Aquaculture, 34 (1983) 115-143 115

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

NUTRITIONAL VALUES OF LIVE ORGANISMS USED IN JAPAN FOR

MASS PROPAGATION OF FISH: A REVIEW

TAKESHI WATANABE’, CHIKARA KITAJIMA’ and SHIRO FUJITA’

’ Laboratory of Fish Nutrition, Tokyo University of Fisheries - Konan, Minatoku, Tokyo
108 (Japan)
2 Nagasaki Prefectuml institute of Fisheries - Matsugae-cho, Nagasaki 850 (Japan)
(Accepted 31 October 1982)

ABSTRACT

Watanabe, T., Kitajima, C. and Fujita, S., 1983. Nutritional values of live organisms used
in Japan for mass propagation of fish: a review. Aquaculture, 34: 115-143.

The mass production of zooplankton, in particular the rotifer, Brachionus plicatilis,

and the brine shrimp, Artemia salina, is considered to be of vital importance for the rear-
ing of larval fish (up to 30-50 mm total body length) in Japan. Data on the proximal,
mineral, protein and essential fatty acid (EFA) contents of live food organisms are re-
viewed: the EFA content chiefly determines the dietary value for fiih larvae. The EFA
content of rotifers supplied with yeast during the culture period was less favourable for
larval fish growth than that of rotifers given marine Chlorella. The nutritional value of
yeast-fed rotifers may be improved either by the use of the recently developed w-yeast
(indirect method) or by feeding with a mixture of homogenized lipids and baker’s yeast
(direct method). Artemia could be classified into two types, marine - containing a high
content of 20:5w 3 (an EFA for marine fish), and freshwater - containing a high content of
18:3w 3 (an EFA for freshwater fish). The fish mortalities sometimes encountered with
Artemia may be related to this difference. Either type may be used for freshwater fiih
larval nutrition. For marine fish, the marine Artemia type is adequate but the freshwater
type should be fed together with marine copepods or should be enriched by feeding on
lipids with high w 3 HUFA contents.

INTRODUCTION

Recently, rearing techniques for various kinds of fish, and methods for the
mass production of living feeds have advanced markedly and, partly as a con-
sequence, the number of fish species in commercial production increases
every year. However, very few fundamental studies have been carried out on
the nutritional value of living feeds used in the production of juvenile fish.
Among various species of zooplankton, the rotifer, Brachionus plicatilis,has
been used most extensively as a live food for rearing various kinds of small
marine and freshwater larval fishes. The use of rotifers was pioneered by
Japanese workers some time ago (Ito, 1960). The most suitable diets for var-

0044-8486/83/$03.00 0 1983 Elsevier Science Publishers B.V.

116

ious developmental stages of some fish, according to our present knowledge,

are outlined below (Fujita, 1973, 1979).
The food schedule used most extensively in the production of larvae of
various fish in Japan is shown in Fig. 1. In hatched fish of body length
greater than 2.3 mm, rotifers are given as the initial diet and this is continued
for about 30 days after hatching. When fish reach 7 mm or more in body
length, marine copepods such as Tigriopus, Acartia, Oithona and Paracalanus
or, in their absence, Moina and Daphnia of freshwater origin, are fed to lar-
vae together with rotifers, as rotifers are slightly small for larvae of 7 mm.
Brine shrimp, Artemia salina, distributed commercially are very frequently
used as a food for larvae of many marine fishes when there is a shortage of
marine copepods. Larvae larger than 10 to 11 mm are fed on minced fish,
shellfish and shrimps or an artificial diet. When juveniles attain 30-50 mm
in total body length, larval production is considered to be finished.

Minced fish

Artemia
TigriopUS, Paracalanus
CoFPod’ Acartia. Oithona
6. plicatilis

6 5 lb 20 ;o 4b 5b Days
3.4 4.2 z3 9.0 15 30 mm

Fig. 1. Food schedule used most extensively in the production of larvae of various fish in
Japan.

As is seen from this schedule, rotifers have been used most extensively
and are very important as the initial live food for rearing larval fish. At
present, without the mass culture of rotifers, larval rearing of marine fishes
would be virtually impossible. Thus, this important organism, systematically
mass produced, has made fish larval production possible. However, some
problems have been encountered regarding the dietary value of these living
feeds. One of these problems concerns rotifers and the other Artemia.
Rotifers were mass-cultured by using marine Chlorella as a feed organism,
until baker’s yeast, Saccharomyces cereuisiae, was also found to be very
suitable for this purpose (Nozawa et al., 1972; Ohara et al., 1974). When
baker’s yeast was used as a food for rotifers, the culture density of the roti-
fers reached about 10 times that obtained by using marine Chlorella. Thus,
recently, the yeast has been used increasingly as the food for rotifers as the
production of juvenile fish has increased progressively in Japan from year to
year.
However, the rotifers cultured with yeast frequently resulted in sudden
heavy losses of larval fish (Kitajima and Koda, 1976; Fujita, 1977; Fukusho,
1977; Kitajima, 1978; Kitajima et al., 1979). It was found that these high
mortalities could be prevented by culturing the rotifers with both yeast and
marine Chlorella, or by culturing rotifers with yeast and then feeding them
 117

