.
2) Fertilized eggs.
The eggs which are fertilized hy mixing the
unfertilized eggs with sperm and after they have taken in
water.
Sucking in of water was completed according to progress
of time. As shown in Fig. 2, the space between the egg capsule
and yolk membrane is filled with perivitelline fluid.
Eggs
63
6.
became elastic and the surface became hard.
3) Appearance of body.
The fetus continuously grew through the division
period and finally the white string-like body could be seen
with the naked eye. Eggs obtained a reddish color at this
time and the whole of the eggs became pinkish. Only the
notochord_could be seen and there was no sign of a blackcolored element for the eye (Fig. 1-C).
•
4) Appearance of eye.
Fish which was notochord-shape obtained its usual
fish shape. Eyeballs with black dot could be seen clearly
but there was no sign of movement in the eggs.
(Fig. 1-D)
5) Eye appearance period.
Period between appearance of eye and spawning.
Young fish grew gradually inSide the membrane.
Heart and
blood vessel could be clearly seen. Blood circulation
became clear, and the body obtained movement. (Fig. 1-E).
6) Hatching.
Young fish swam out to the open by breaking the
capsule.
Yolk called "SAINO" was attached to the body as
can be seen in Fig. 1-F; it was hanging from the body and
thé surface was covered with blood vessels.
7) Yolk absorbed.
Yolk in "SAINO" was gradually absorbed and thus
• "SAINO" became smaller.
The shape of the young became that
7.
of rainbow trout and the body color became darker. (Fie. 1-G)
-a
. 7Z )
8) Swim-up-fry.
"SAINO" disappeared and the color lightened.
Spots
characteristic of trout started to appear. There was very
active movement. Feed was not yet provided. (Fig. 1-H)
It is believed that the eggs metabolized by using
the compositions stored in the body of parent fish up to
this stage.
IL
Changes in general components of eggs during egg
development.
(1) Foreword
As shown in Fig. 1-A; B, eggs of rainbow trout
suck in water gradually and formation of perivitelline fluids
between egg capsule and yolk membrane occurs.
The egg capsule
becomes hard and thus protects the inner contents from.autside
impact. The egg capsule is semitransparent. Although water
and inorganic ions can pass through the egg capsule,
substances such as protein, lipid and organophospho compounds
which are relatively large in size cannot pass through the
capsule, therefore any changes occurring in the substances
mentioned above are believed to be changes from within during
the development period.
There are a large number of studies being carried
out on the changes in egg composition during the development
as a first step of chemical research on development.
8.
There is a research article on fish eggs during
the period after the appearance of the eye by Ha yes ll on
Salmo salar. In Japan, there are studies by Suyama et al l2
on the changes of rainbow trout after fertilization, Ono"
et al 13 on the changes in crude fat and amount of phosphoric
acid in rainbow trout in the period of one year after
fertilization and Yamamura et al 9 on the disappearance of
Vitamin A during the period between fertilization and swim
up in Oncorhynchus keta.
According to these studies, large changes in
general components occur after hatching. However, the steps
before hatching are the most important and interesting period
from the development chemistry point of view since the period'
includes division of fetus, differentiation and formation
of shape. During this period the fetus uses up part of the
yolk composition and synthesizes protein, therefore it is
believed that there is some consumption of energy at this
stage.
It is believed that lipoprotein in yolk composition
is the compound used for the above mentioned change and
therefore analysis of the compound is obviously expected.
Studies in this report consist of general analyses
of eggs during the development together with acetone-soluble
lipid which is mainly made up of neutral fat and acetoneinsoluble lipid, mainly of phospholipid.
These compounds
6/,
9.
were separated and the changes in quantity were obtained.
Further, several chemical changes in acetone-soluble
compound were investigated.
(2) Experiment
1) Materials:
Rainbow trout eggs including unfertilized eggs,
eggs with appearance of fish ( 9 days after fertilization),
eggs immediately befOre the appearance of eyes (13 days
after fertilization), eggs with appearance of eyes (19 days
after fertilization), eggs immediately before hatching
(26 days after fertilization)
and fry immediately after
hatching (34 days after fertilization), fry with half of
the yolk absorbed (47 days after fertilization) and swim-up-fry
(58 days after fertilization) were collected from
Nagano
Fisheries Directional Centre in February of 1958 and 1959.
Water on the samples collected was removed with filter paper.
The samples were then placed in polyethylene bags, frozen with
dry ice and brought to the research centre. The samples were
kept in a freezer at -23 degrees C. until they were used for
the experiment.
The size of unfertilized eggs for both years
was about 5.5mm and they weighed 90.5mg.
2) Method of analysis:
Unfertilized eggs were defrosted in running water
while they were still in a polyethylene bag.
They were
converted into a milky condition in a homogenizer while the
1 0.
mass was cooled in ice.
Since egg capsules of fertilized
eggs were hardened, and it was difficult to convert them
into a milky condition, the eggs were ground in a mortar.
Due to the hardening of bones in the samples after hatching
and because it was difficult to obtain homogeneity, the
analyses were carried out on whole fish except in the case
of extracting total lipid.
•
Analyses of water and ash were done by the usual
method.
Total nitrogen content was determined by using the
microKjeldal method. 1 4
Determination of total lipid content
iv.as done by the Sperry method. 15 This method is a simple
method for extracting lipid from samples with a high water
content. This method consists of adding the homogenised
sample to purified anhydrous ac%tone and break up any
solid protein by shaking while adding acetone gradually
and dry acetone and water under reduced pressure at a
temperature below 40 degrees C. on a water bath while passing
nitrogen through the sample acetone mixture.
Next extracting
the lipid with 10 times the volume of a dry sample of
chloroform:Methanol (2:1) mixture in a nitrogen atmosphere
while stirring with a magnetic stirrer for 6 hours. By
repeating this method three times, total lipid was extracted.
The combined solution was dried under reduced pressure and
again new solvent was added and the lipid was purified by the
Filch et al method. 16 That is by adding 1/5 the volume of
11.
water to the solution and shaking vigorously and centrifuging
for 21 minutes at 3,000 rpm, separating water-methanol upper
layer and adding methanol up to the original volume so as to
dissolve the white matter which appeared at the surface of
the chloroform layer, and obtaihed purified lipid solution.
Solvent was removed under reduced pressure and the total
amount of _lipid was determined. Separatioh of acetone-soluble
and insoluble lipid was done as follows. Lipid obtained
from the previous step was first dissolved in pet-ether and
about 20 times the volume of previously cooled acetone was
added to the solution. Solution was shaken and left overnight
in a freezer and acetone-insoluble material was separated.
This procedure was repeated to obtain complete separation of
acetone-soluble and insoluble lipid.
Each lipid was dried
after separation and the amount was determined.
Acid value for acetone-soluble lipid and neutrolization value for fatty acid were determined using the usual
methods. 17 Saponification value and elements were determined
using semi-micro determination. 18 Further determination of
,
unsaponifiable matter and separation of fatty acid were
carried out using the Hilditch method. 19
The above mentioned extractions and handlings of
lipid were done under nitrogen atmosphere.
The separated
samples were stored under atmospAere of nitrogen at -20 degrees
C. in order to avoid oxidation by air.
12.
3) Results and Discussion:
1) Changes in general composition.
Analytical values for water, ash, total nitrogen
and total lipid during development are shown in Table 2-1.
Changes in weight and reduction of each ingredient related
to weight changes per egg are shown in Table 2-2.
Numerical
values in the tables are average values of two years.
The
results are almost the same as the results already known. 12,13
The results show that there are no unusual changes in the
general ingredients during a normal development process.
Fig. 2-2 shows the reduction of each ingredient
during the development.
A large increase in weight which
can be seen after fertilization is due to the phenomenon
of the egg sucking in water. A large reduction in weight
after hatching is due to the loss of perivitelline fluid
after occurrence of the breakage of the egg capsule.
The weight gain due to sucking in water by the egg
and the increase in water content are both at 13 mg per egg,
therefore it is believed that the weight gain during this
period is due only to weight gained in water sucked in by
the eggs. From the fact that the weight loss due to hatching
was 12 mg and only 4 mg of the total loss was due to loss in
water, the water content of swim-up-fry is about 80%.
Assuming
that the water content of unfertilized eFg is eaual to the
water Content of yolk, then it is 60%.
Except for the change
in weight due to sucking in water up to the hatching period,
66
13
there is no appreciable weight gain in egg, water and dry
matter.
The above fact shows that the fetus forms the body
by utilising only the water sucked in by the eggs up to the
time of hatching.
As shown in Table 2-1, there is a parallel
between increases in egg weight and water weight.
One can
say that the increase in weight of the egg after . hatching is
solely due_to the increase in water weight'if one relates
the fact that the weight of ash decreases during this period.
If one calculates the amount of yolk ingredient
per egg used for changes in body structure and'for basis of
various energy source, one finds that 19 mg of dry matter in
yolk is used for body formation and the remaining 9 mg is
used for energy source, for the following reasons. Because
the weight . ratio of body to yolk at the time of hatching was
22.3% and 77.7% as shown below, Table 2-1, 71 mg out of a
total weight of 92 mg for the egg after hatching was made.up
of yolk and the remaining 21 mg was the weight Of the body.
Dry matter was calculated to be 28 mg and 4 mg respectively
and therefore the amount of dry matter in the body increases
from 4 mg to 23 mg up to the time of swim-up-fry. There are
not many studies concerning the steps of growth after hatching
but there is evidence, of an increase in the amount of ash
value after hatching.
This shows that the body takes in
inorganic materials from outside.
67
2) Changes in lipids composition.
Changes in the amount of acetone-soluble lipid,
acetone-insoluble lipid and unsaponifiable matter contained
in acetone soluble lipid are shown in Table 2-1.
The weight
changes of the above compounds for each egg during the
development are shown in Fig. 2-2.
According to the table,
1/3 of total lipid is spent before hatching and the remaining
2/3 is spent after hatching.
A detailed study shows that 1/2
of the acetone soluble lipid contained in an unfertilized egg
is spent before hatching and there is no spending of the lipid
after hatching.
Acetone-insoluble lipid on the other hand
shows no decrease in amount during the period from fertilization
to hatching but it is spent rapidly after hatching and at the
time of the swim-up-fry period the amount is reduced to about
1/2 the oriFinal amount.
The changes in the unsaponifiable
matter contained in acetone-soluble lipid are the same as
those of acetone-soluble lipid.
The main ingredient for the egg yolk is lipoprotein.
About 50% of total lipid is said to be phospholipid. 2° If
the formation of protein in the body is done by decomposition
of lipoprotein during the growing process of the young, it is
obvious to think that there will be changes in the amount of
lipid. It is not yet known how the protein in yolk changes
into body protein or how the protein acts. It can be said
from this experiment that at least lipids, especially
phospholipids, go through changes and take an important part
15.
in continuing the development.
The fact that lipid becomes the source of energy
during the development stages prior to hatching was shown
by Hayes 6 in rainbow trout. This theory was deduced from
the fact that the breath quotient is low and if the protein
was to be the source of energy, there must be an increase
in the amount of nonprotein nitrogen. Smith 5 too, basing
his argument on the same reasoning as Hayes, states that
lipid and protein either separately or together form the
source of energy during the early part of development.
One
can safely say that the source of energy for the early stages
of development for rainbow trout eus is phospholipid, for
the above reasons.
There is a study by Yamamura et al 9 on the changes
of acetone-soluble and insoluble lipid in the development of
salmon eggs.
The method is similar to this experiment, and
the results too are similar.
The reason for the increase in the amount of
acetone-soluble lipid before the appearance of the eye shown
in Table 2-1 and 2-2 is unknown. Lipid decreases after the
appearance of the eye and the rate of decrease is rapid,
especially after hatching.
This is believed to be due to
high energy requirements by the active young fish. The reason
for the small decrease in the amount of acetone insoluble
lipid after hatching is believed to be the fact that the lipid
became constructive materials for the organs and cells of the
fish.
16.
3) Several chemical characteristics of acetone soluble
lipid.
The main ingredients making up the acetone-soluble
lipid are glyceride, unsaponifiable matter and free fatty
acid.
Among these ingredients, the characteristics of fatty
acid are very important.
Several chemical characteristics
of acetone.soluble lipid and mixed fatty aCids which are
obtained from acetone soluble lipids are shown in Table 2-3.
The iodine value decreases slightly after fertilization and
increases after hatching.
The fact that there is no
appreciable change in the average molecular weight of fatty
acid shows that fatty acid is spent without discrimination
as a base of energy. That iS to say that there is no
selective use of one particular fatty acid during the
development period but fatty acids are used equally for
creating energy. If one takes the changes of iodine value
into account, one can see that there are weight changes in
unsaturated fatty acid but the amount is not large enough
to alter the average molecular weight of fatty acid as a
whole.
There is a relatively high iodine value for fatty
acid, therefore the presence of a large amount of unsaturated
fatty acid can be predicted. However, it is slightly lower
than the values for salmon and trout eggs which are 217 and
192 respectively. 21 The iodine value for the oil from the
body of rainbow trout is around 130. 21 ' 22 The reason for
1 7.
the high iodine value in egg oil is believd to be the
content of highly unsaturated fatty acid which is required
for the development process. The suitable development
temperature for salmon, trout and rainbow trout is below
10 degrees C.
However, it is believed that the temperature
should be high enough to keep the lipid in liquid state in
the living body.
It is clear from the table that there is no large
-change in the amount of free fatty acid before the hatching
but fatty acid shows a rapid increase after hatching.
The
amount of free fatty acid, assuming that it did not occur
during the extraction process, in acetone soluble lipid
was calculated basing the calculation on mean molecular
equivalent and compared with the remaining glyceride and
unsaponifiable matter and the result is shown in Table 2-3.
It is questionable that so much free fatty acid is present
in the living body. If phospholipid disintegrates and
produces free fatty acid it will obviously be in acetonesoluble matter. It is believed that the decomposition
power of the enzyme over fatty acid becomes stronger as the
young fry's internal organs develop.
For these reasons,
even though some decomposition occurred up to the time of
determination, it is believed that the main source of free
fatty acid is the enzyme decomposition of glyceride.
III.
Changes in total lipid ingredients during the development.
(1) Foreword
The fact that lipid becomes the source of development
energy during the development of the egg is shown by LOVTRUP 4
the eggs of Siredon mexicanum. He bases his arguments using
on the fact that there is a decrease in the amount of lipids
during the_development period up to the tiMe the body acquires
-a tail. Mathur 23 sh .0 wed that lipoprotein and phospholipids
are absorbed by the fetus up to the blastula period using the
eggs of Salmo trutta. Ohman 24 has commented on the
.
relationships between the changes in the amounts of free
lipids and phospholipids which are combined with protein, in
the fertilized and unfertilized eggs of Echinocardium cordatum.
The results of the amount changes in the acetonesoluble and insoluble lipids during the development of.
rainbow trout eggs Were already mentioned in the previous
chapter. It was also determined that the acetone-insoluble
lipids were spent before the hatching and there was almost
no utilization of acetone-insoluble lipids after hatching.
*
It was assumed that phospholipids have an important part in
the development process of the fetus from the above results.
The purpose of this chapter is to further subdivide
acetone-insoluble lipids and to know the individual decreases
in components during the development.
One can roughlY divide complex lipids into glycerylphospholipids and sphingolipids. The former consist of
69
19.
lecithin, cephalin, plasmalogen, etc. All of these compounds
contain a glycerine base in the molecule.
The latter consist
of sphyngomyeline, cerebrosides etc. which have a sphyngosine
base in the molecule. There are many more lipids known other
than those mentioned above but since the amounts of these
lipids are small and there is no method of determination, it
was assumed that the acetone-insoluble lipids are made up of
the five species mentioned above, in this experiment.
Since there is no set method for quantitative
determination of individual components in this type of
experiment, a method which involves determination of the
chemical base which makes up each compound and calculating
the amount of the compound relatively from the weight of the
base was used.