on marine Chlorella secondarily before feeding them to fish (Kitajima and

Koda, 1976; Fukusho, 1977; Kitajima et al., 1979). This was one of the
most important findings in the mass production of juvenile fish.
Our recent investigation on the relationship between the nutritional
quality of living feeds and the culture organisms used has demonstrated that
the content of essential fatty acids in the living feeds is the principal factor
in their dietary value.

PROXIMATE AND MINERAL COMPOSITION OF LIVING FEEDS

Proximate and mineral compositions of Brachionus, Artemia, Tigriopus,

Acartiu, Moina and Daphnia are shown in Tables I-IV (Watanabe et al.,
1978a). Culture media such as baker’s yeast and freshwater or marine
Chlorella greatly affected the proximate composition of Brachionus, but not
the mineral composition. When rotifers cultured with baker’s yeast were
compared with those cultured with Chlorellu, the water content was higher
in the former and the lipid content in the latter, suggesting a high gross
energy content in rotifers cultured with Chlorella. There were also no
marked differences in the mineral compositions of Tigriopus and Moina due
to the differences of culture media, although a slight change was observed in
the contents of trace elements, Artemiu produced in different locations
showed a similar mineral composition regardless of their origin, except that
the iron content was several times higher in Artemiu from South America
and Canada than in those from San Francisco. Roeder and Roeder (1966)
reported that a low dietary value of Artemiu as the sole feed was due to its
low iron content. However, in our analyses the iron content of Artemia
raged from 28 x 10-3to 295 X 10s3 mg/g and this may satisfy the iron re-
quirement of fish, although the content of iron differs from lot to lot or
from location to location.
A slight seasonal variation was observed in the content of trace elements
of Acartia caught in May, June and November.
These results show that minerals are not the principal factors in the diet-
ary value of living feeds.

NUTRITIONAL EVALUATION OF PROTEINS OF LIVING FEEDS

The nutritional quality of living organisms as protein sources was investi-

gated by determining their amino acid composition, digestibility, protein
efficiency ratio (PER), and net protein utilization (NPU) (Watanabe et al.,
197813). As shown in Tables V and VI, there were no marked differences in
amino acid composition of various kinds of living feeds, except for a low
threonine content in Artemia. Artemia nauplii are reported to be deficient
in histidine, methionine, phenylalanine, and threonine, whereas adult brine
shrimp are rich in all essential amino acids (Stults, 1974; Gallagher and
Brown, 1975; Claus et al., 1979).
TABLE I

Proximate and mineral compositions of the rotifer, Brachionus plicatilis, cultured with baker’s yeast, Saccharomyces cerevisiae, and
marine chlorella, Chlorella minutissima, at Nagasaki Prefectural Institute of Fisheries during 1975-1977 (wet basis)

Yeast Yeast + Chlorella’ Chlorella

1975 1976f 1977 1975 1976’ 1977 1975 1976’ 1977

I II I II I II

Moisture (%) 90.8 91.8 - 89.6 87.9 89.0 - 89.1 86.4 86.7 - 87.6
Crude protein (%) 6.0 5.5 - 7.2 7.7 7.4 - 7.9 7.9 8.0 - 7.8
Crude lipid (%) 1.4 1.7 - 2.3 2.8 2.2 - 2.3 3.7 4.2 - 3.8
Crude ash (%) 1.0 - - 0.4 0.7 - - 0.4 0.9 - - 0.5
Ca (mglg) 0.16 0.18 0.27 0.12 0.11 0.10 0.10 0.26 0.15 0.11 0.11 0.21
Mg (mg/g) 0.23 0.14 0.20 0.14 0.20 0.17 0.14 0.17 0.31 0.14 0.16 0.14
P (mg/g) 1.11 1.20 1.13 1.48 1.38 1.31 1.26 1.44 1.54 1.36 1.39 1.37
Na (mg/g) 3.27 0.16 0.21 0.41 1.51 1.29 1.51 0.30 2.06 0.26 0.29 0.29
E (mg/g) 0.61 0.19 0.19 0.35 0.60 0.54 0.45 0.12 0.62 0.53 0.37 0.23
Fe (rg/g) 26.0 25.8 38.2 15.9 28.6 9.5 11.0 52.5 27.4 9.7 17.5 43.3
En (rg/g) 8.6 7.6 7.8 7.4 9.8 6.9 7.6 9.8 9.9 4.3 6.9 8.2
Mn (rg/g) 0.7 0.6 0.9 0.4 0.8 0.8 1.2 1.1 1.1 1.1 0.7 1.1
Cu (rglg) 0.7 0.8 0.4 1.1 0.8 0.4 0.5 1.5 0.9 0.8 0.5 1.7

’ Rotifers cultured with both marine chlorella and yeast (1 g of yeast/lo6 cell per ml sea water per day).
’ The culture was conducted in duplicate 200 ton tanks (I and II).
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978a.
TABLE II

Proximate and mineral compositions of Artemia salina from San Francisco, South America and Canada (wet basis)

Larvae of San Francisco Eggs’ Larvae*

Brand’
San South San South
Tokyo Nagasaki Gifu Francisco America Canada Francisco America Canada

Moisture (%) 90.1 89.3 85.7 - - - 89.7 90.9 88.2

Crude protein (%) 5.8 6.6 8.7 54.4 51.5 47.5 6.1 6.5 6.8
Crude lipid (5%) 1.9 1.8 3.7 6.4 10.5 4.8 2.0 1.6 2.1
Crude ash (%) 1.0 1.0 0.8 6.3 13.0 15.3 1.2 1.0 1.5
Ca (mg/g) 0.21 0.22 0.45 3.73 2.21 1.41 0.23 0.24 0.41
Mg (mg/g) 0.24 0.35 0.26 2.80 2.53 5.59 0.44 0.20 0.68
P (mg/g) 1.51 1.53 1.75 7.60 6.95 7.63 1.33 1.21 1.44
Na (mglg) 1.56 1.54 0.61 6.13 31.91 28.58 4.02 1.43 4.93
K (mglg) 0.73 1.52 0.80 5.73 5.34 7.12 1.08 0.96 1.16
Fe (pelg) 40.3 28.4 40.4 1298.0 1276.6 1021.6 52.2 294.6 287.3
Zn (rglg) 18.5 16.0 18.3 91.2 96.0 61.4 16.1 21.1 24.1
Mn (rglg) 0.2 1.2 1.5 98.3 50.9 14.8 2.1 2.6 3.7
Cu (rglg) 1.6 2.9 3.2 10.6 9.1 15.9 0.6 1.1 1.9

1 Larvae, hatched out at the laboratory of Tokyo University of Fisheries (Tokyo), Nagasaki Prefectural Institute of Fisheries (Nagasaki)
and Mino Branch of Gifu Prefectural Fisheries Experimental Station (Gifu) respectively in 1975.
z Eggs from San Francisco, South America and Canada in 1977 and their larvae just after hatching.
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978a.
120

TABLE III

Proximate and mineral compositions of Tigriopus cultured under various conditions (wet basis)

Cultured under Tigriopus japonicus cuItured with

..
~~roa;ttc~e~~ Yeast Yeast + Yeast + S Yeast* Soy sauce
Chlorella formula cake
Nov.-Feb.
diet of
1975-1976
prawn
Dec. Nov. 1977’ Jun. Nov. Nov. 1977 Dec. Nov.
1976 I II 1976 1977 1976 1977

Moisture (96) 88.6 87.1 87.3 87.1 86.0 87.3 86.3 87.2 87.8 84.8
Crude protein (96) 8.1 8.5 9.2 9.1 9.1 9.0 9.8 8.7 9.2 9.6
Crude lipid (W) 2.6 3.2 2.2 2.4 3.6 2.8 3.1 2.6 1.7 3.0
Crude ash (%) 0.5 0.6 0.5 0.6 0.5 0.5 0.5 0.6 0.6 0.6

Ca (me/e) 0.11 0.26 0.25 0.18 0.30 0.15 0.25 0.40 0.36 0.50
Mg (mrlg) 0.23 0.35 0.21 0.26 0.28 0.23 0.27 0.33 0.38 0.31
P (msle) 0.94 0.92 1.27 1.39 1.24 1.31 1.39 1.41 1.46 1.42
Na (msle) 0.73 1.57 0.31 0.52 0.47 0.61 0.42 0.46 1.98 0.56
R (mglr) 0.66 1.07 0.63 0.77 0.89 0.84 0.63 0.68 0.96 0.86
Fe @g/g) 94.1 20.3 41.9 26.5 36.8 33.8 22.3 33.5 25.4 44.7
Zn &z/e) 11.4 9.4 32.0 15.8 8.1 12.3 40.6 24.5 8.5 40.4
Mn (w/g) 2.1 0.9 1.0 0.8 1.5 1.0 1.3 1.3 1.1 1.9
Cu trsle) 1.8 3.0 3.1 2.5 2.3 2.4 5.2 2.7 1.9 8.2

’ Tigriopus cultured using a 1 ton (I) or a 200 ton tank (II).