The basic structures of the five compounds mentioned
above are as follows:
Lecithin: glycerine, phosphor, cholin (one molecule each), .
fatty acid (two molecules);
Cephalin:
glycerine, phosphor, ethanolamine or serine
(one molecule each), fatty acid (two molecules);
Plasmalogen:
glycerine, phosphor, long chain aldehyde,
fatty acid, cholin or ethanolamine (one molecule each);
Sphyngomyeline: sphingosine, phosphor, cholin fatty acid
(one molecule each);
Cerebrosides: sphyngosine, galactose fatty acid (one molecule
each).
20.
For this reason, if one calculates total nitrogen, total
phosphor, total fatty acid and their average molecular
weight, total cholin, lecithincholin, glycerine, long chain
aldehyde, galactose and sphyngosine, one can calculate the
weight of each compound.
(2) Experiment
1) Materials:
Uniformly sized eggs from three year old fish were
collected at the hatching ground belonging to Tokyo
University in Yàmanashi Prefecture durinF December of 1960
and 1961. Part of the eggs Were left unfertilized and the
rest were fertilized and brought back to the lab.
The
fertilized eggs were developed in the apparatus shown in
Fig. 3-1 at room temperature'up to the time of the.swim-up-fry
period.
The extractions, purifications, and separations of
lipids were done as in the previous chapter at various stages
of development.
-
The hatching apparatus consisted of a large glass
container covered . with black .paper around the side and the
.top covered with a wooden cover. The wooden cover contained
holes. These holes were used for water inlet, outlet and
air inlet. Tap water was used for this experiment.
A
dechlorination apparatus was attached between the tap and
the container so that tap water was dechlorinated before
entering the glass . container.
by using siphoning.
Removal of water was done
The tube from the container was stoppered
21.
with a pinchcock and by opening the cock the water was
maintained at a uniform level. Aeration was done with the
aid of a compressor, according to the circulation of water.
Although the circulation of water was unsatisfactory, the
development of the eg!D-s was Iuite uniform.
The amount of eggs was determined so that about
of acetone-insoluble lipid could be obtained from the
eggs, basing the determination of the amount on the previous
experiment.
2) Extraction of lipids
Eggs were ground into a milky liquid as in the
previous experiment.
The liquid Was then added to a large
amount of acetone with vigorous stirring.
The mixture was
then stirred with a magnetic stirrer for 6 hours, the water
was removed from the mixture and at the same time extraction
of lipids was carried out.
This process was repeated three
times. Next, the residue was extracted with chloroformmethanol mixture (2:1) twice. Both extracts were dried
under reduced pressure.
Lipids in the former solution were
transferred to pet-ether and after drying the pet-ether
solution with anhydrous sodium sulfate, pet-ether was removed
and the residue was redissolved in chloroform-methanol solvent
and mixed with the latter solution.
was done by the Folch et al method. 16
Purification of lipids
Next, a large part of
the solvent was removed under reduced pressure and the sample
was diVided into acetone-soluble and insoluble lipids by
70
22.
adding acetone to the sample.
The acetone-insoluble part
was dissolved in chloroform-methanol solvent and the acetone
was separated from the mixture. The acetone-insoluble lipids
were dissolved in chloroform and were used for the solution
for the determination of each compound.
3) Method
a) Total nitrogen content:
The Micro-Kjeldal method was used.
The B method
in "method . in Enzymology"'was used for decomposition
determination.
b) Tàtal phosphor:
Allen's method was used.
c) Total fatty acid and average molecular weight:
Materials were saponified with 0.5N alcoholic KOH
for 6 hours (need for long hours for saponifying fatty acid
which is bonded to amide in sphingomyerine) and the solution
_
was acidified with 6N HC1.
was determined.
The amount of fatty acid separated
The average molecular weight was determined
from the neutralization value.
d) Glycerine:
Analysis was based on the Blix method. 27
This method
"consists of converting glycerine into isopropyliodide by ,
reacting glycerine with red phosphor and hydroiodic acid,
. distilling it and titrating iodine with sodium thiosulfate.
Neumann's apparatus 28 for determination of the methoxy group
was modified and used for this experiment. (see Fig. 3-2)
2 3.
e) Aldehyde:
The Leupold method 29 was used for this determination.
Only the long chain aldehyde was determined here through
commercially available plamitylaldehyde purified by reduced
pressure distillation at 20mm Hg and the temperature between
192-193 degrees C was used as standard. This method consists
of adding-the test solution to the hydroly:sed lipids and
developing color, transferring the colored material to
isoamylalcohol and measuring the absorbancy at 547 mu. The
sanie procedure was repeated for the standard and the amount
of long chain aldehyde was determined relatively.
f) Cholin:
There are many techniques in use tO free cholin
from sphingomyerine since it is a very difficult process.
In this experiment however, the Thanhouser et al method 31
• which was recommended by Artom 3 ° was used.
This method
consists of hydrolysing the compound with 6N HC1 in methanol
for 3 hours, removing HC1 and methanol under reduced pressure,
washing the hydrolysed material with water, forming cholinReinecke salt by adding amonium-Reinecke salt to the hydrolysed
material, filtering the crystals formed and washing the
'crystals with n-propylalcohol 32 , followed by dissolving the
salt in acetone and measuring the-absorption at 526mu. Cholin
hydrochloride was used as a standard for this experiment.
g) Lecithin:
The selective hydrolysis method of Schmidt et al 33
was used.
This method frees cholin from glycerylphospholipids
2 4.
but it does not free cholin from sphingomyerine. For this
'reason it is believed that the cholin obtained in this
experiment is the sum of the cholin from lecithin cholin
and plasmarogen-cholin.
h) Galactose:
Fatty acid and sphingosine resulting from the
71
hydrolysis of lipid with 3N ethanolic sulfilric acid was
extracted with ether.
The sulfuric acid solution was kept
for experimental solution after the extraction.
Color was
developed using Scott et al method 34 which utilizes anthrone
and absorbanqy was measured at 625mu.
Galactose was used
for a standard.
i) Sphingosine nitrogen:
The Mckibbin, Taylor method 35 was used. Lipids were
hydrolysed with a saturated Ba(OH) 2 solution and it was further
hydrolysed using 1N HC1 solution. Sphingosine was extracted
from the resulting solution and its nitrogen content was
determined using the micro-Kjeldal method.
4) Methods for calculating compositions.
-
Each one of the deterMined values for the compounds
mentioned above was divided by its molecular weight and the
number of moles for each compound per 100g of acetone-insoluble
lipids was obtained.
The number of moles for each compound in
the lipids was calculated according to the idea mentioned in
the introduction.
Each value was multiplied by the assumed
molecular weight of each compound to obtain the weight in 100g
72
25.
of the lipids and the results were shown in terms of percentages.
Average molecular weight was used for calculating
the assumed molecular weight of fatty acid.
(3) Results and Discussion.
The analytical results are shown in Table 3-1.
The results of the number of moles calculated in 100g of
acetone-insoluble lipids are shown in Table. 3-2.
The number'
of moles for each ingredient calculated from Table 3-2 is
shown in Table 3-3.
The weights of each ingredient calculated
from Table 3-3 are shown in Table 3-4 and their percentages
are shown in Table 3-5.
•
There is a direct determination method3 6 for
determination of the molar concentration of cephalin by
determining ethanolamine and serine but the molar concentration
was obtained by calculation in this experiment.
This method
consisted of subtracting the weights of 4 ingredients obtained
from total nitrogen, total phoSphor, total glycerine and total
fatty acid groups from the.' total weight of the ingredients and
the remaining weight was assumed to be the weight of cephalin. The average values were used and the results are shown in
Tablé 3-6.
The values of lecithincholin shown previously are
believed to be the sum of lecithincholin and plasmarogencholin
but since there is no quantitative method for separating these
two, it was calculated as cholin from lecithin. However, since
each one of the compositions had a total amount of ingredients
,
•
26.
over 100g, it was rectified that the salt group of plasmarogen
is made up with cholin and the results are shown in Table 3-L.
The error arising from this type of calculation is
believed to be concentrated in the division of cephalin but
since the value obtained by multiplying molar concentration
by molecular weight came out to almost 100g, it is . believed
that there_is no appreciable error in the estimated values.
When there is a lyso form present, which is the
glycerylphospholipid from which one molecule of fatty acid
is removed, the amount of total fatty acids in acetone-insoluble
becomes higher since the fatty . acids « separated from the lipids
stay with the acetone insoluble matter, when compared to the
result which has no lyso form. As a result of this, the
cephaIin calculated from the total fatty acid becomes smaller.
However, there, is no confirmation of the lyso form present
from the table. One could see, however, the presence of
lysolecithin when the experimenal material was separated
with thin layer chromatography (silica gel 5B Takeda was used
for coating and chloroform-methanol-water-acetic acid
•
65:35:4:1 ratio was used for solvent), in order to study the
73
lipids which contain cholin, in both fertilized and unfertilized
'eggs.
•
The investigation re the presence of lipids which
contain serine as a salt group was carried out with unfertilized.
eus using the Nojima et al method. 36 Ethanolamine and
serine were investigated together but there was no serine
2 7.
extracted. Since there was no investigation carried out
with fertilized eggs, the appearance of serine during
development of the eggs was not known.
The changes in relative content of acetone-insoluble
lipids mentioned in the previous chapter during development
are shown in Fig. 3-3.
The changes in the content of five
compounds mhich made up the acetone-insoluble lipids during
the development are shown in Fig. 3-4.
The amount contained
in unfertilized eggs was taken to be 100g.
The following points can be observed from Fig. 3-4.
i) There is a parallel relationship between the decrease in
the amount of acetone insoluble lipids and the amount of
lecithin and cephalin. A decrease in lecithin can be seen
before hatching but the amount of lecithin remains almost at
a constant level after hatching. Cephalin on the other hand
continues to decrease even after hatching. Both of these
compounds contain two molecules of fatty acid in a compound.
It is believed that these compounds are used as the basis
for energy before the hatching. Although it is only
qualitative, the fact that there is some lysolecithin present
shows the presence of an enzyme which frees fatty acid from
lecithin.
The assumption can be made on the fact that the
freed fatty acid becomes one of the basic ingredients for
respiration of the fetus. However, there has been no
investigation carried out on the presence of such an enzyme.
Further, there is no research article saying that there is
28.
such an enzyme present. Ohman37 has shown that one can
obtain a standard fertilized membrane by reacting lecithinade
which he obtained from bee poison with unfertilized eggs,
75
using sea urchin eggs. It is believed that the above
phenomenon occurs as a result of fertilization in the case of
rainbow trout eggs.
ii) The amount of sphingomyerine is small Compared to the
rest of the compounds. It decreases rapidly at the time of
hatching.
The large changes occurring during the hatching
are the breaking of the egg capsule and the loss of the
perivitelline fluid.
There is no confirmation of the fact
that these materials contain phospholipids up to now but in
this experiment, it is believed that there is some relationship.
iii) The plasmalogen increased between fertilization and
appearance of the eye but it decreased rapidly after that.
There is a study by Thiele 38 on changes of plasmalogen during
the development of chicken eggs. It shows the increase in
the amount of plasmalogen after fertilization.
The reason for
the increase is said to be due to lecithin. Up to now only
•
plasmalogen present in the brain is said to possess cholin
as their base. For this reason it is correct to assume that
plasmalogen in rainbow trout eggs too contains cholin as its
base.
iv) Cerebrosides belong to the carbohydrate lipids. Its amount
decreases rapidly at the time of fertilization and almost the
total amount has been spent at the time of hatching.
In this
29.
experiment, only galactose was determined as carbohydrate
but the carbohydrates other than galactose are known so
the determination in this experiment cannot be most accurate.
However, the . fact that almost all the carbohydrate lipids
are spent at the start of the development shows that the
lipids containing carbohydrate are spent firét for energy
for development.
This parallels with the Tact that the
order of compounds Used as development energy sburce is
hydrocarbon-lipid-protein. So even in lipids, one containing
carbohydrate as a base is spent first, and the order as
mentioned above is believed to exist.
IV. Changes in fatty acid composition of lipids during the
-development of rainbow trout eggs.
(1) Foreword.
Several chemical changes in- acetone insoluble
lipids were determined in the previous studies of rainbow
trout eggs during their development.
The assumption was made
from the degrees of unsaturation and average molecular weight
of fatty acids, that the changes occur among the unsaturated '
fatty acids in lipid containing fatty acids. Also it is
believed that the changes occur in the fatty acid structure
.
in the combined lipids from the composition changes of the
lipids.
The purpose of this experiment is to study the
changes in fatty acids composition of glycerides and the
fatty acids composition of phospholipids which are the main
ingredients of acetone-insoluble matter and to know how
30.
important these compounds are during the development.
There are only a few reports on the fat composition
of fish eggs. .Especially concerning the development, there
are only- studies by Lovern 39 on the changes of fatty acid .
composition during the development of salmon eg7s and Lasker
et alW on the changes of.fatty . acid composition and the
relationetip between the feed and fatty acid . composition
in sardine eggs obtained froffi the Pacific Ocean. There is
no study on the .,continuous development after fertilization
of fish eggs. There is only a study by 3chjeide et al l° on
the fatty acid composition of liver cells during the
development of chickens.
• It was made clear with many sup 12orting . studies
that the fatty acid composition of animal fat is generally
affected by the composition of fatty acid in the feed oil.
It is believed that fat taken in by an animal is decompOsed
into fatty acid and glycerine or fatty acid and mondglyceride
inside the digestive organs under the influence of an enzyme
which has fat decomposition properties, and the compounds
decomposed are again converted into fat inside the intestinal
wall and sent to the storing areas by the blood.
For this
reason it is obvious that . the fat in feed has a large
influence on fish eggs, which obtain fat from the parent fish
while the eggs are developing inside the fish.
There is a
study illustrating this point. 4. 0 However, lipids in eggs
which are ejected from the parent's body have no way, other
31.
than from the sperm, in which they can obtain a further
supply of fat.
Lipids in the egg are spent as a source of
development energy and also as a basic structure for the
formation of body lipids in a young fish.
How do the fatty
acids stored in an egg while it was under the influence of
feed oil during the process of maturation of the oocyte,
show decrease due to spending during the development of the
egg?
If there is a fatty acid especially needed for the
development, unrequired materials should remain behind.
Furthermore even the fatty acid not required at the time of
development, which thus remained unused may be used during
the time between the swim7up-fry period and when the young
fish first feed on their. own.
The question arises for each
one of these points mentioned above.
This experiment will
take these points into consideration.
(2) Experiment.
1) Materials:
The rainbow trout eggs used for this experiment were
collected in December of 1965 from the hatching grounds
belonging to Tokyo Fisheries University.
Several three
year old fish were used as sources of eggs. Eggs with a
diameter of 5.5mm were used.
Part of the - collected eggs
were left unfertilized and the rest were fertilized and
brought back to the lab. The development of the eggs occurred
as mentioned in the previous chapter.
50 eggs were taken out
of the container every 5 days and extraction of lipids was
76
32.
carried out.
•
2) Extraction of lipids, purification and separation.
Extraction of lipids.
Purification of lipids was done according to the
method used in the previous chapter, i.e. acetone extraction
followed by Chloroform-methanol (2:1) extraction and further
purified by using the Floch et al method. • There was a
requirement for complete separation of neutral fat and
complexed lipids since this experiment was directed at
studies of fatty' acids which make up both lipids. Thin
layer chromatography was used in this experiment.
The
disadvantage of this method is that it takes a.relatively
long period of time and therefore the unsaturated compounds
are in contact with air for a long time. For this reason
there is always . the fear of unsaturated compounds becoming
oxidized.
In order .to avoid this, the air in the container
was replaced with nitrogen and an antioxidising agent,
butylated hydrooxyanisole, was added to thé material to be
separated in TLC. Since the antioxidant vaporizes at 100
degrees C, there is no danger of this compound interfering with fatty acids while carrying out gas chromatography.
TLC was carried out according to Stahl.