‘Baker’s yeast Saccharomyces cereuisiae supplemented with cuttlefish liver oil.
Reproduced with permission from Jpn. Sot. Sci. Fish.; Watanabe et al., 1978a.

TABLE IV

Proximate and mineral compositions of Acadia, Daphnio and Moina (wet basis)
Moina sp. cultured with
Acartia clausi Daphnia sp.
Yeast Yeast + Poultry
poultry manure
manure
May Dec. Jun. Jun. Feb. Oct. Dec.
1976 1976 1977 1975 1976 1976 1976
Moisture (W) 86.9 87.8 88.1 89.3 87.2 89.0 81.9
Crude protein (%) 9.3 8.6 8.5. 7.5 8.8 8.6 8.2
CNde lipid (46) 1.1 1.5 1.3 1.4 2.9 1.3 3.3
Crude ash (%) - 2.1 0.7 - -

Ca (m/g) 0.60 0.39 0.39 0.21 0.12 0.12 0.23

Mg (w/e) 0.91 0.90 0.76 0.12 0.12 0.11 0.18
P (m/r) 1.53 1.52 1.48 1.46 1.85 1.23 1.57
Na Owls) 6.91 5.66 6.63 0.74 1.09 1.46 0.56
K (m/n) 2.90 3.05 2.21 0.72 0.92 1.03 0.90
Fe trek) 37.3 61.0 11.5 72.2 46.4 38.0 175.8
Zn Wg/g) 98.2 130.4 39.0 12.8 10.0 9.4 17.2
Mn (m/g) 0.8 1.0 0.2 13.2 0.5 0.7 3.5
cu W&a!) 12.2 8.6 2.8 1.1 6.8 2.8 3.8

Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.: Watanabe et al., 1978a.

The digestibility of the protein of rotifers was as high as W-94%, irre-

spective of whether the culture organism used was baker’s yeast, freshwater
or marine Chlorellu; the digestibility of the Artemiu protein was slightly
lower than that of Brachionus for both carp (83%) and rainbow trout (89%)
 121

TABLE V

Amino acid composition of rotifer. Brachionue plicatilis, cultured with baker’s yeast, Saccharomyces
cerevidae and marine chlorella. Chlorella minutissima (Nagasaki), or freshwater chlorella, Chlorello
regularis (Gifu) (g/100 g crude protein)’

Nagasaki’ Gifus

1975 1976 1975

Yeast Yeast + Chlorelka Yeast Yeast + Chlorello Yeast Chlorella’
ChZorella4 ChZoreZZa4
lsoleucine 2.9 2.8 3.1 4.4 4.0 4.0 3.2 3.4
Leucine 5.5 5.3 5.6 6.9 6.1 6.2 6.2 6.1
Methionine 0.8 0.8 0.8 1.0 0.9 0.9 0.9 0.8
Cystine 0.7 1.1 0.8 0.7 0.7 0.7 0.9 0.6
Phenylalanine 3.5 3.4 3.5 4.5 4.1 4.1 3.9 3.9
Tyrosine 3.0 3.0 3.2 3.0 2.8 2.9 3.2 3.1
Threonine 3.5 3.1 3.4 4.0 3.5 3.4 3.4 3.2
Tryptophan 1.1 1.2 1.2 1.1 1.1 1.2 1.2 1.2
Vahne 3.6 3.5 3.8 4.4 4.0 4.2 4.0 4.2
Lysine 5.7 5.8 5.8 6.6 6.0 6.0 5.5 6.1
Arginine 4.2 4.5 4.6 5.2 4.6 4.8 4.4 4.6
Histidine 1.4 1.4 1.4 1.7 1.5 1.7 1.5 1.5
Alanine 3.2 3.2 3.7 3.9 3.5 3.5 3.9 3.8
Aspartic acid 7.7 7.5 8.0 9.8 8.9 8.8 8.5 8.0
Glutamic acid 8.9 8.8 9.3 10.1 9.7 9.5 10.1 9.8
Glycine 2.9 2.9 3.1 3.6 3.1 3.2 3.1 3.1
Proline 5.2 5.9 5.8 5.0 4.8 4.9 6.1 6.7
Serine 3.7 3.7 3.9 3.7 3.6 3.7 4.2 4.0

Total 67.5 67.9 71.0 79.5 72.9 73.7 74.2 74.1

’ Each sample was extracted twice with 80% ethanol. followed by diethyl ether before hydrobsis.
’ Rotifea cultured at Nagasaki Prefectural Institute of Fisheries.
s Rotifers cultured at Mino Branch of Gifu Prefectural Fisheries Experhnental Station.
’ Rotifers were cultured with both marine chlorella and yeast (1 g of Yeast/lo6 cell per ml sea water
per day).
’ Rotifers cultured with yeast were secondarily cultured with freshwater chlorella per 36 h.
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978b.

(Table VII). The value8 for PER and NPU of living feeds determined with
carp and rainbow trout were also high. The values for NPU of Bruchionus,
Artemia, Tigriopus, Moina and Daphnia were 70-80% of those obtained
with casein, indicating that these living organisms have a high feed value as
protein in sources for fish. These results agree well with the conclusion of
Ogino (1963) that various kinds of natural zooplankton are valuable pro-
tein source8 judging from their amino acid compositions. The gross energy of
the living feeds was proportional to the lipid content of the living organisms:
high in Brachionus cultured with marine Chlorella, and low in Acartiu.

NUTRITIONAL VALUE OF LIVING FEEDS FROM THE VIEWPOINT OF ESSEN-

TIAL FATTY ACIDS FOR FISH

Recent studies on essential fatty acid8 (EFA) in fish have demonstrated

that the EFA requirements of fish differ considerably from species to
to species. Rainbow trout require fatty acids of the linolenic family (~3)
122

TABLE VI

Amino acid composition of living organisms (g/100 g crude protein)

Amino acid Artemia’ Acartia Trigfiopus Moina

salina clausi iauonicus SP.
Isoleucine 2.6 3.5 2.5 2.5
Leucine 6.1 5.5 5.0 6.0
Methionine 0.9 1.5 1.1 1.0
Cystine 0.4 0.8 0.7 0.6
Phenylalanine 3.2 3.7 3.5 3.6
Tyrosine 3.7 3.6 4.0 3.3
Threonine 1.7 4.2 3.8 3.8
Tryptophan 1.0 1.1 1.1 1.2
Valine 3.2 4.5 3.3 3.2
Lysine 6.1 5.4 5.7 5.8
Arginine 5.0 4.3 5.2 5.1
Histidine 1.3 1.9 1.6 1.6
Alanine 4.1 5.4 4.9 4.9
Aspartic acid 7.5 9.0 9.0 8.3
Glutamic acid 8.8 9.5 10.8 9.8
Glycine 3.4 4.6 4.5 3.7
Proline 4.7 4.6 4.8 4.2
Serine 4.6 3.3 4.3 4.0

Total 68.3 76.4 75.8 72.6

‘Larvae just after hatching.

Reproduced with permission Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978b.

as EFA (Caste11et al., 1972a; Watanabe et al., 1974a; Takeuchi and Watana-
be, 1976), whereas carp, eel and chum salmon require not only linolenic
but also linoleic acid for good growth (Watanabe et al., 1975a, 1975b;
Takeuchi and Watanabe, 1977a; Takeuchi et al., 1979, 1980). On the other
hand, these fatty acids did not meet the EFA requirements of marine fish,
and highly unsaturated fatty acids, such as 20:5w3 and 22:6w3, had to be
supplied as EFA for them (Yone, 1978; Yone and Fujii, 1975a). Based
upon these results the dietary value of living feeds was investigated from
the viewpoint of EFA for fish.

Ro tifers

The relationship between nutritional quality of rotifers as a living food

and their culture organism, e.g. baker’s yeast or Chlorellu, was investigated
from the viewpoint of EFA for fish (Watanabe et al., 1978c). Table VIII
shows the fatty acid distribution of rotifers cultured with baker’s yeast,
marine Chloreka or both organisms, during 1975 to 1977 at Nagasaki Pre-
fectural Institute of Aquaculture. The same tendency was observed in the
fatty acid distribution of the rotifers each year. The most striking difference
was the content of EFA. The rotifers cultured with yeast were quite low in
TABLE VII

Nutritional evaluation of protein of various kinds of living feeds

Brachionus plicatilis fed on Tigriopus Dophnia Moina Artemia Acartia

japonicus sp. SP. salina clausi
Casein Yeast Yeast + Chlorelta
Chlorelh’

Digestibility of
proteins ’ (%)
Carp 90.3 92.1 89.1 82.9
Rainbow trout 1 g6-98 93.8 94.1 94.2 89.3
PER’
Cnrp 3.2 2.8 2.8 2.6 2.5
Rainbow trout 6.0 2.9 3.2 3.1 3.6 3.9 2.6
NPU’
Carp 100 78 76 73 66
Rainbow trout 100 68 72 16 IO 74 70
Gross energy 6.3 5.2 6.4 6.1 6.3 5.2 4.1
(k&/g dry sample)