That is,
"Waco-F,eri B5 (silica gel containing.5% adhesive) waS
spread over the surface of a 20 by 20cm glass plate at a
thickness of 300um. The plate Was then dried and activated
at 100 degrees C for 30 miniltes. The sample was applied to
33.
one end 2cm from the bottom and developed with pet-ether
(bp. up to 60 degrees C)-ether-acetic acid (90:10:1).
When the - solvent front moved 18cm (developing time of one
hour) the plate was removed .from the solvent container and
it was sprayed with rhodamine 6G (0.05% in ethanol) and the
positions for triglycerides and phospholipids were determined.
The compounds were scraped Off and the forMer was dissolved
and extracted with pet-ether-ether (85:15) and the latter
was dissolved and extracted with chloroform-methanol (2:1).
The ingredients of lipids obtained from the above method were
cholesterolester, triglyceride, free fatty acid, free
cholesterine and phospholipid which was found on the original
spot. The'order of the above mentioned compounds is from the
solvent front on downward.
Extraction solvents . were removed . under reduced
pressure. Triglycerides were added to about twice the
amount of benzol and 4 times the amount of 1% methylsulfate
and both compounds were esterified for 5 hours on a water
bath under refluxing condition while bubbling nitrogen
through the sollition.
After termination of the esterification
process, the solutions were transferred into separatory
-
funnels with a large amount . of water. Esters were extracted
with pet-ether and washed with water until the washing solvent
was neutral to methylorange.. The pet-ether ester solution
was dried with sodium sulfate and a:large part of the solvent
was removed.
The remaining solution was set aside for analysis
3,4
under nitrogen atmosphere and at a temperature of minus 23
degrees C.
3) Analyses of fatty acid composition.
Analyses were done with gas chromatography.
The
instrument used in this experiment was a Shimazu model GO -1C
and f011owing are the particular details.
Column:
3mm inside diameter, 3 U-shape columns 375 mm in
length, one straight column 2,625 mm in length, made of
stainless steel.
Detector: Heat conductance model detector.
Carrier gas: . helium.
Rate of flow:
40m1/min. . •
Column pressure:
2Kg/cm 2
Column temperature:
77
205-210 degrees C.
Detector temperature:
250 degrees C.
Temperature of vaporization chamber:
275 degrees C.
Recorder: 'Electron tube style automatic recorder.
BridFe . current: 80mA
'Chart speed:
5mm/min.
•
Amount of material injected:
0.5-1u1 (no solvent included)
”Shimaliten 80-100 mesh (manufactured by Shimazu Co.) was
used for the packing.
The liquid phase was made by converting
diethyleneglycol and succinic acid into diethyleneglycolsuccinatepolyester using the Ito et al method, 43 The packing
material was covered with 15% (v/W) of the above liquid before
use.
35.
The column was changed since the separation of
stearate and oleate became gradually worse.
The column ,
was changed when the ratio of retention time of stearate/
oleate became 1.05.
The determination of Peaks was done by comparing
them with standard peaks.
The standard used for this
determination was obtained by esterifying commercially
available lauric acid (C12) to C
acid (all even-numbered
22
carbon atoms) to obtain methylesters of corresponding acids
and distilling under reduced pressure to purify the compounds.
The standards Were dissolved in hexane before they were used
as standards.
Among the standards for unsturated fatty
acids, standards for oleic acid, linoleic and linolenic
acids were prepared by using the urea adduction process.
It
was difficult to obtain other unsaturated acids recuired
for the standards in this experiment commercially, therefore
methylester of mixed fatty . acids of triglycerides in
unfertilized eggs was treated.with mercuric acetate according
to the Jantzen, Andrea method" so as.to convert unsaturated
acid esters into the mercury adduct product. The mixture was
then separated into acid esters and unsaturated acid ester
mercury compounds using TLC according to the Manfr,old et al
method. 45 The latter was further divided into one double
bond (monoene), two double bonds (diene) and three'double
bonds (triene). Each spot on TLC was scraped off.
Saturated
acid ester was extracted- with pet-ether-ether (50:50) solvent.
36.
Unsaturated acid ester mercury adducts were extracted with
5% HC1-methanol solvent, and ester was recovered by adding
water to it. The compounds were separated using the same
column and hydrogen flame ionization detector and the peaks
were compared with peaks on record for mixed fatty acid esters.
Furthermore each peak was relatively determined while
comparing to known peaks for C 1 8 series.
The compounds
separated with the above procédure were used for standards.
It was confirmed that there is a straight line relationship
between retention time and number of carbons in a compound 46
saturated fatty acids, monoene and diene series.
on
The
compounds which could not be determined by gas chromatography
due to overlapping of peaks were analysed using the above
mentioned method. When the diethyleneglycolsuccinatepolyester .
column was used, the retention time for the compound with the
larger number of double bonds is said to be longer than for
the compounds with more carbon atoms but a smaller number of
double bonds.
It is also said that the separation of C 18:3
and C 20 . 1 is-not possible 47 but the separation of C
and
0 20 and the separation of 0 20:5 and C 22:I were not'possible
in this experiment.
'As a supplemental method for separation
of compounds which were inseparable by gas chromatography due
to the overlapping of peaks, mixed esters were hydrogenated
using pladium black as a catalyst and oxidative - removal of
unsaturation in acetone was carried out with potassium
permanganate. The degree of unsaturation was obtained for
37.
esters with the same number of carbon atoms from the above
experiment and from the ratio of unsaturation obtained, the
calculation was made and the distributions of compounds were
made.
The record for each compound was transposed onto a
thick copying paper and peaks were cut out. The ratio was
obtained according to weight determination' and the results
are shown in percents.
The compounds with contents below
0.1% were discarded. Furthermore it has been said that the
sensitivity of the detector becomes low when the number of
,
double bonds increases or the number of carbons increases; 48
however, since a heat conductance type detector and helium
•
gas as carrier were used in this experiment, there was no
need for correction of the results obtained.
78
(3) Results and Discussion
The changes in composition of fatty acid compositions
in triglycerides and fatty acid compositions in. phospholipids
during the development process are shown in Table 4-1 and
Table 4-3.
Since only the composition of fatty acids is
shown and the total rise and fall was difficult to determine
from these data, these data were combined with the rise and
fall of acetone-soluble lipids and insoluble lipids obtained
in chapter 2, the changes of fatty acid which was originally
contained in the unfertilized eggs were calculated and the
results are shown in Tables 4-2 and 4-4.
38.
It is quite difficult to match the results of this
experiment concerning triglycerides and phospholipids with
those of chapter 2 since the methods used were different,
but since the main ingredient of acetone-soluble lipids
was triglycerides and furthermore the amount of fatty acid
in acetone insoluble part was about the same in both cases,
it is believed that there is no large error involved.
The following points became clear from the experiments.
1) Differences in fatty acid composition of triglycerides
and phospholipids.
The relative ratios of fatty acid composition in
triglycerides and phospholipids of unfertilized rainbow trout
egg by their unsaturation obtained from Tables 4-1 and 4-3
are 'shown in Table
Remarkable différences can be seen on saturated,
81
monoene, diene and hexaene acids. Generally the amounts of
saturated and unsaturated fatty acids in phospholipids of
land animals are the same° but in fish the amount of
saturated acid in phospholipids is about 20%, like the
amount of fatty acid in triglycerides. The amount of
saturated fatty acid in rainbow trout eggs was higher than
'average, at 30%. Saturated fatty acids, low unsaturated
fatty acids and highly unsaturated fatty acids were about
the same at 1/3 of the tôtal content for each group. On
the other hand, the monoene content was 50% of the total in
triglycerides.
•
39.
If one compares individual fatty acid composition,
one can obtain the results shown in Table 4-6. The main
ingredients for saturated fatty acids in triglycerides and
phospholipids are C 1 6 acids.
The amount of C
in phospholipids is muéh higher•than in triglycerides.
acid
Unsaturated acids in triglycerides are mainly made up of c 16:1 ,
and ..C 1
and highly unsaturated acids such as C
C 18 •1
226
were found mainly in phospholipids.
Generally the unsaturated fatty acid composition
of body lipids for fresh water fish is mainly made up of
C
and C
. There is some• C
20 acid but it is said that
16
, 18'
21
there is no C
However, rainbow
22 acid in the lipid.
trout eggs, although they are from fresh water fish, contain
over 10% of highly unsaturated fatty acids such as C 22: 6•
If one observes the rainbow trout eggs one finds that .
their composition is closer to that of sardines or salmon
from salt water. As shown in Table /4.-7, since the feed
used for this experiment was mainly made up of Isaza,
it is believed that there is some eUect of its linid
•
fatty acids on the composition of eggs, however, the amount
of highly unsaturated fatty acid compositions in triglycerides
and phospholipids are:
Triglycerides C 2 0:5 5%' C 226 1 3 °3
'
'
Phospholipids C 205 5%; C 22:6 25 %
'These results are almost the same as the résults from
this experiment's analytical values. It is therefore
believed that there is no effect from the fatty acid of
Isaza, but that the effect is related to the feed oil added
to the feed system.
Among the feed oils, there are many species
belonging to fish liver oil residues. ,The analytical values
for these are shown in Table 4 7. Even with the eggs which
-
received the influence of these oild, the composition of
both lipids of rainbow trout eegs is considerably different
from the others.
It is believed that the parent fish store
the fatty acids required to develop the eggs selectively.
2) The decrease in the fatty acid composition. of triglycerides during development.
As shown in Table 4-1 and Fig. 4-2, the fatty acid
compositions in triglycerides of rainbow trout eggs • are
made up of 23 peaks and 25 ingredients.
The composition is .
as complicated if not more complicated than that of any other
fish oils.
The majority of these ingredients remain in the
body up to the swim-up-fry period. C21 1»: 2? in the table
was estimated from . C 2:0
Which was made clear by hydrà4
genation and calculated, assuming that the compound overlaps
with C 22:6 •
The changes in main fatty acid composition (
equivalent) of 100g of mixed fatty acids of triglycerides
during develôpment of rainbow trout eggs are shown in Table
4-2.
As shown in this table, there is no change in fatty
acids other than C 18:2' 0 20:5 and
022:6 fatty acids, up
4 1.
to the time of appearance of the eye. '411en the eggs enter
the latter part of appearance of the eye, the young fish in
the eggs start the body movements. From this time on the
gradual decrease in fatty acids other than C 1 8 :0 starts.
It is believed that triglycerides are spent as sources of
energy for the movements. After hatching, the movements of
83
the young_fry become rapid and therefore the decrease in the
amount of fatty acids becomes considerable.
As mentioned in chapter 2, there are large decreases
. in the amount of fatty acids other than C 18:2 , 0 20:5 and
0 22:6 fatty acids which : increased during the • ame time.
There must be some special reasons for the increase in
the'above mentioned acids. There is not enough reason to
say that highly unsatùrated fatty acids form during the first
period of development. It may be due to the transformation
of the part of glycerylphospholi .Pids into glycerides since
às shown in Table 4-4, the above mentioned fatty acids in
phospholipids show a large decrease and as shown in the
•
previous chapter, lecithin and cephalin show a considerable
decrease during this period.
The order in amount of fatty acids spent up to the
swim-up-fry period is as follows: C is : , > 0 161
> C160 >
18:2> C 226 > C 20:5 >C14' C 18:0 shows a slight increase.
It was reported previously that lipids are manufactured from
.yolk protein, 51 therefore it is believed that there is time
for synthesis of fatty acids. It is not possible to determine
42.
the order of importance of fatty acids from these tables,
hoWever, it is clear that the above mentioned fatty acids
become the basis of energy for the movements after the
hatching.
3) Disappearance of fatty acids in phospholipid compositions.
Table 4-4 shows the disappearance of main fatty
acids by eombining the results from Table 4-3 and the results
of changes in the amount of phospholipids from chapter 2.
As shown in this table, the degree of disappearance of fatty
acids is C 226
C 205 > C 201 . C 22:6'
C 20:5 and C18:1 show slight-increases after hatching.
It is assumed that synthesis of fatty acids is carried out
as mentioned previously, during the developing stages
after hatching, since the internal organs of young fish .
are gradually developing. Since the fatty acids compobition
in triglycerides shows a rapid decrease, it is believed that
triglycerides may be converted into-phospholipids as a contrary
action to the process at the beginning of the development
process. The results showing the comparisons on disappearance
of the fatty acids of triglycerides and phosPholiPids are
shown in Fig. 4-1. ›
As shown in chapter 3, since lecithin, cephalin, etc.
which are glycerylphospholipids,.hold thé greater part of the
total spending of phospholipids, the main fatty acids which
make up the lipids in rainbow trout eggs are C 226 ,. C 16:0'
18:1 and C 18:0 . Fatty acids of phospholipids in fish contain
L3
.
a larger amount of long chain highly unsaturated fatty acids
than those in land animals. 52 It is reported that
3e
of
the lecithin in matured river trout is made up of 0 22: 6. 53
It is believed that the large concentration of highly
unsaturated fatty acids is characteristic
in fish.
•
of phospholipids
It has already been shown in chapter 3, that there
is a large, amount of 0 24 fatty acids obserVed in unfertilized
eggs and they decrease to an undetectable amount by the eye
appea.rance period and increase again at the latter part of
the development period. This can be compared to the disappearance of carbohydrate lipids which are said to contain
C 24 fatty acids.
(4)
Summary
If one assumes the order of importance of fatt y
acids spent during the development period from the above
results, it becomes as follows:
C 18:1 -> 0 22:6
0 16 >016:1>
However, it is believed that there is no selective
C18:2*
spending of fatty acids as mentioned in chapter 2 and as
further showri in Tables 4-1 and 4-3, there are no large
changes in the composition ratio of fatty acids at each
stage of the development. This evidence supports the previously
advanced theory that there is no selectivity in the spending
of acids. There is a report on the' effect of oil in the feed
system of parent fish on the fatt ÿ acid composition of the
eggs. 4° As far as there is disappearance' of fatty acids as
shown in this experiment, there is a need for addin7 fat which
44.
contains large amounts of fatty acids to the feed.
The composition of added oils used for this purpose
at present is shown in Table 4-8. These are not enough for
the formation since they are used separately. There is a
need for suitable distribution of oil in the feed.
It is
known that oxidized oil has some bad effects on several
nutritiong. 54 For this reason,- if one wishes to add highly
unsaturated fatty adidsi there is a great need for care to
avoid oxidatiOn.
V. Ultracentrifugal studies on yolk protein of rainbow trout. 55
(1) Foreword.
The disappearance of acetone-soluble and insoluble
lipids was studied up to the previous chapter using rainbow
trout eggs.« It was learned from these studies that there
were no appreciable changes in lipids before hatchine but that
they were spent quickly after'hatching. It was also learned .
that acetone-insoluble lipids were decreased to about half
during the development stages up to hatchine.
It was already stated that the composition of fish
egg is mostly yolk protein and lipids if the water is removed.
There are small amounts of carbohydrate and inorganic matter
also contained in the composition.
Lipids are present in
liquid oil form and in combined form as lipo-protein. This
lipo-protein makeS up the main part of yolk protein.
contains phosphor.
It.
It is very important for the development,
as a source of energy and as a source of body protein. It was
11 5.
-
shown by Smith 57 that it is difficult to study these
phenomena mentioned above through chemistry alone.
For
this reason, there is a need for physical studies using
new techniques: There are studies by Young et al 58 on
yolk protein of Atlantic salmon and by Monroy 59 on the changes 83
of egg protein before and after the fertilization of sea
urchin eggs using electrophoresis. There are studies by
" 61 on
Cook et al 60 and by Sugano
chicken egF yolk protein
using centrifugal analysis. A study on frog eggs was
reported by Schjeide. 62
However, there is no study concerning
the changes in yolk proteinduring the development of eggs,
using a physical technique, as far as dan be seen.
•
In this chapter, the main ingredients' several
physical characteristics were made clear by studying how
the egg yolk protein'S ultracentrifugal composition goes
through changes during the period between fertilization
and young fry and further by studying.to see 'whether there
is a new compound appearing or not, and combining these
results.