’ BMChiOnUs plicatilia fed both marine chlorelln and baker’s yeast (1 g of yeast/lo’ cell per ml sea water Per day).
‘Experiments were conducted using carp and rainbow trout weighing 50-100 g at water temperatures of 23-24OC in
carp and of 18-19’C in rainbow trout.
‘Carp and rainbow trout weighing l-2 g were fed on diets containing living organisms as Protein SoUl’CeS.at B 20% Pro-
tein level for 15 days at 22-26°C in carp and at a 30% protein level for 18 days at 18-19’C in rainbow trout, respectively.
‘The total amount of metabolic N and endogeneous N was calculated as 9 mg in carp and 16 mg in rainbow trout, re-
spectively. The values for NPU were expressed as the relative values to those obtained with casein.
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.: Watanabe et al., 1978b.
124

TABLE VIII

Certain fattyacids of total lipids from rotifer Brachionus plicatilis, cultured with baker’s yeast,
Saccharomyces cerevisiae. and marine Chlorella at Nagasaki Perfectural Institute of Fisheries during
1975 to 1977 (area %)

Fatty acid November 1975 May 1976 May 1977

Yeast Yeast + Chlorella Yeast Yeast + Chlorella Yeast Yeast + Chlorello

Chlorella Chlorello Chlorelfa
16:0 6.1 4.2 14.4 7.1 13.2 19.4 8.7 11.7 16.8
16: 1~7 27.2 26.7 20.4 26.5 22.6 22.4 24.2 16.6 24.3
18:0 3.8 4.4 2.2 4.3 3.6 1.9 4.8 6.0 1.7
18:lw9 26.8 25.8 10.1 29.1 21.5 11.0 33.9 22.8 10.1
18:2w6 8.9 5.1 4.7 6.9 6.3 3.4 5.8 10.4 3.2
18: 3~3 0.6 0.6 0.1 0.2 0.5 0.2 0.6 2.2 0.4
20: 1 3.6 3.4 1.7 4.2 4.1 2.3 6.0 3.3 2.4
20:3w3
20: 4~6 2.0 2.3 4.1 0.9 3.0 4.2 0.4 2.3 4.4

20:4w3 0.4 0.6 0.2 0.4 0.4 tr 0.5 0.6 0.2

20: 5w3 -1.9 11.8 27.7 s4 22.8 l.ou 24.1
22:l 0.9 2.1 1.8 0.9 0.4 0.4 1.7 1.5 1.3
22: 5w3 0.3 1.8 3.0 tr 2.9 3.4 0.2 1.7 3.8
22:6w3 0.5 0.5 tr tr tr tr 0.5 0.9 0.5

Zw3 HUFA 3.1 14.7 30.9 2.1 14.4 26.2 2.2 11.3 28.6

Lipid % 1.4 2.8 3.7 1.7 2.2 4.2 2.3 2.3 3.8

03 highly unsaturated fatty acids (w 3 HUFA) such as 20:5w3, and high in

monoenoic fatty acids such as 16:l and 18:l. Those cultured with marine
Chlorellu were found to contain a high amount of 20:503, which is one of
the EFA for marine fish (Yone and Fujii, 1975b). The rotifers fed on both
yeast and Chlorellu showed average values. For larval production of red sea
bream at this Institute rotifers cultured with both yeast and Chlorella are
generally used. These results may explain why rotifers cultured with yeast
are always inferior to those cultured with marine Chlorella in their nutritio-
nal quality as a living feed.
Table IX also shows certain fatty acids of total lipids in rotifers fed on
yeast and freshwater Chlorellu at Gifu Prefectural Fisheries Experimental
Station. These rotifers were used in the production of juvenile sweet fish
(ayu), Plecoglossus ultiuelis. The rotifers cultured with yeast were additional-
ly fed on freshwater Chlorellu for 1.5 days before feeding them to ayu. The
same fatty acid distribution was observed in the rotifers cultured with yeast
at this station as in those at Nagasaki. On the other hand, the fatty acids of
the rotifers cultured with freshwater Chlorellu were quite different from
those cultured with marine Chlorellu. The rotifers fed on freshwater
Chlorellu were high in 18:206 and 18:3w3 and low in w3 HUFA, indicat-
ing that marine, rather than freshwater, Chlorellu should be used as culture
organisms for rotifers intended as a living feed in the production of juvenile
marine fish (Watanabe et al., 1978c). These differences in the concentration
of 20: 503, an EFA for marine fish, were also found to be attributable to
 125

TABLE IX

Certain fatty acids in the total lipids from rotifers cultured with baker’s yeast, freshwater
chlorella, Chloreila regularis, and photosynthetic bacteria (PSB) at Gifu Prefectural
Fisheries Experimental Station