(2) Experiment
1) Materials:
Rainbow trout eggs, both fertilized and'unfertilized,
were obtained from the Kaizawa hatching grounds at the
beginning of January 1962.
The fertilized eggs were developed
in the laboratory as shown in chapter 3.
(Fig. 3-1) Eggs
.used for the experiment were taken out every three days.
14, 6.
They were washed with phosphate buffer solution (pH 7.0 and
ion strength of 0.2) and the contents of the eggs were pushed
out into a precooled phosphate buffer solution by breaking
the membrane. The material produced by the above technique
was placed dn . a centrifuge at 10,000 r.p.m. for 20 minutes
at a temperature of 0-5 degrees C.
The • ight yellow clear
solution obtained from the middle layer waà used for the
analyses.
2) Analytical Method:
The centrifuge used for this experiment was a SPinco
.ultracentrifuge E model_with 59,780 r.p.m.
For the sedimentation -
coefficient, Svedberg unit Sw.20 was used.
The viscosity and
density of the solution which were needed for determination of
the sedimentation coefficient were measured using the Oswald
viscometer and. picnometer'reSpectively.
The values obtained
. at a teMperature betweeri 10-20 degrees .C. were shown in the
graph and they were used for the calculation.
For the partial
(literal translation) specific volume, the usual value of
0.750 (20 degrees C.), which ds generally used for protein,
was not used, since the main ingredients of yolk protein are
combined with lipids. Instead, the value of 0.771 (20 degrees
.
C), obtained by actually measuring it with the picnometer, was
used.
The viscosity and the density of water were obtained
from the constant table. ".
The determination of the diffusion constant which
was needed for deterMination of the molecular weight was done
•
4.7.
on a Hidachi HTB-2 model with NOilarts (literal) diffusion
cell. The molecular weight of the compound was obtained
by combining the value obtained above with the sedimentation
coefficient or sedimentation constant. The concentration of
protein required for the above determinations was obtained
by determining the total nitrogen content with the microKjeldal methoa; 25and multiplying the value by 6.25.
Quantitative determination of phosphor was according to
Allents color determination metbod. 26Total lipids were
determined by the Folch method 63which'uses chloroformmethanol (2:1): for extraction. Relative concentrations of
ingredients of yolk protein were determined from the areas
of each peak on the sédimentation pattern.
(3) Results and Discussion.
1) Discussion on the ingredients of yolk protein.
1.5% solution of unfertilized egg yolk ingredient's
ultracentrifuge pattern is shown in Fig. 5-1. As shown,in
the figure, there are two peaks.
The number of peaks
remained the same even though the concentration was increased.
The sedimentation coefficient of the main peak (ingredient I)
•
obtained from 1.5% protein Solution throughout the development
'period was 8.6 ± 0.4 3 .
The sedimentation coefficient at zero
concentration of protein was 9.4 ± 0.4S. '(Fig. 5-2).
The
sedimentation coefficient of ingredient II which had slower
sedimentation values than ingredient I was 3.1 ± 0.2S.
A -
sedimentatiOn constant for ingredient II could not be obtained
since the peak flattened out as the concentration of protein
became low.
The ratio of ingredients I and II calculated
from the area of peaks in *unfertilized eggs was 90:10.
Ingredient III which sedimented quicker than
ingredient I appeared as a shoulder after the appearance
of the eye stage as shown in Fig. 5-1(2).
coefficient was 11.2 ± 0.3S.
Its sedimentation
The sedimentation constant
could not be obtainèd as in ingredient II, since the peak
flattened out with low concentration of protein.
From the above statements, it is believed that
ingredient I is the main composition of yolk protein and
important in the development.
For this reason an attempt
. was made to Separate ingredient Ï to know several physical
and chemical properties, but.separation of ingredients I
-
and II was not possible. The method used for this separation
was dialysis of protein against water with 10% NaC1 solution
in the presence of ether and dilution of the protein so
obtained with phosphate buffer solution. The separat ion
a Hidachi centrifuge was also carried out without using
success. The sample was treated in the centrifuge machine
for 10 hours at 40,000 r.p.m. and the upper, middle and lower
slayers were taken and it was tried to analyse ingredients I
and II but this process too failed.
The sample was divided
into ingredient II and ingredient I containing a small amount
of ingredient II uSing a separation cell, and several
determinations were done on each part.
49.
The mole ratio of phosphor to nitrogen in
ingredient II was 1:8 and in ingredient I 1:20.
The value
for ingredient I was closer to the value obtained by
Chargaff 65on lipovitelline which had a ratio of 1:19.
The sedimentation constant of lipovitelline was shown by
Jaubert et al 66 to be 11.1S. The sedimentation constant of
a-lipovit ■alline was shown . by Sugano to be 20 3. b-lipovitelline on the other hand had values of 123 and 3.9S,
made up of two peaks.
The total lipid content of ingredient
I was 20% which corresponds to the amount of lipid in
•
, 67The reason for
lipovitelline which is 20.270.
the difference
between the sedimentation . coefficient of ingredient I and
lipovitelline from the chicken eggs, although there are
similarities in the values obtained by chemical analysis,
is believed to be due to the difference in shape and size
of the molecules.
The determined value for the diffusion constant for
ingredient I, under the assumption that ingredient II which
is mixed with ingredient I does not change the diffusion .
constant too much, since the content of ingredient I is
above about 90% of yolk solution of the unfertilized ego's,
is 4.1 ± 0.2 (fig. 5-4).
This is almost the same as the
diffusion Constant for lipovitelline, which is 3.81.
The
molecular weight calculated from the sedimentation constant,
9.4 3 and the diffusion constant, 4.1 was 240,000.and the
axle (axis) ratio (literal translation) was 1.2.
The molecular
weight of lipovitelline is 260;000 if 3.8 is used as the
50.
diffusion constant and the axle ratio is 1.3 .. As can be
seen from the above data, there is only-a small difference
between thé result of ingredient I and the lipovitelline.
A value which is more accurate than the above could not be
obtained since the complete separation of ingredient I was
not possible but it is believed that there is no large error .
in the reaults above.
The approximate molecular weight of
ingredient I is much smaller than the value obtained by
Bernardi et al 67 for a- and b-lipovitelline which was
listed as 400,000.
The Thosphor content Of ingredient II was much
86
higher than that of ingredient I. However, the mole ratio
of phosphor to nitrogen was 1:8 and it was much below the
value for PHOSBITIN (unable to confirm this expression) lelich
was 1:27. 68,69 Furthermore the sedimentation coefficient of
ingredient II was 3.1 5 and it is close to the value shown by
Bernardi et al 67 for lipovitelline in chicken egg yolk
which is 3.4S and the one shown by Sugano 61 which is 3.9 5.
.Flichinger et al 70 have carried out ultracentrifugal analysis
on yolk of frog eggs and obtained two ingredients represented
by 11S and 6S.
However there were ingredients which quickly
. sedimented and had a sedimentation coefficient which is siMilar
to ingredient I of the rainbow trout and'there is another
which had a sedimentation coefficient about twice that of
ingredient II of rainbow trout. It is very difficult to
discuss ingredient II any further unless a large amount of
•
51.
ingrPdient II can be separated.
2) On alkali denatured ingredients I and II
The test solution for unfertilized egp.;s was diluted
with caustic potash carbonate buffer solution to the
concentration of 1.5%. This solution was used at various
hydrogen ion concentrations and the changes in the ultracentrifugal .pattern were investigated. Results are shown
in Fig. 5-1(3).
Curve lines C, t
,
E, F are obtained by
superimposing the picture obtained 30 minutes after the start
of the revolution and bar angle of 65 degrees. These lines
show the changes of ingredient I and II - clearly. Line C
showing ingredient I at pH 7.0 shows no chan7e after 4 hours
of pH adjustment and a pH of up to 11 but between a pH. of
11 and 12, the appearance of ingredient IV which has slightly
slower sedimentation can be seen. There are an increase of
ingredient IV and a decrease of ingredient I to be observed
at a pH over 12 (curves D, E). At pH 13 and after 24 hours,
ingredient I has completely disappeared and only ingredient
IV iS present (curve F)..
The sedimentation coefficient of ingredient II
becomes smaller gradually when the sample is made alkaline but
no change can be seen similar to that of ingredient I.
Since there is no other peak to be seen and curve F.
is symmetrical when the curve shows only ingredient IV, it ià
believed that.the sample.contains a single compound ultracentrifugally speaking.
The sedimentation coefficient of .
52.
protein at a concentration of 1.5
is 4.9
0.53 but the
value for ingredient I was 8.6 3 which is slightly lower than
half the value for protein.
From the above observation, ingredient I was divided
into (a) two subunits by alkaline denaturation. (b) 2 and 3 .
dimensional structures of ingredient I were broken up and
sphere-like protein showing an axle (axis) . ratio of 1.2 was
unravelled and became, coil-like and was converted into
ingredient which sedimented slower than the original protein.
Two points above are conceivable. (b) phenomenon seen from
ultracentrifugal pattern can be shown by the slow movement
of the normal curve. 71 Further, in the case of phenomenon (a),
one peak moved to the other without going through the middle
peak position.
In Fig. 5-1(3) curve C changes without creating
the intermediate stage, therefore it is believed that
phenomenon (a) is acceptable. This idea can be confirmed
by determining the molecular weight of ingredient IV which .
was produced from ingredient I:by alkali denaturation of
ingredient I and comparing the value '‘,dth the one that
belongs to ingredient I.
Ingredient I which was converted into ingredient . IV
by keeping ingredient I at a pH of 13.0 for 24 hours.
This
ingredient had a diffusion-constant of 4.2 ± 0.2 (Fig. 5-4).
The calculated molecular weight using 4. 9S as sedimentation
coefficient 72 was about 120,000. The axis ratio of this
88
53.
ingredient was 1.5. From these data it was concluded that
ingredient I was made up ,of two subunits. This fact
coincides with.the Bernardi et al 73
observation on lipo74
'
vitelline in chicken egg yolk which was divided into two
subunits with the aid of alkaline or 4mole urea. However,
it is believed that the resistance against alkali varies
with the animal because chicken egg ingredient showed first
separation at a pH of 9.0 whereas rainbow trout egg. did not
start the separation until the pH reached 11.0; Ingredient II
is believed to be phenomenon (h) since it is different from
ingredient I and the sedimentation coefficient gradually
decreased and with the conditions when ingredient I was
completely changed to ingredient IV, it showed a sedimentation
coefficient of 1.4S.
3) Changes in thé ingredients.of protein during the development.
10 eggs per 20m1 of phosphate buffer solution
were used as material for studying the changes of yolk
protein ingredients during the development.
This number
was increased as the yolk sucking was advanced.
The
quantitative Changes in each ingredient during the development
were shown by the areas of peaks of the ultracentrifugal
pattern.
Furthermore, weight equivalent to about 10 fry wei-e
used for the analysis of the hatched samples.
are shown in Fig. 5-3.
The results
The total amount of yolk protein was
shown by the total area of each ingredient. Further, since the
-
progress of development in this study was the same as in the
5h.
previous,studies, their conditions were shown above the
figure.
Different up and down aspects of ingredients I
and II during the development-are shown by the curves
and especially the following points became clear.
(a) Ingredient II shows its maximum amount at about 10 days
after the notochord of the fish becomes clear and it is
opposed to the decreasing period of ingredient I.
The same
trend was obtained on a different group during the
development. Genetically the period between fertilization
and this period is the period just after the egg injury
(literal translation) and blastula and therefore it is a
prosperous period for the differentiation of cells.
For
this reason, this period requires more ingredient I than II
as a source of differentiation energy or for the ingredients
required for the changeover to body protein.
(h) Ingredient I decreases rapidly after hatching.
This is
the period when small fry swim out into the world, breathe
freely and all the metabolism becomes prosperous. There is
no change in ingredient II at this time but ingredient I is
still important even at this stage.
(c) ingredient III appears after the èye appearance stage and
the amount remains unchanged. Ingredient III is believed to
be the protein in the blood since at this stage the
appearance of small blood vessels on the surface of the egg
membrane occurs and the blood circulation starts in the latter
55.
part of this stage. Schjeide 62 has reported the appearance
of new protein in the blood of froF eggs at the time of
tail appearance.
is 16S.
This new prOteinTs sedimentation constant
The sedimentation constant of ingredient III was
11.2S and it is smaller than the above value but this
ingredient is related to the new protein shown by Schjeide.
(4) Summany
The following things car be observed if one combines
the results of this chapter and the rise and fall of acetoneinsoluble and soluble lipids from the previous chapters.
The fetus uses phospholipids which are combined with yolk
protein to shape the body of fish during the period between
fertilization and the eye appearance period. After the
eye appearance period, the fetus uses mainly yolk combined
with triglycerides to complete the body.
During this period,
lipids combined with protein and liquid lipids are used for
various energy sources and at the same time part of the
lipidS are changed into body lipids.
The molecular weight
of protein bonded to yolk is about 800.
It is rather small
compared to the molecular weight of protein and there is
no difference ultracentrifugally therefore separation could
not be accomplished even if there are structural differences
in the compounds.
VI. Influence of feed oil on the fat composition of body
oil of young,fry.
(1) Foreword
The modes of consumption of fatty acids in lipids
a
•
56.
stored up in rainbow trout egps by the parent fish up to
the start of development, species and composition of fatty
acids are made clear from the previous chapters. It is
concluded that it is quite probable to formulate the oil in .
.feed if the added oil causes some effects on the composition
of fatty acids, and thus improve several feed systems
preSently_in use.
Many studies confirmed the effect of feed oil on
the composition of body oil in the past. There are species
more difficult•to be influenced by the oil in the feed and
there are also speciès easily influenced by the feed oil,
among the animals. Fish is believed to belong to the
species which are easily influenced by the feed oil.
There are studies by Tujimoto 75 , Oshima 76 , Lovern 77 on
eels, Kelly et al 78 on North American fresh water fish. and
Reiser et al 79 on Mugil japonicas, on this subject. There
are also studies by Toyomizu et al 22 on rainbow trout
and Niima et al e) on ayu (sweetfish). All these studies
show the effect of feed oil on the composition of fatty
acids of the body oil.
As mentioned in the introduction,
when one uses white meal which has a low fat content, one
can estimate that it lacks fat as well as phospholipids when
compared to the fresh fish feed used in the past.
The purpose of this experiment is to confirm the
effects of feed oil composition on the fatty acids composition
of the fish.
57.
(2) Experiment
Two experiments listed below were carried out in
this chapter.
Experiment I.
Body fat from skin and meat, internal organs except
liver, and liver were extracted and converted into methylesters and the composition of the fat was Obtained using
gas chromatography.
The results of the chromatographic
analyses were compared.
In this experiment the controlled
fish used was eel fed with raw saira for the period of one
year and the experimental fish, also eel, was fed with an
artificial feed mixture (white meal, a-starch and mixed
vitamins) supplemented with 10% feed oil.
Experiment II
Eels fed with raw saira in the previous experiment
were changed to the artificial feed system mentioned in
experiment I.
One group was fed with the feed system
supplemented with 10% vegetable feed oil M (trade mark)
which has a high C 18:2 fat content. The other group was fed '
with the feed system supplemented with 10% animal oil,
feed oil P (trade mark) which has a low C18:2 fat content.
Both groups were kept on the specific feed system for 70
days. Oil was extracted from various areas as in experiment I
and separated into triglycerides and phospholipids using TLC
and the results were compared. Methods for extracting lipids,
esterification, and gas chromatographic analyses were the same
58.
as those used in chapter IV.
(3) Results and Discussion.
90
The results of experiment I are shown in Table
6-1 and those of experiment II are shown in Table 6-2.
Analytical values of saira oil shown in Table 6-1 were
obtained from the results of Ito et al. 43 The degree of
unsaturation and the corresponding amounts Wère &lawn in
the bottom table.
As shown in the table, if one divides
the oil from various parts of the body of eel into saturated
and unsaturated oil, one finds that there is no difference
in the ratio of saturated to unsaturated for any particular
part of the body but they are almost uniform.
The ratio of
saturated to . unsaturated fat in experiment I was 28:72.
The
ratio for experiment II was 32:68. The difference between
the two experiments is believed to be the difference of the
surrounding conditions at the time of breeding.
The ingredients
of saturated fat were C 16:0 followed
by C
and C 180'
.
14:0
The amount of C14:0 oil in the controlled fish was almost
the same as the amount of
C14:0 oil in the fish fed with
the artificial feed system in experiment I. The amounts
were similar to that of feed system.
The amount of C
14:0
in experiment II was similar for both,fish although the
amount of C 14:0 oil in the feed system varied. For C
16:0'
there were some differences between the phospholipids of
the controlled fish and the phospholipids of fish fed with
the artificial feed system but the ambunts of triglycerides
-
91
59.
were 'similar in both cases which were sirriilar to that of the
feed system in experiment II.
The fish fed with the
artificial feed system had a higher content of the above
mentioned ingredients than the controlled sample in experiment
I although the contents of these ingredients in the feed
systems were about the same.
Since the artificial feed
systems were supplemented by 23% of a-starch and vitamins 81
(Table 6-3) it is quite obvious to believe that there are
some differences between the stored fats and the body
compositions of the controlled fish and the experimental
fish.
However, the amount of C 18 . 1 of the fish fed with the
artificial feed system was the same as the one which was fed
raw saira. This is believed to be due to the fact that there
is some process for manufacturing, fatty acids from carbohydrates in the body.
.
There were differences in the composition of
unsaturated acids.
This is believed to be caused by the
composition of the feed oil.
C 16:1' C 18 .:1' C 20:1 ' C 20:5
The main unsaturated acids were
and C 296 .
There was also a
relatively large amount of C18:2 oil in feed oil M in
experiment II. Among these oils, the amount of C
161
and C 18:1 showed a higher content ratio in fish than the
ratio in the feed oil but the amount of oils was comparable
to the amount of oils in the feed oil. ,There was a large
difference in the amount of C 226 between the feed oils in
experiment I. This difference was also shown in the fat
60.
from various Parts of the bodies of the eels which were
fed with corresponding feed systems.
The differences in
the amount of feed oil ingredients were shown for 0 16:1 ,
93
C18:2 and 0 20:5 in experiment II. These differences were
also shown in the amounts of triglycerides and phospholipids.
C 182 is said to be one of the essential acids and it is
said that_the amount of this acid does not . increaSe in the
body unless it is brought in from the outside.
This
theory is believed to apply for the eels also.
If one
compares the triglycerides and phospholipids in the fatty
acids composition in experiment. II, one finds that there
are large. amounts of highly unsaturated phospholipids such
as C 20:5' 225 and C 22 :o
,. The above data coincided with
the previnusly known theory that the highly unsaturated
fatty acids are easily taken in by phospholipids. 82
83
Le Breton
has reported that there is selective combination
of C18:0 in blood phospholipids of the rat. This phenomenon
took place in eels also since the amount of C 18:0 is higher
than the amount of triglycerides when compared.
As can be seen from the above data, it is clear
that the fatty acid composition of phospholipids in the feed .
'system causes some effects on the fatty acid composition
of eels. Its effect is largest in the unsaturated fatty acid
composition, especially in high molecular highly unsaturated
fatty acids. Sometimes some differences can be seen in
saturated fatty acids according to the presence or absence
(1.
of carbohydrate in the feed system but the differences are
small. There is the question to see whether these differences
can be seen not only in body composition but also in eg7s
Lasker et a1 40 have concluded that there is a direct influence
of feed oil on eggs because the fatty acid composition of
phospholipids in blood is similar to that of eggs. Generally
it is beli.eved that the egus are influenced by the oil in
the feed system during the maturation of eggs. •
.
•
VII Conclusion.
If one summarises the above information, one can
see that the feed system of fresh water fish is chani7ing
from raw fish with a•high•fat content to the artificial
feed mixture consisting mainly of white fish meal with a low
fat content as mentioned in the introduction.
It is believed
that the composition of the artificial feed mixture
not only influences the growth and the health of the fish
but also the qualities of the eggs. The purpose of this
experiment is to obtain a reasonable feed system for obtaining
healthy eggs and to control the parent fish. As a basic
study l rainbow trout eggs were used and Compositions of
lipids stored in the eggs and biochemical changes in the
compositions during the development were studied and the
following points were made clear.
1) Results in changes in water content, ash, total nitrogen
and total lipids in this experiment coincide d . with the
reàults of the other studies.
The consumption of total
62.
lipids was lower during the period before hatchinp; when
compared to the period after hatching. Lipids were
divided into acetone-soluble which consisted mainly of
triglycerides and.acetone-insoluble which consisted mainly
of phospholipids and the consumption of each ingredient
was observed during the development.
Acetone-insoluble
.substances were the main part of lipids consumed during the
period up to the time of hatching and there was almost
no consumption of acetone-soluble materials during this
period.
This process was reversed during the period
after hatching. Acetone-insoluble matter showed a weight
increase immediately after hatching. From the average
molecular wei.ght and iodine values, it was shown that some
changes occurred in unsaturated fatty acids.
2) Acetone insoluble matters which were consumed before
the hatching were mainly made' up of lecithin and cephalin
and there were small amounts of carbohydrate lipids, plasmalogen
and sphingomyerine present.
The main ingredients consumed
were lecithin and cephalin.
There wasa decrease in the amount
of carbohydrate lipids after fertilization.
The decrease of
sphingomyerine associated with the breaking of the egg
membrane and the loss of perivitelline fluid was considerable.
3) The differences in the fatty acids which made up the
triglycerides and phospholipids were: mono-unsaturated fatty
acids.were the principal ingredient of the former compared
to saturated, mono- and di-unsaturated fatty acids and highly
unsaturated fatty acids each making up 1/3 of the total
63.
ingredients for the latter. The order of consumption for each
acid in triglycerides during the development was C 18:1'
C 16:1
C16 --,- C 18:2 >C22:6 and 0 18 had shown an increase
during this period. It was C 22:6
for phospholipids.
C 16 ,> C:1
C 20:5
C 18
The decrease of C 22:6 'was especially
large.
4) Centrif.mgally, the yolk protein was made up of two
ingredients during the period between unfertilized egg to
the eye appearance period. They were the ingredient with a
'sedimentation constant of 9.4S and the ingredient with a
sedimentation coefficient of-3:.1S.
The appearance of a new
ingredient with a sedimentation coefficient of 11.2S was
observed after the eye appearance period.
The ingredient
with 9.4S had a diffusion constant of 4-1, a molecular weight
of 240,000, P/N = 1-20 and contained 20% lipids. This
the ingredient with a sediingredtwasubivdento
mentation coefficient of 4.9S, a diffusion constant of 4.2
and a molecular weight of 120,000 by alkaline.
The ratio of
ingredient with 9.4S to 3.1S in unfertilized eggs was
9:1.
The ingredient with 9.4 3 showed tWo phases (up to the
eye appearance period and after hatching) of consumption.
T/N = 1/8 for the ingredient with 3.1S.
high phosphor content.
This ingredient has a
The 11.2S ingredient was believed to
be related to blood.
5) Fatty acids which mak e. up a large part of the lipids
were influenced by the oil which was added to the feed
94
6)..
system.
The unsaturated acids were easily influenced by
the added oil.
This phenomenon . was considerable in highly
unsaturated acids. Phospholipids showed that they used
highly unsaturated acids selectively for constructive
compounds.
The concluding remarks were divided into the five
points above but if these points were combined thJ changes in
the ingredients of rainbow trout eggs can be divided into the
periods before and after hatching.
The ingredient which is
bound with phospholipids, mainly consisting of lecithin, was
used for the development of the fetus before hatching.
The
ingredient which is bOund . to triglycerides was used by the
young fry for growth and sources of energy and for the
construction of body protein after hatching. During this
period, fatty acids were used as fuel for various development
energy.
Part of the fatty acids was converted into young
fry lipid ingredient. Highly unsaturated fatty acids in
phospholipids and monoene fatty acids in triglycerides
made up the main part of the ingredient of fatty acids
consumed. These acid compositions wereinfluenced by the
composition of the fattY acids added to the feed system.
It is believed that the above discussion can
become a basic indicator for preParing the feed system for
young fry.
Acknowledgements:
This study was carried out at Tokyo Suisan University
and Tokyo Kogyo University Biology Departments.
I would like
65.
to thank Prof. K. Shibata, of Tokyo Kogyo University, for
direct assistance.
I also want to thank Honorary Prof.
Sarutani of Tokyo Suisan University, Prof. Nakano and Okada
of Tokai University for their valuable asistance. I further
extend my thanks to Profs. Funagi and Shibata of Tokyo
Kogyo University. and Profs. Suginb, Mizuguchi, Nagahisa
and Suzuki for valuable guidance while completing this
report.
In addition I ,extend my thanks to Prof. Nakamura
of Showa University and the people in the Biology Department of
Tokyo Kogyo University for valuable assistance in operating
analytical instruments.
I also thank Mr. Tanizaki, the
director of Nagano Prefecture Fisheries and Mr. Okawa of
Tokyo Fisheries Testing Grounds for obtaining the materials
used in this expetiment.
66.
Bibliography (Japanese items only; see also below)
7) M. Suyama; Nihon Suisan Gakkaishi (Japanese Fisheries
Bulletin) 23, 789, 1958.
8) J. Ishida; Shishitsu Kagaku (Lipid Chemistry) 2,
Kyoritsu Shippanshà 1958, p158.9) Y. Yamamura, S. Muto; Tohoku Suiken Kenkyu Pokoku
(Tohoku Fi:sheries Research Report) 19, 179, 1961.
10) K. Yamanoto, I. Ota, K. Takano, T. Ishikawa; Nihon
Suisan Gakkaishi (Japanese Fisheries Bulletin). 31, 122,
1956.
12) M. Suyama, C. Ogino; Nihon Sùisan Gakkaishi (Japanese
Fisheries Bulletin) 23, 7e5, 1958.
17) Ushi Kagaky Binran (Oil Chemistry Manual) Hihon Yukagaku
Kyakai Hen, Maruzen 1958, p17.
22) M. Toyomizu, K. Kawasaki, I. Tomiyasu; Nihon Suisan
Gakkaishi (Japanese Fisheries Bulletin) 29; 957, 1963.
43) Y. Ito, K. Fukusumi; Kogyo Kagaku Zasshi (Journal of
Industrial Chemistry) 65, 1963, 1962.
4 8 ) R. Amamiya; Gas Chromatography, Kyoritsu Shuppansha.
56) K. And°, Nihon Suisan Gakkaishi (Japanese Fisheries
Bulletin) 28, 73, 1962.
72) K. Watanabe, Y. Kawaide, K. Iso; Tanpakushitsu Ka.q. aku
(Protein Chemistry) . Editor:
S. Akabori, S. Mizushima.
2, Kyoritsu Shippansha 1961, pp 357-360.
75) M. Tsujimoto; Tokyo Kogyo Shikenjo Hokoku (Report of
Tokyo Industrial Testing) 26, (10) 1931.
67.
76) K. Oshima; Suisan Shikenjo Hokoku (Fisheries Research
Report) 6, 221, 1935.
•
80) Y. Niima, S. Taguchi; Nihon Suisan Gakkaishi (Japanese
Fisheries Bulletin) 30, 918, 1964.
68.
Bibliography
Chemical Embryology. Cambridge Univ. Press. 1931.
Biochemistry and Morphogenesis. Cambridge Univ. Press. 1942.
3)
The Biochemistry of Development. Pergamon Press Ltd. 1960.
4) S. LrtivTaup and B. WERDINIUS: The J. Exp. Zoology. 135, 203 (1957).
5) S. SMITH:
The Physiology of Fishes. M. E. BROWN (Editor) Vol. 1. Academic Press Inc.
1)
J. NEEDHAM:
2)
J. NEEDHAM:
J. BRACHET:
6)
7)
8)
New York. 1957, p.323.
F.R. HAvEs: The Quarterly Review of Biology. 24, 281 (1949).
pei_411j,
23, 789 (1958).
eAWek 1 958 , 158 W.
Terediget 19 , 179 ( 1961 ).
9) al ;Ii;15 • egiii —Pg:
W*e44- ere., 31 , 123 ( 1965).
10) 1111: 1,- --- 111: • 'Mil g.'P, • jiJiflhJ • 5.)11M:::
11) F.R. HAvEs: Biochem. J. 24, 735 (1930).
12)efil I 17-1"-:=1.: • V;V.M*: Fl**.eeff:eil 23, 785 (1958).
ertILIZ-.1"- -a*:
-f-HEI1M:
13) T. ONO, J.
14) R.
SENO,
BALLENTINE
•W. M. SpERBY:
F.
and K. HIKOTA: J. Tokyo Univ. Fish., 45, 79 (1959);
Anal. Chem. 19, 281 (1947).
Biochemical Analysis. D. GLICK (Editor) Vol. II. Intersci. Pub.
NAGAYAMA
and J.R.
GREGG:
Methods of
Ltd. New York. 1957. p. 83.
16) J. FoLcu, M. LEES and G.H. SLOANESTANLEY:
15)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
J. Biol. Chem. 226, 497 (1957).
JIS 1958, 3381.
•
iiiiljrn-f-feed::
ibefECi:: H 7l.1Ceelt: 51, 120 (1948).
The Chemical C9nstitution of Natwal Fats. Shapman and Hall Ltd.,
T. P. HILDITcH:
London 1947.
Y. ITO and T. FUJII: J. Biochemistry. 52, 221 (1962).
958 , 171.
i11111 I'fbreftrd:
C:7.KTISLI • )110eriti. • éltlÉ: H **e'4.-"erkt 29 , 957 ( 1983).
D.K. MATHUR: Abstract. Dissertation. University of Cambridge. p. 28.
L.O. ÜHMAN: Ark. Zool. 36A, (7) (1944).
R. BALLENTINE: Methods in Enzymology; S.P. COLOWICK and N.O. KAPLAN (Editor): Vol. 3 ,
Academic Press Inc. 1957. p.984.
R.J.L. ALLEN: Biochem. J. 34, 858 (1940).
G. nix: Microchem. Acta. 1, 75 (1937).
F. NEUMANN: Ber. Dtsch. Chem. Ges. 70, 734 (1937).
F. LEuPOLe and H. BiiTTENER: Z. Physiol. Chem. 292, 13 (1953).
C. ARTOM: Methods in Enzymology. Vol. 3, p.358.
S.J. THANNHAUSER, J. BENOTTI and H. REINsTEIN: J. Biol. Chem. 129, 709 (1939).
D. GLicK: J. Biol. Chem. 156, 643 (1944).
G. SCHMIDT, J. BENOTTI, B. flEasiimAN and S.J. THANNHAusEa: J. Biol. Chem. 166, 505(1946).
T.A. Scon and E.H. MELVIN: Anal. Chem. 25, 1656 (1953).
J.M. McKinBIN and W.E. TAYLOR: J. Bio. Chem. 178, 29 (1949).
S. NOJIMA and N. UTSUGI: J. Biochemistry. 44, 565 (1957).
•
L.O. ÔHMAN: Ark. Zool. 39A, (11) (1947).
D.W. THIELE: Z. Physiol. Chem. 299, 151 (1955).
J.A. LOVERN: Biochem. J. 30, 20 (1936).
R. LASKER and G.H. THEILACKER: J. Lipid Research. 3, 60 (1962).
■
Ae 1
40)
41) O. A.
42)
SCHJEIDE:
Progress in the chemistry of fats and other lipids; R. T. HOLMAN
W.O. LUNDBERG and T. MALKIN (Editors): Vol. VI. Pergamon Press. 1963, p. 286.