Fatty acid November 1975 December 1976

Yeast Chlorella Yeast Chlorella Yeast + PSB
I II
16:0 6.7 8.9 6.8 9.3 9.9

5.9 18.5. 19 3 36.5

18~3~3 0.6 10.2 1.2 3.7 7.7 2.1
20: 1 0.6 0.3 4.9 3.4 2.9 3.0
0.6
20~4~3 0.4 1.1 tr
20~5~3 2.0 1.9 2.0 1.9
22~5~3 - 0.3
22~6~3 1.0 - - -

the different fatty acid compositions of yeast and marine or freshwater

Chlorella (Watanabe et al., 1978c).
Tables X and XI list certain fatty acids in the total lipids of the culture
organisms. The baker’s yeast used for the mass culture of the rotifers is
commercially distributed by three companies in Japan. Each yeast shows a
very similar and simple fatty acid distribution. They contain a fairly high
amount (52-82s) of monoethylenic fatty acids, 16:l and 18:1, and no
w3 HUFA. On the other hand, marine Chlorella contained a high level of

TABLE X

Certain fatty acids of total lipids from baker’s yeast, Saccharomyces cereuisiae

Fatty acid Kaneka Kyowa Oriental

1975 1977 1977 1977
14:o 0.3 2.2 1.1 3.1
16:0 8.3 16.8 11.2 20.0
16:lw7 38.2 32.8 14.2 27.2
18:0 4.1 3.4 8.4 4.7
18:lw9 43.9 28.5 38.0 26.1
18:2w 6 2.8 7.6 15.1 10.9
18:3w 3 0.5 1.8 6.4 3.2
2O:l 0.2 tr 1.6 0.8
126

TABLE XI

Certain fatty acids of total lipids from marine Chlorella

Fatty acid Nagasaki Yamaguchi Hiroshima Kumamoto

Chlorella minu tissima Chlorella Chlorella Nannochloris
SP. coccoides
Sept. 1977 May 1976 Jul. 1980 “uzgaris
14 :o 4.8 4.3 6.9 4.8 5.2 5.6
16:0 26.1 22.5 20.6 20.2 19.7 11.1
16:lw7 26.3 22.3 30.7 29.5 30.5 25.2
18:0 1.1 1.0 0.1 tr 0.7 0.1
18:lw9 6.2 3.1 2.5 8.6 2.7 3.5
18:2w6 2.3 3.4 3.6 4.1 2.4 2.5
18:3w 3 0.2 0.1 0.1 tr 0.2 0.1
20 :l 0.1 0.1 0.1 - - tr
20:3w3
20 :4w 6 I 3.9 4.7 2.9 2.4 3.6 4.9
20:4w3 - 0.1 - tr
-205~3 -24.8 31.8 27.3 26.6 27.8 237 8
22~5~3 - - 1.7 -
22~6~3 - - -. - 0.3 -

20:5w3. These results suggest why the rotifers cultured with yeast always
contain few w3 HUFA whereas those fed on marine Chlorellu always contain
high levels of w3 HUFA. On the other hand, the fatty acid composition of
freshwater Chlorellu is quite different from that of marine Chlorella. The fresh-
water Chlorella contained high amounts of 18:2w6 and 18:3w3, but waslow
in 03 HUFA (Table XII). Consequently, rotifers cultured with freshwater
ChZorelZa had high levels of 18:2w6 and 18:3w3, directly affected by the
fatty acids of the Chlorellu. At first we postulated that the 18:3w3 contain-
ed in the freshwater Chlorellu is converted to 20:5w3 in the rotifers, as ob-
served in freshwater fishes (Caste11 et al., 1972b; Watanabe et al., 1974b;
Takeuchi and Watanabe, 1977a, 1977b), but this is not true, as we demon-
strated later (Watanabe et alJ979).
These results have shown that the fatty acid composition of rotifers is
readily affected by the fatty acids of the culture organism, and that the con-
tent of w3 HUFA in the rotifers chiefly determines their dietary value as a
living feed. Further experiments were conducted to verify the relationship
between the dietary value of rotifers and their w3 HUFA content by feed-
ing them marine Chlorellu or freshwater ChZoreZZa(Kitajima et al., 1979;
Watanabe et al., 1979). Fig. 2 shows the effect of secondary culture with
marine Chlorellu on the fatty acid distribution in the total lipids in rotifers.
When marine Chlorellu was used as culture organism, the low level of w 3
HUFA in the yeast-fed rotifers increased in proportion to the length of the
culture period due to incorporation of 20:503 from marine ChZorelZu;the
 127

TABLE XII

Certain fatty acids of total lipids from living and dried freshwater Chlorella

Fatty acid Living Dried sample

Chlorella Chlorella Chlorella
regularis regularis sp.
1976 1975 1976 1977 1976
14:o 0.5 1.0 0.7 0.3 1.2
16:0 16.9 11.1 6.3 23.5 13.0
16:lw7 2.7 1.7 3.5 2.0 23.5
16:2 18.0 15.4 26.3 12.3 3.7
17:o
16:4w 3 68
-_ 37.7 37.3 7.0 2.3