E. STAHL: Thin-Layer Chromatography. Academic Press Inc. 1965, p. 152.
43) fpeite•Ntt—m: meibseagt
45)
65, 1963 (1962).
and H. ANDREAS: Chem. Ber. 94, 628 (1961).
H.K. MANGOLD and R. KAMMERECK: Chem. & Ind. (London) 1032 (1961).
44) E.
JANTZEN
and
60.
46)
47)
48)
49)
50)
51)
52)
53)
54)
55)
56)
57)
E.C. HORNING, A. KARMEN and C.C. SWEELEY: Progress in the chemistry of fats and other
lipids; R.T. HOLMAN and T. MALKIN (Editors): Vol. VII-2. Pergamon Press. 1961.
B.M. CRAIG and N.L. MURTY: J. Am. Oil Chemist's Soc. 36, 549 (1959).
, eAwret.
-e e' -9 7 4
rire.a.E: 9/.7.
G. B. ANSELL and J. N. HAWTHORNE: Phospholipids. B. B. A. Library Vol. , 3. Elsevier Pub.
Co. 1964, p.34.
E.H. GRIIGER, Jr., R.W. NELSON and M.E. STANSBY: J. Am. Oil. CheMist's Soc. 41, 662 (1964).
F.R. HAYES: Biochem. J. 24, 735 (1930).
F. B. SHORLAND: Comparative Biochemistry; M. FLORKIN and H. S. MAsoN: (Editors) Vol.
III. Acadetnic Press. 1962, p. 56.
G.B. ANSELL and J.N. HAWTHORNE: Phospholipids. B.B.A. Library Vol. 3 Elsevier Pub. Co,
1964, p.422.
T. ONo, F. NAGAYAMA and T. MASUDA: J. Tokyo Univ. Fish., 46, 97 (1960).
K. ANDO: Canadian J. of Biochemistry. 43, 373 (1965).
28, 73 (1962).
r.k- jr#--:
S. SMITH: The Physiology of Fishes; M. E. BROWN: (Editor) Vol. 1. Academic Press. Inc.
New York. 1957, p. 323.
E.G. YOuNG and T.L. PHINNEY: J. Biol. Chem. 193, 73 (1951).
A. MONROY: Exp. Cell Res. 1, 72 (1950).
J.E. VANDEGAER, M.R. REICIIMANN and W.H. COOK: Arch. Biochemi. Biophy. 62, 328 (1956).
H. SUGANO: J. Biochem. Tokyo. 46, 417 (1959).
O.A. SCHJEIDE: Progress in the Chemistry of Fats and other Lipids; R.T. 1101,NtAN (Editor):
Vol. VI. Pergamon Press Ltd. 1963, p.251.
J. FoLcu, I. Ascom, M. LEES, J.A. MEATH and F.N. LEBARON: J. I3iol. Chem. 101, 833(1951).
E. CHARGAFF: J. Biol. Chem. 142, 505 (1942).
E. CHARGAFF: J. Biol. Chem. 142, 491 (1942).
F.J. JOUBERT and W.H. Coox: Can. J. Biochem. Physiol. 36, 389 (1958).
G. BERNARD! and W.H. Coox: Biochim. Biophys. Acta. 44, 96 (1960).
ILL. FEvOLD'and A. LAUSTEN: Arch. Biochem. 11, 1 (1946).
D.K. MEECHAM and H.D. OLCOTT: J. Am. Chem. Soc. 71, 3670 (1957).
R.A. FLICKINGER and O.A. SCHJEIDE: Exptl. Cell Res. 13, 312 (1957).
K. KURIHARA and K. SHIBATA: Arch. Biochern. Biophys. 22, 298 (1960).
: Vol. 2 ,
; 'iMzihilK1(l5 • 7JUi
1,
e=rert f
igt.''M
•
**e*.eitt
•
58)
59)
60)
61)
62)
63)
64)
65)
66)
67)
68)
69)
70)
71)
72)
e:Atilieu: 196
e • inumE
p. 357360.
73) G. BERNARD! and W.H. Coox: Biochim. Biophys. Acta. 44, 86 (1960).
74) G. BERNARD! and W.H. COOK: Ciochirn. Biophys. Acta. 44, 105 (1960).
75)
76)
77)
78)
±:1IA: 31i31OEM.V.Mtgei 26, ( 1 0) (193 1 ).
: 7.Keieep-mm 6 , 221 ( 1935).
J.A. LOvERN: Biochem. J., 32, 1214 (1938).
P.B. KELLY, R. REISER and D.W. Hoot): J. Am. Oil Chemist's Soc., 35, 503 (1958).
79) R. REISER, B. STEVENSON, M.
Chemist's Soc., 10, 507 (1963).
80)
81)
82)
83)
KAYAMA,
R. B. R.
CHOUDHURY
and D. W. HOOD: J. Am. Oil
FI*Açjr&:: ;,!ii.b: 30, 918 (1964).
ïij- imm;—n5 • al n
J.E. IIALvNa: J. Nutrition., 62, 225 (1957).
Phospholipids. B.13. A. Library. Vol. 3 Elsevier Pnbl.
G. B. ANSELL and J.N.
Co. 1964, p. 194.
E. Le. BRETON and M. PASCAUD: In '+,,istion, Absorption Intestinal et Transport des
Glycerides. Editions C.N.R.S., Paris, 1961, p.179..
7 0.
Summary
Until recently,' chrysalis of silkworm, raw fishes' and mysid shrimp had been used
as foods for fish•culture in Japan, but these raW Materials have recently been replaced
gradually by formulated diet made of white fish meal, carbohydrates, oils and vitamins.
The composition of formulated diets used, however, are not always suitable for
the nutritional requirements of fishes. Thus, it is considered that the chemical
composition of diet may have some important effects upon the quality of 'fish-eggs
and their development after fertilization. From the reasons mentioned above, the. study Of chemical composition of fish egg
and their changes during development are considered -to be useful for improvement
of the cliet for fish culture.
There are a considerable literature on egg proteins, but little is known on the
lipids. so far. The purpose of the present study was to investigate the composition
of lipids and their changes during the development of eggs and to make a contribution
to the preparing of the diet for fish culture.
The analyses of chemical composition were made on eight stages of samples during
the development. The -changes of whole egg weight, Moisture, ash, total nitrogen
and lipids 1,vere analyzed. The total lipids were• fractionated into acetone insoluble
and acetone soluble-lipids and the characteristic changes of these lipids were clarified.
Chemical properties of acetone soluble lipids and their fatty acids were measured.
Acetone insoluble lipids were considered to be consist, of five components: lecithin,
cephalin, plasmalogen, sphingomyelin and cerebrosides, and their composition changes
were calculated from the data obtained frOrn the analyses of the functional groups.
After fractionating phospholipids and triglycerides by thin layer chromatography, fatty
acids of each lipid were converted into methylesters and were analysed by gas-liquid
chromatography. The differences of fatty acids composition between these lipids and
the changes in content of each fatty acid were determined.
Yolk proteins were studied by means Of ultracentrifugal analysis, and some physicochemical properties of the components and .their changes during the development were
studied. The influences of different dietary fats upon the fatty acid composition of
'cultured eels taken from eel culture-pond were studied.
The restilts obtained in the observations of the changes of .chemical composition
during the development of rainbow trout eggs were essentially similar to those published
in literature. It was found, however, that acetone insoluble lipids were consumed to
one half of the content in unfertilized eggs. On the other hand, acetone soluble lipids
s;vere not consumed before hatching. The major components of acetone insoluble lipids
were lecithin and cephalin. These were consumed itnifermly before hatching, but the
minor components were consumed non-uniformely. Especially, cerebrosides ‘vere
consumed largely before the eye stage of the egg, and a considerable amount of
-
71.
sphingomyelin was lost by hatching.•
The differences of fatty acid composition between triglycerides and phospholipids
in unfertilized eggs were as follows: the former was composed mainly of monoenoic
acids (50%), and the composition of the fatty acids in phospholipicls were saturated,
mono- and dienoic, and polyenoic fatty acids, each being one third in content of the
total fatty acids. The order in the rate of decreasing the content of the fatty acids
in triglycerides during development was Ci8 : 1>C16 : 1>C16 :o>'Ci8:2 and that of the fatty
acids in phospholipids Cz2:6> Cis:o> C16: 1 .
The ultracentrifugai patterns of unfertilized yolk proteins showed the existence of
tl,vo components. The major one was the lipoprotein combined with 20% lipids, and
the sedimentation, diffusion constants and the molecular weight calculated from both
constants were 9.4S, 4.1 and 240,000, respectively. This component was split by
alkali into two subunits. The minor component was 3.1. S and a new component of
11.2 S appeared at the beginning of the eye stage. During development, the 9.4S
component decreased in two stePs, before the eye stage and after hatching, respectively.
When eels were fed for a long period on different dietary fats, the fatty acid
composition of eels were altered with the difference in fatty acid composition of
dietary fats.
It was concluded from these results that, after fertilization of eggs, embryo grows
to larva with spending phosphOlipids combined to lipoprotein and, after hatching, larva
grows to young fish with consuming fat droplets and triglycericle combined t'o lipoprotein. The easily consumable fatty acids in development 3.vere monoenoic acids in
triglycerides and polyenoic acids in phospholipicls. The fatty acid composition of fishes
can be altered by dietary fat.
r yok
rminal disc
\,\.- Egg capsule
merntwane
A, Unfertilized
Egg
B, Fertilized
Egg
Top Water Inlet
Air Inlet
Doenlorinotion
Siphon
C, Larva
appeared
D, Eye
appeared
E, Just before
Hatching
Apparatus
Incubation
Troy
Le_
Wo or Outlet
Fig. 3-1, The equipment of incubation
for development of rainbow trout
eggs in laboratory.
F, Just after
Hatching
G,
1/2 Yolk
absorbed
H, Swim up
Fry
Fig. 1. The morphological transformation
of embryo in the development of
rainbow trout egg and their names
given in this article.
Hatching 1/2.Yelk Swim up
Unferti- Larva Eye
absdrbed fry
lized appeared appeared
Unferti- Larva Eye
Hatching 1/2 Yolk Swim up
lized %peered appeared
obsored
fry
4
i
4
(mg)
II
I0
9
801—
8
ture
6
601—
5
4
40t
Dry Matter
3
a El -c:r
2
I
10
20
1
30 40 50 60
Days after Fertilization
The changes in weight contents
(mg.) of total weight, moisture and
dry matter in one rainbow trout egg
during development.
•Fig. 2-1,
10 20 30 40 50 60
Days after Fertilization
Fig. 2-2. The changes in weight contents
(mg.) of total lipids, acetone soluble
and acetone insoluble lipids in one
rainbow trout egg during development.
Table 2-1.
Sample
No.
■
li
Ill
IV
V
VI
VII
yul
Chemical analysis of the rainbow trout eggs during development.
Time
after
fertilization
(days)
Development stage
Unfertilized egg•
Larva appeared
Just before.eye stage
Eyed egg
Just before hatching
Newly batched fry*
•
Fry, 1;2 yolk absorbed'
Swim up fry
1
Ash
Moisture
Total
Total
lipids
(%)
(%)
(51 )
(%)
Acetone soluble Acetone
lipids
insoluUnsapo- ble
nifiable liPids
(%)
matter
(%)
3.5
0.6
0.6
2.6
0.6
2.0
0.5
1.9
1.5
0.5
0.5
1.6
0.3
1.4
1.2
0.2
Total
(%)
59.3
64.1
64.0
64.1
64.5
67.8
75.9
79.7
9
13
19
26
34
47
58
4.6
4.1
4.1
4.2
4.0
3.6
2.6
2.2
1.6
1.3
1.3
1.4
1.4
1.4
1.1
1.2
8.3
7.2
7.4
7.1
7.2
7.0
5,0
3.2
11.8
9.9
9.4
9.0
8.7
8.7
6.4
4.4
* Yolk 77.7%, Larva 22.3%
Table 2-2.
Sample
No.
Changes of chemical constituents in one egg during development.
Average
weight
Moisture
of one
(mg)
egg
Total
Dry
matter
Ash
(mg)
(mg)
Nitrogen
(mg)
(mg)
i Acetone Soluble Lipids
Total Acetone;
Lipids insol. 1
UnsapoiI
"
Lipids I To tal 1 Tr'gly
(mg)
fiable
I (mg)
(mg, , cerides . Matter
(mg)
(mg)
i
-
'
I
II
III
IV
V
VI
VII
VIII
53.6
66.4
66.1
66.3
66.8
62.6
91
101
103
103
104
92*
t
10882.0
,
113
90.1
1
36.9
37.1
37.1
37.1
36.8
29.8
26.0
23.0
1.4
1.4
1.4
1.4
1.5
1.3
4.2
4.2
4.1
4.3
4.2
3.3
1.2
1.4
2.8
2.5
10.7
10.2
9.7
9.3
9.0
8.0
6.9
5.0
3.2
2.7
2.1
2.0
1.6
1.5
1.5
1.4
7.5
7.5
7.6
7.3
7.4
6.5
5.4
3.6
:
,
■
6.9
6.9
7.0
6.8
6.9
6.0
I
5.1
'
:
3.3
0.6
0.6
0.6
0.5
0.5
0.5
0.3
0.3
Table 2-3. Chemical properties of acetone soluble lipids and their fatty acids
and composition of acetone soluble lipids calculated from acid values,
mean molecular equivalent of fatty acids and unSaponifiable matter.
Sample
No.
II"
III
Iv
yr
VII
VIII
Acetone soluble lipids
Acid
Iodine
Value
Value
5.6
5.7 '
6.1
7.5
9.1
14.3 t
25.6
42.5
156
149
151
159
159
162
165
175
Saponification
Value
175
170
171
173
170
172
170
173
Composition of Acetone
soluble lipids
Nlean
Free
;
Tri _
Unsaponi:
Iodine Neutrali- Molecular Fatty glyceride liable
zati "
E•
Value
Value
ci ty%r a " A LI'cl ,
lent
Mixed fatty acids
170
163
161
172
177
180
185
194
195
194
193
193
192
193
. 194
194
287
.
288
291'
291
992
291
289
289
1_(%)__.
(%)
1
89.1 .
89.1 '
89.0
88.6
88.2 j
85.2
80.3
70.9
2.9
2.9
3.2
3.9
4.7
7.3 '
13.2
21.8
8.0
8.0
7.9
7.5
7.1
7.4 .
6.5
7.2
9
160
1
270
I0%, •
Ne-Acetcde-
Acidic
Acid
Gois 60
biz
inf. I
35 Hydriodic Acld
f Red Phoipborus
Fig. 3-2.
The apparatus for glycerine
determination in lipids modified
from Nettmann's apparatus for
methoNY irrotIp dote rtnino t
Unferti- Loryo
Eye
fized appeared oppeared
1 1
Hatahina 1/2 Yolk Swim up
absorbed fry
1
,
Hatching 1/2 Yolk Svom up
Unferti- • I. row Eye
gad appeared appeored
absorbed fry
(gr.)
100
4
4
4
4
4
(%)
90
100
80
90
<I 70
80
<1 60
6
70
Î 50
5
60
40
50
30
(gel
40
-1
("1:1
40
\
- 3 11
—111 —
30
7
2
1 0 110
20
30
40
50
60
Days otter Fertilization
Fig. 3-3. The changes in relative content of
acetone soluble and acetone insoluble
lipids during development of rainbow
trout egg. Values are calculated by
considering the contents at unfertilized
stage is 100%.
10 20 30 40
50 60
Days dfl2r Ferflization
Fig. 3-4; The changes in the content of
lecithin (curve 0... e), cephalin
(curve A—G), plasmalogen (curve
•—À), sphingomyelin (curve CI-12)
and cerebrosides (curve D—D) in
100 gr. of acetone insoluble lipids
during the development of rainbow
trout egg.
e
Chemical analysis of acetone insoluble lipids during development.
Table 3-1.
Fatty Acids
Time
.
! Total Total
after
Sam- Development fertiStage
ple
HzaNo.
p
N
:
; tion
Mean
Total
(%)
mol.
,
(alys)
10
Larva
appeared
20
III Eye. appeared
32
IV Newly
, hatched
V Fry absorbed 48
.
1/2. york
60
VI Swim up fry 1
II
1
Cc)
_
A* 1.79 3.64 70.1 ,
0
I . Unfertilized
Equiv.
Leci- SphynGlyc Tot al thin gosine Galac-AldeYlos Cho.
tose hyde
N
line Hne : (5)
(5) (51)
(%) ;
B** 1.83 3.68 68.9
A 1.76 . 3.69 70.3
1.84 ; 3.73 70.3
/£ 1.80 : 3.79 1 71.0 :
1.84 ' 3.85 71.0 1
A 1.78 3.76 ! 70.3 ;
1.80 3.78 ; 70.1 I
A 1.801 3.76 70.0 I
1.75 3.60 ' 69.7
A 1.80 3.82 71.4 'I
1.82 , 3.76 71.2
10.5 . 11.9 10.3 0.14 1.24 0.59
300
10.3 11.2 10.3 0.18 1.32 0.57
300
10.6 11.7 10.7 0.13 0.58 0.82
300
10.8 11.8 10.9 ' 0.14 0.64 . 0.86
301
ld.6 12.9 12.0 0.14 0.12 i 0.33
299
10.7 13.1 12.1 0.13 0.14 1 0.31
300
10.7 13.4 12.6 0.07 1 0.1010.17
297
299
10.6 ' 13.1 12.7 ; 0.07 0.10 i 0.15
298
10.7 ; 13.9 13.3 0.12 0.20 ! 0.40
10.6 13.6 12.9 '0.15 0.22 : 0.48
299
296
10.81 14.5 1340.08 • 0.30 0.50
297 ; 10.8 ! 14.3 13.5* 1 0.10 0.34 0.58
I
* Dec. 1960: ** Dec. 1961
r.ffile 3-2.
Sample
No.
Molar concentration of each chernical group in 100g acetone insoluble
lipids calculated from the data Table 3-1.
Total
Nitrogen
I Total
phos _
phor
SphyingoI
Total Lecithin ,
.Galactose Aldehyde
sine
Fatty plycerine .Choline
, Choline
N
açids I
Total
_
I
A
II
A
B
B
•
HI A
B I
IV A I
B 1
A 1
B 1
VI A
13
V
0.128
0.131
0.126
0:131
0.129
0.131
0.127
0.129
0.129
0' 125
0.129
-
0.130
0.118
0,119
0.119
0.120
0.122
0.124
0.121
0.122
0.121
0.123
0.123
0.121
0.233
0.233
0.235
0.234
0.238
0.237
0.237
0.234
0.234
0.233
0.241
0.240
0.114
0.112
0.115
0.118
0.115
0.116
0.116
0.115
0.116
0.115
0.117
0.117
0.092
0.093
0.097
0.097 ;
0.106
0.108
0.110
0.108
0.115
0.113
0.120 I
0.118 ;
0.085
0.085
0.088
0.090
0.099
0.100
0.104
0.105
0.110
0.106
0.112
0.112
0.010
0.012
0.009
0.010
0.010
0.009
0.005
0.005
0.009
0.007
0.006
0,005
0.002
0.002
0.003
0.003
0.001
0.001
0.001
0.001
0.002
0.002
0.002
0.002
0.007
0.007
0.003
0.004
0.001
0.001
0.001
0.001
0.001
0.001 .
0.002
0.002
Table 3-3. Molar concentration of lecithin, cephalin, plasmalogen, sphyngomyelin
and cerebrosides in 100g acetone insoluble lipids calculated from the data
Table 3-2.
Sample
No.
I
A
II
B
1£
B
III
A
IV
A
V
A
IV
A
B
B
B
B
Lecithin
Cephalin
Plasmalogen
Cerebrosides
0.083
0.083
0.085
0.086
0.098
0.100
0.104
0.104
0.108
0.105
0.110
0.110
0.026
0.026
0.024
0.025
0.015
0.015
0.012
0.012
0.006
0.008
0.005
0.005
0.002
0.002
0.003
0.003
0.001
0.001
0.001
0.001
0.002
0.001
0.002
0.002
0.007
0.007
0.003
0.004
0.001
0.001
0.001
0.001
0.001
0.001
0.002
0.002
SphyngornyeHn
0.005 •
0.007
0.007
0.007
0.008
0.008
0.005
0.004
0.006
0.006
0.006
0.006
7
76
Amount of each lipid component calculated from the data Table 3-3.
(Molar concetration x presurned molecular weight, i.e. lecithin 840,
Table 3-4.
cephalin 800, rdasmalogen 800, sphingornyelin 750 and cerebrosides 780,
respectively.)
Sample
No.
I
Lecithin
(mg)
A
B
II
A
III
A
IV
A
B
B
B
✓
A
VI
A
B
-
B
Table 3-5.
68.0
68.4
70.1
71.1
80.6
82.4
84.7
85.7
88.9
85.7
89.9
88.6
Cephalin
Plasmalogen
(mg)
20.6
20.3
18.7
19.4
11.6
11.8
9.1
9.5
4.8
6.3
3.6
4.3
1.4
1.5 .
1.7
Lecithin
(%)
Cephalin
(%)
68.5
67.8
70.8
70.2
81.0
80.9
85.8
86.5
88.5
87.4
89.3
89.0
20.7
20.1
18.9
19,2
11.5
11.6
9.2
9.6
4.8
6.4
3.6
4.3
II
A
III
A
IV
A
✓
A
B
A
VI
B
i
I Sphingomyelin
(mg)
5.3
5.6
2.4
2.7
0.5
0.6
3.8
4.9
5.3
5.5
6.9
6.1
0.5
0.5
0.9
0.9
1.3
1.4
3.9
2.9
4.6
3.8
4.3
3.6
Lipid composition in per cent by weight of acetone insoluble lipids.
,No.
A
(mg)
' 1.7
1.7
2.5
2.5
0.9
0.9
0.5
0.5
Sample
I
Cerebrosides
(mg)
Plasmalogen
(%)
•
1.7
1.7
2.5
2.5
0.9
0,9
0.5
0,5
1.2
1.4
1.5
1.7
;
;
Cerebrosides
(%)
Sphingomyelin
5.3
5.6
2.4
2.7
0.5
0.5
0.5
0.5
3.8
4.8
5.4
5.4
6.1
6.0
3.9
2.9
4.6
3.9
4.3
3.6
0.9
0.9
1.3
1.4
Table 3-6. Molar concentration of cephalin calculated from total nitrogen, total
phosphor, glycerine and total fatty acids listed on Table 3-2.
Calculated from
Sample
No.
Total N
I
A
II
A
Ill
A
IV
A
✓
A
VI
A
0.026
0.025
0.020
0.024
0.012
0.014
0.012
0.016
0.005
0.006
0.003
0.007
Total P
0.023
0.027
0.024
0.024
0.015
0.016
0.012
0.014
0.005
0.011
0.005
0. 5
Glycerine
0.029
0.026
0.027
0.028
0.017
0016
0.011
0.010
0.007
0.009
0.005
0.005
Average
FattY acids
0.027
0.026
0.025
0.028
0.016
0.014
0.012
0.011
0.008
0.008
().005
0.006
0.026
0.026
0.024
0.025
0.015
0.015
0.012
0.013
0.006
0.008
0.005
0.006
77.
Table 4-1.
Changes of fatty acid composition of triglycerides in per cent during
development of rainbow trout egg.
Number of C
atoms and
Double bonds.
Days•after Fertilization
0
5 ! 10 I 15 ' 20
1
•
I 25 30 35 1 40 I 45 I 50 55 '.60 65
0.1 i 0.1 0.1
0.1 011 0.1' 0.1 ; 01,01 0.1: 0101
.
2.6. 2.61 2.6: 2.3; 2.3 2.3 , 2.8i 2.5, 2.6 2.4. 2.4 2.3: 1.8 1.7 1.2
0.2, 0.21 0.2 0.2i 0.2 0.2 0.2: 0.2 0.2 0.2' 0.1 1 0.2 0.1 0.2 0.1
I
I
0.3: 0.31 0.3 0.3 0.3' 0.3 0.3 0.2 0.2 0.2
H).3 0.3 0.3 : 0.3!
I
'
13.7,13.6,12.413.0i14.14.013.713.514.013.8,13.8 . 12.5:12.812.816.4
11.111.1110.340.2i0.310.0 9.5' 9.4 9.0
8.1 8.910.210.5 8.2
I
'
0.7 0.51 0.5 0.51 0.5, 0.5 0.6, 0.6 0.7 0.4, 0.4 0.5 0.4 0.4 0.2
0.4 0.4 0.5 0.7; 0.7: 0.6. 0.7 0.6 0.6 0.6 0.6 0.5 0.4 0.5 0.3
;
1.5 1.5 1.8 1.7, 1.7 1.6 1.5 1.3 2.8 3.5 3.7 3.9 4.2 4.1 4.4
32.232.01.531.021.230.930.630.931.231.630.730.230.228.830.7
9.1 9.2 9.4 9.3' 9.3 9.310.0 9.6 9.6 9.0 8.8 8.6 8.0 7.8 7.0
1.5 1:5' 1.8 1.7: 1.7 1.6 1.5 1.3 1.4 1.4 1.4 1.3 1.1 1.1 0.9
0.8 0.71 0.8 0.8' 0.7 0.6 0.5 0.3 0.3
1
0.3 0.4 •0.4 0.41. 0.6 0.4 0.5 0.2 0.2 0.3 0.3 0.2 0.2 0.2 0.2
I
C12
C14
C15
'
C17
Cia
.:1
:2
:4
C19:1
Cua
:2
:3
:4
:5
•
:5
•
Cl24 ,r
2,2 1.8 1.6 2.1' 2.1 2.1 1.9 2.3 2.2 2.4 2.5 2.8 2.9 2.7 2,6
1.5 1.1. 1.4 1.4. 1.5 1.4 1.5 1.5 1.4 1.4* 1.5 1.7 1.4 1.7 1.0
0.7 0. 6•0.9 1.1i 1.0 1.2 1.2 1.0 1.1 1.1 1.0 1.2 0.8 1.0 0.8
1.5, 1.6 1.6 1.6, 1.5 1.5 1.3 1.4 1.1 1.1, 1.0 1.1 1.4 1.6 1.2
0.4 0.7. 0.5 0.5 0.5 0.5 0.5 0'.5 0.2 0.2 0.2 0.3 0.4 0.9 0.8
3.2 4.3, 5.4 5.0 4.2 4.2 4.5 4.7 6.4 5.2 5.2 5.2 4.8 3.4 2.8
1.4 1.4 1.0 1.0 1.1 1.4 1.1 2.0 2.5 2.5 2.8 2.1 2.2 1.8 1.8
0.2 0.1 0.2 0.2 0.1 0.2 0.2
I 2:4 2.1 , 2.6 2.4 1.8 2.6 2.1 1.8 1.1 1.5 2.0 2.0 1.5 2.0 2.3
1.1.7l1.912.312.912..612.512.612.812.012.513.314.014.916.216.5
0.3 0.3 0.3 0.2 0.2 - 0.2
.
0.2 0.2 0.2 0.3 0.3
78 .
'j'able42. Changes of main fatty acid composition (g) equivalent to 100g of mixed
fatty acids of triglycerides during development of rainbow trout egg.
Days after Fertilization
Nualleaof C
- ---
atoms
Unferti-
and
double •
bonds ;
C1 4
C16
.
C18
I
.2
I
. I
:3
C20:1
:0
•
.-• -
Larva
Eye
appeared
1appeared
,
1
10
'
20
Hzed
1
0
2.6
2.7
13.7
13.7
11.1
11.1
1.5
1.5
32.2
32.2
9.1
9-.2
1.5
1.5
2.2 , 1.8
4.3
3.2
2.413,5
10.6
1.4
32.2
9.7
1.8
2.2
5.2
1.4 ' 1.4
Cna
:0
1
:0
1.0 •
2.4 ! 2.1 ;
12.0
11.7
2.5
13.4
j
Increase
Hatch- 1/2 york Swim up Before
or
ing
absorbed ; fry
feeding . decrease
30
2.4
2.4
14.4
13.0
10.3
9.1
2.41
1.7
31.9
29.8
96 1 . 9.3
1.3
« 1.7
2.2
2.2
- 4.3
4.3
1.4
1.9
.
2.7 '
1.7
. 13.0 . 12.3
35
i
2.4 '
50
1.5
8.2
12.7
8.1 1
5.8
,.
2.52.6
19.8
28.2
5.6
8.7 j
1.3
0.9
2.0
1.9
4.3
3.4
2.2
1.7
1.0
1.3
10.8
9.2
!
60 '
0.8
6.2
5.1
2.0
14.0
3.8
0.5
1.3
1.6
0.9
1.0
7.9
65
I
0.5
6.6
3.1
1.8
12.3
2,8
0.4
1.0
1 .1
0.7
0.9
6.5
-
2.1
- 7.1
- 8.0
+ 0.3
-19.9
- 6.3
- 1.1
-
1.2
-
2.1
0.7
1.5
5.1
Table 4-3. Changes of fatty acid composition of phospholipids in per cent during
development of rainbow trout egg.
Number of C
atoms and
double bonds
C44
C15
. C16
a
•
'2
Cut
C119
• 11
:2
'3
:4 .
C101
C20 : 1.
:2
c
,3
, e,
Cna
'8
16
C24:T
Days after Fertilization
.
01 1! 5 1 10 15 1 20 ; 25 1 30 1 35 1 40 1 45 1 50 55 ' 60 65 «
i
'
•
0
1.0 1.0, 1.111 1.3 1.1 1.3 1.5 1.6: 1.2; 1.2 1.6 1. 31 .91
1208
,
1
,
1
0.3 0.2 0.3' 0.3 0.3 0.3 0.2 0.2 0.4 - 0.5, 0.2 0.1; 0.2 0.1
!
.
!
,
17.717.216.518.518.919.7
17.218819.719.617.5,19.518.619.619.9
2.71 2.8 ! 3.0 3.3 3.4 2. 8 3.11 3.0: 2.5 4.3 4.9 3.2 3.3 3.3 3.3
,
1
. ~3 - ~ 31 -, 0.2 0.3' 0.2 0.4 0.3 - - -: - 1 -- ,
I
I
-0.4 0.4 0.31 0.4 0.6 0.9 0.5 0.5 0.3 0.6 0.6 0.8 0.4 0.6 0.5
■
i
: 9.5 8.5 9.210.3 9.9 10.5'11.1 10.3 7.0 8.2 7.8,. 8.4: 9.0'1 8.4! 8.0
I
.
, 14.914.816.216.014.7i17.2 15.7 15.018.418.8,17.815.516.4 16.316.7
2.7 2.7: 2.5 2..i 3.1 2.3 2.9 1 3.6 ,1 2.41, 4.81 2.5 2.9
. . 2.0 2.1:' 3.3!
1
0.2 0.4 0.3 0.3, 0.6 0.6 0.5' - - --, - --I - - 0.3 0.4 0.3 0.3 0.5 0.2 0.2 0.2 0.2 . 0.1 0.4. H 0.3 0.2 0,2-
■
' 0.41 0.4 0.3, 0.3 0.6! 0.2 0.2 0.2 5.4 0.4' 0.5 0.4 0.1 0.1 -
; 3.5 3.8! 4.1 4.9 4.8 1 3.6 4.5. 3.7 4.7 4.0 4.4 3.6 3.1 3.2 2.2
, 1.9 2.6 3. 1 3.4 2.9, 3.1! 3.2 2.4 2.4, 2.6 2.6; 1.9; 1.4 1.5 1.0
1.1 1.5: 2.62.6 1.6 2.3' 1.7 1.8 1.5 2.0 1.71 1.4 1. 0.7 0.8 -.
1
•
' 2.5 3.3 1 3.71t
3.3 2.5: 2.5, 2.6 1.8
1 3.2:
! 2.3. 3.5 , 3.7 3.8 2.9;2.4
•
:
i
1
; 5.9 4.6:! 3.0 3:t 3.8' 3.8 3.6 4.6 3.4 4.0, 3.6 5.6 5.4 . 6.2 6.6
1.0 3.2 3.6 3.t 3.i 3.8' 3.6 4.6 3.6 2.1; 2.9 2.11 1.6 1.0 1.0
1
1
1
i
1
'
1
'
. ' 2.9! 2.6 3.1 , 2.a 2.4 1.7; 2.0 2.8 2.0 2.5' 1.7; 2.tE 1.51 2.6 2.0
'
i,
'
1
'
1
30.829.226.4 123.725.7:22.8 24.825.9 27.625.024.6,29.330.1 ,29.2 32.2
1.
1
i
I
:
1
I
I 0.6 , 0.5 0.31 0.31 0.2, --, H - 4 -i H 0.2: 0.4 0.5 0.8
7 9.
Table 4-4.
Changes of main fatty acid composition (g) equivalent to 100g mixed
fatty acids of phospholipids during development of rainbow trout egg.
Number
of C
atoms Unfertiand
lized
double
bonds
0
1.0
C14
Ci0-
Days" after Fertilization
Eye I
Larva
appeared appeared '
1
10
2.7
:9
9.5
14.9
2.0
0.2
3.6
2.5 -
;11
5.9
-2.7
8.5
14.6
2.0
0.4
3.7
3.3
4.5
C29:1
0.9
2.9
30.8
3.2
2.6
28.9
:1
Cie
:1
:2
" :3
C20:
:8
:6
1.0
1.0
16.9
17.7
Increase
•
14.7
Hatch- 1/2 York ,Swinitq) Before
or
i ing . absorbed ; fry feeding decrease
1
2.6
8.2
12.7
2.1
0.2
3.9
2.5
2.5
2.5
1.8
18.8
0.6
0.6 '
0.5
9.4
1.2
8.9
1.5 :
8.9
5.2
7.5
1.6 ;
3.3
8.8
1.1
3.8
6.8
1.1
3.8
7.4
1.1
-I
-2.2
1.4
1.6
0.8
11.9
0.8
9.4 ,
1.5
6.4
2.3
1.6
1.0
13.8
35
I
10.4
1.5
0.4
1.8
2.1
.
60
30. '
1.7
II
.
20
1.9
1.9 ;
2.3 ;
0.9
1.4
13.0
I
I
50
:
1.5
71.4
1.2
2.8
1.6
1.1
2.5
1.0
1.4
1.0
13.2
65
0.4
1.2
13.2
1.2
13.3
0.4
- 0.6
9.0
1.5
- 8.7
- 1.2
3.5
- 6.0
7.6
1.3
1.0
0.8
3.0
-
0.5
- 0,4
0.9
- 2.0
-16.1
14.7
Relative ratio of fatty acid composition in triglycerides and
phospholipids of unfert il ized rainbow trout egg by their unsaturation.
Phospholipid
Triglyceride
Fatty Acid
(51 )
(5)
30
19
Saturated
'
25
50
Monoenoic
5
10
•
Dienoic
3
3
Trienoic
trace
'1
Tetraenoic
5
• 7
Pentaenoic
30
12
Flexaenoic
Table 4-5.
Table 4-6.
Lipids
Triglyceride
Phospholipid
Fatty acid composition of triglycerides and phospholipids
in unfertilized rainbow trout egg.
hiaturutsd Aulds
(5)
C14
C16
Cie
3
1
14
18
2
10.
Table 4-7.
Unsfiturated Aelds
Clea , Cia:t •
.30
10
3
15
Çiii:2
10
3
C.:.°:i
2
4
Composition of foodstuff for rainbow trout
before spawning' season (October-December)
Formulated Diet
Isaza
1964
70
. 20
Sen.
Skim dry milk
V itamin Mixture '
Fee'd Oil
0
2
. 10
1965
30
70
0
. -10
2
10
C20:5
4
4
Cmel
12 '
30
7.3
0.7
0.2
2.6
1.7
2.9
8C •
•
Lintel, Larva Eye
used appeared appeared
Hatching
Swim up
fry
Hatching
Unferti- Larva Eye
lized appeared appeared
4
i
(911
Swim up
fry
10
3
Cie
30
25
20
15
10
Cie:i
10
10
20
Days
lized
(ar)1
30
40
50
Larva Eye
Flatching
appeared appeared
1
60
10
70
20
39
40
50
60
Days offer Fertilization
atter Fertilization
Swim up
Unferli- Larva Eye
Hatching
lized appeared appeared
1•
(gr)
f ry
1
10
Swim up
try
3
ClEP2
30 Jà-
h\
C22 6
•
\
Czo
À
•
10[
10
20
30
40
50
O
60
Days cf ter Fertilization
10
20
Days
•
30
40
50
60
of ter Fertilization
The changes of individual fatty acid contents between triglycericles
and . phospholipids during development of rainbow trout egg. In ezich
figure, the curve 0-0 and
represented triglycericle and phos-
Fig 4-1.
•—•
' pholipid, respectively.'
•
-•
<3
Gas-chromatogram of mixed
fatty acids prepared from the
triglycerides of unfertilized
rainbow trou't egg.
Fig. 4-2.
Table 4-8.
Feed-oils
Fatty acid composition of some feed-oils for fish culture.
C18
Pollack liver oil
(Residue of molecular
distillation)
Finhack whale oil
Cuttlefish , liver oil
Feed oil A.*
Feed oil B.*
Soybean oil
C18
C18:1
C18:1
C18:2
C20:1
C20:5
C22:1
C22 . 5
C2218
13.2
2.5 10.9
25.7
1.5
11.5
9.4
4.5
1.7
6.1
13.6
15.8
13.5
14.1
10.0
2.5 12,5
3.3 6.6
2.7 9.5
2.3 8.2
2.7 0,5
31.8
16.6
22.3
25.3
19.6
3.0
2.1
2.0
L7
60.1
3.1
10.9
9.6
9.3
0.5
3.3
8.3
11.7
8.6
0
2.2
8.0
6.0
8.1
0
3.1
1.7
2.9
1.7
0
2,8
12.0
5.5
3.9
0
* Commercial name
Native (unfertilized)
1 '
AlkaH Denoturdfion
A
■
Fig. 5-1. The sedimentation patterns of
native and alkali-denatured yolk
proteins. Curves A and B are the
patterns obtained for native yolk
proteins in unfertilized eggs and
in the eggs in the eyed stage, re.
spectively. Curves C to F show
the process of alkali denaturation
of component 1; curve C, native
yolk proteins in unfertilized eggs
at pH 7.0; and curves D, E, and F,
the proteins denatured at pH 12.0
for 2 hours, at pH 12.5 for 2 hours,
and at pH 13.0 for 24 hours,
respectively.
4
82
10 -
9
Sm
HatchEye
Lana
Fertiup Fry
lization appeared opperred ing
1•Eystccit -e-±-Sac Fry Stage --*1
9
"A
8
6
71_
0
.
1.0
05
.
.
1.5
Protein Concentration (gm/100m1) -
.
_
Fig. 5-2. Sedimentation constant at
zero protein
concentration for unfertilized york protein
of rainbow trout egg.
/•
cr,
•
.2 4
c.)
—3
IF;
o
a)
2
-a)
T
f
I
•
1
I
4—Unfertilized Yolk
Derdrttred Yolk
r-1
5
•?e
•
55
2
2
•
—
o-1
02 .03 0-4 0-5 06 0-7 09 99
Protein
Concentration
(em/looml.)
Diffusion constants at zero protein
concentration for both unfertilized
and its alkali denatured yolk protein
of rainbow trout egg.
Fig. 5-4.
I
0
40 5 ■5 60
30
Days after Fertilization
10 20
'
Fig. 5-3. The changes in relative content of
components I, II, and III during the
development of embryo from fertilization to the swim-up fry stage. The
values are estimated from the peak
areas in the sedimentation patterns
for the yolk protein solution (20 m/.
of phosphate buffer, pH 7.0) from 10
eggs. The sedimentation analyses
were made at 59,780 r.p.m. and bar
angle = 75 0 .
83
go
Table 8-1. Fatty acid composition of eels.fed for a year with raw mackerel pike (A)
and with formulated diet added 10% feed oil (B).
oil
-
_
a
4.2.
10.5
3.5
12.4
23.4
42.4 .
2.3
2.3
6.8
10.1
12.0
28.0
43.9
'2.8
2.5
3.1
9.0
10.5
27.5
43.2
2.8
2.5
2.3
. 8.3 '
12.4'
5.1
:1
:3
C17
2.4
10.1
2:3
2.3
5,9
13.4
C15
u.
:3
,
:4
Czou
13
0.9
8.4
- 13.8
:4
' :5
Cnn
:3
:5
:s
C24:t.
Saturated
Monoene
Diene
Triene
lretreene
Pentaene
Iiexaene
'
era
Body oil
Visc
Liver oil
_
1.7
12.0
0.7
0.7
13.4
Cm
Liver
on Feed oil
-
5.5
0.5
0.8
18.6
- 6.2 .
0.9
1.3
,
1.3 •
24.8
1.9
0.9
0.7
7 .8
2.6
1.6
4.8
3.9
_
Cly
. oil
6.8
• 0.6
1.9
15.6
7.2
0.9
1.1 "
" 2.6
24.7
1.9
1.4
1.1
6.7
1.1
2.0
4.8'
4.7
6.9 ' "
C14
I . 'Viscera
•
double bonds pikè - body Body oil
.
B
A
Number of C '
atoms and Mackerel
2.8
0.4
-0.2
21.4
3.9
1.1
0.8
1.3
42.1,
1.8 .
0.6
3.3
3.9
0.3
0.3
12.2
9.7
1.3
1.9
1.6
22.8
1.1
0.6
2.0
10.9
2.2
4.1
1.6
2.3
1.2
10.9
9.3
2.0
10.4
1.9
'5.8
26.5
51.3. 2.9 ,
2.3
2.8
6.1
.
10.4
19.9
53.0
2.4
0.6
3.2
12.8
5.8
, 3.9
0.8
0.4
22.5
5.5
0.7
. 0.7
1.0
37.3
1.5
1.0
0.9
7.9
0.8
0:9
4.1
3.4
j
I
.
2.2
4.5 1
28.5
54.9
2.2
1.8
1.8
6.3
4.5
4.1
0.4
0.7
21.4
5.3
0.7
1.2
1.3
32.7
2.0
0.8
0.6 j
5.6
2.4
0.2
6.3
3.2
3.1
0.8
0.3
20.9
4.5
0.7
1.3
46.9
1.1
0.5
0.4,
5.5
0.9
3.0
2.0
.
2.7
. 8.4
1.7
.6.4
28.7
47.2
2.7
3.3
0.8
9.0
8.4
26.3
59.7
1.1
. 0.5
1.3
4.7
6.4
81, .
.111
nib
Table 6-2. Eatty•acid composition of eels fed for 70.days on formulated diet mixed
lvith 10% feed ôil P.
Feed oil
double bonds
Body oil
Ca *
.
C15
Cm
.:1
C11
.
'
.
C1e
.
,1
:2
Cmt
"
:a
• '
.
:5
:0
Viscela
Liver oil
oil
: 0.1
3.9
0.5
0.4
0.7
' 0.4 .
20.0
21.2
8.7 ,
11.0
1.2
• 1.1*
1.0
* 0.3
4.5.
'4.0. •
36.1
37.2
1.3
2:2
* 0.2
'
0.5
6.8
7.3 .
..
0.7 •
0.5
3.3
3.6
3.1
2.5
'
1.9
2.4
3.1
3.3 '
0.2
.
C14
:2
Phospholipids
T ri glyc eri cies
lquniber of C
atorns and
•
Saturate'
iVionoene
.
IMene
. j
Triene
,
Tetraene
Pe.ntaene
flexaene
:
4.8
0.3
0.6,
16.8
12.7
: 2,6
0.3 .
2.8
. 26.7
1.5
0.5
10.6
0.9
8.4
.
6,7'
1.1
: 2.7
4.8.j
'
.
' . 30.9
57.5
' 2.4
0.9 1
25.2
57.0
4.1
1.4
29.2
56.1
3.4 '
1.0 • !
-•
'
9.5
2.7
•
5.2
3.1
'
I
6.0 '
3.3
, Body oil Viscera oil Liver oil
• 0.1
4.7
0.4
0.4
21.5
8:8
0.2
3.8
0.5
21.5
10.0
.
0.2
5.1 •
46.6
3.6
0.5
3.6 .
. 0.8
1.7
1.9
:
1
0.6
1
3.4
!
5.3
38.3
0.9
1:
26.3
1.7.
8.7
•
5.8
1.5
7.5
2.4
3.3
9.0
2.6
3.9
1.4
4.2
30.8
61.5
30.7
60.1
0.9
0.5
5.3
1.9
i
4.0
4.2
0.1
3.3
0.3
0.5
17.9
8.6
0.2
'
4.6
;
0.6
0.6 .
20.8
8.1
1.7 •
0.6
'
0.7
5.7
41.7
0.9
'
I
- 5.1
1.5
4.5
1.3
1.2
6.7
32.1
43.2
3.4
1.5
28.2
57.0
0.9
1.5
10.8
9.0
5.7
6.7
,
••‘‘è :9+
•
Continued. Peed oil M.
Table 6-2:
Number of C
atoms and
double bonds
(45
•
C18
:1
5.6
.
A.2
22.0 ,t1
C18
.3
33.2
18.5
0.9
C20:1
6.0
:2
A
20 0
7.0
0.3
0.5 ‘1.1 ‘'.
0.2
0.4
' 4.0
3.6
40:3
41.0
5.8
6.0
0.7
0.8
5.3
5,8
• 0.3
:1
4.6
0.3
•
0.3
A
Cl/
Viscera
oil
Body oil
0.1
2.6
0.2
- 0.2
20.1
• 4.9
C12
• CI.4
•
0.3
0.5
2,2
2.0
1.3
3.6
A
• •
:4
.C22:1
2.5
2.0
1.9
1.1
2.4
25.4
48.5
19.2
0.9
31.9
55.4
6.5
0.7
3.5
4.2
:5
A
Saturate
Monoene
Diene
Triene
Tetraene
Pentaene
Iiexaene
Phospholipids
Triglycerides
Feed oil
:
3.5
2.5
•• ,
I
3.1
2:4
4,
0.1
4.0
0.2
3.4
0.4
0.3 :
,21.9 • f
0.2
18,9
8.1
0.7
0.3
0.6
0.6
4.0
47.6
3.8
36.3
5.2
0.5 :
0.6
4.3
0.2
0.7
.26.0
5.8
5.8
24.0
3.5
0.7
24.6
5.3
0.9
0.2
8.3
26,3
4.3
4.9
2.5
4.3
0.5
2.8
4.5
0.7
3.5
10.4
38.1
35.0
5.7
2.8
0.3
5.0
0.4
3.9
4.0
•
4.3
2.4
7.2
5.6
1.1
4.3
9.2
• 27.8'
29.1
56.1 •
1 53.8
4.4
6.1
1..1
• 0.4
0.5
3.5
3.1
3.0
3.6
30.9
48.2
5.9
1.1
36.8
36.3
3.5
4.3
6.7
10.9
9.2
6 mg
4
•Vitarnin B 6-11C1
28
Ca-pantothenate
0.6
Biotin
p-aminobenzoric acid 40
Ascorbic acid .-. 200
•. 4
Menadion
•
Body oil Viscera oil Liver oil
1.5
1.5
1.6
3.0
Table 6-3.
Vitamin B1 -HCI
Liver oil
7.2
Vitamin Mix.
Vitatnin
B2
Nicotinic acid
Inosit
•
Folic acid
Choline chloride
n-tocopherol
Vitamin
B12
20 mg
80
400
1.5
800
40
0.09
,
8.0
10.4