18:O 4.1 0.5 0.8 5.6 6.6

18:lw9 3.5 1.6 0.7 3.5 6.1
18:2w6 37 3
A ;11 1 8.3 32.9 9.7
18:3w3 9.1 14.4 12.0 9.8 13.5
18:4w3 > - -
0.1 0.3 2.6
20:o

2O:l 0.1 tr 0.4 0.1 -

20~3~3 tr -
> 0.6 tr 0.1
20~4~6
20:5w3 0.2 tr 0.3 0.2 1.0

22:l - tr 0.4 0.6 -

“i i “i i 6 .-1 i bh
Oh (II 1.5h (II) 24h IHI

Fig. 2. Effects of secondary culture with marine Chlorella and starvation for 0 (I), 1.5 h
(II) and 24 h (III) after feeding Chlorella in each period on the fatty acid distribution of
the total lipids in the rotifer, Brachionus plicatilis. Reproduced with permission from
Bull. Jpn. Sot. Sci. Fish.; Watanabe et ai., 1979.
128

level of 20:503 reached about 12% in 6 h of feeding. This level is almost

equivalent to that of rotifers cultured with both yeast and Chlorella, which
are used for the production of juvenile fish at Nagasaki. Furthermore, the
dietary value of the rotifers for red sea bream larvae was found to be signi-
ficantly improved by secondary culture with marine Chlorella for more than
6 h, as shown in Table XIII. When secondary culture was continued for 7
days, the concentration of 20:5w3 reached a maximum at around 28% in
2 days of feeding. The percentage of 18:l showed a reverse trend to that of
20:50 3. Thus it was found that rotifers can readily incorporate u3 HUFA
from the culture organisms.
TABLE XIII

Effect of secondary culture with marine chlorella, Chlorella minutissima, on the dietary
value for red sea bream larvae of rotifers cultured with baker’s yeast

Living No. of Total body length Rate of Survival at

feed fish at the end of survival activity test
feeding (mm) (%) @)
Experiment I
Y-rotifer 23 000 5.38tO.53 20.2 9.2’
YlOmC 23 000 6.1820.49 58.7 54.3
Y30mC 23 000 7.68r0.61 71.7 67.8
Y60mC 23 000 7.79kO.76 76.1 60.7
Y2hC 23 000 8.01kO.54 65.4 94.3
C-rotifer 23 000 8.76kO.55 79.8 99.6
Experiment II
Y-rotifer 24 000 4.56kO.30 22.1 45.6=
Y2hC 24 000 5.98+0.52 49.5 79.2
Y6hC 24 000 6.31tO.54 50.9 90.7
Y6hDC 24 000 5.03kO.45 50.8 82.4
Y 12h C 24 000 6.76kO.38 49.6 87.9
Y 24h C 24 000 7.03kO.81 60.4 96.1
C-rotifer 24 000 7.15+0.52 58.1 92.7

‘Survival rate during 24 h after the fish were moved to another aquarium at the end of
the feeding experiment.
*Survival rate during 24 h after 500 fish were lifted out of the water with a scoop net for
5 s and moved to a 30-liter tank.

When freshwater Chlorella were fed to the rotifers, the content of 20:5u3
did not increase but that of 18:2u6 and 18:3w3 did do so in proportion
to the culture period (Fig. 3). These fatty acids also reached a maximum in
a 2-day feeding. This result indicates that 18:3w3 is not converted to
20:5w3 in the rotifers as suggested earlier. Table XIII shows the effect
of secondary culture with Chlorella on the dietary value of rotifers. Larval
red sea bream given the yeast-fed rotifers showed a low growth rate and a
high mortality. The food value of the rotifers fed on yeast was found to be
effectively improved by secondary feeding with marine Chlorella (Kitajima
 129

18:3iu3

0.5 i i ii -’ i
Days

Fig. 3. Changes of fatty acid distribution of total lipids in the rotifer, Brachionus
plicatilis, due to secondary culture with living freshwater chlorella, Chlorella regularis,
for 10 min to 7 days. Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe
et al., 1979.

et al., 1979). Larvae fed on the rotifers cultured secondarily with marine
Chlorella for more than 6 h showed good growth and a high survival rate,
comparable with those of the larvae fed on the Chlorellacultured rotifers.
On the other hand, the dietary value of the yeast-cultured rotifers was not
much improved by secondary culture with freshwater Chlorellu. Commercial
spray-dried freshwater ChZoreZZuwas found to be suitable as a food for roti-
fers (Hirayama and Nakamura, 1976). However, these results show that
marine ChZoreZZashould be used as culture organism for rotifers intended as
a food for marine larval fish. These results also indicate that the content of
w3 HUFA in the rotifers is a vital factor in their dietary value as a living
feed and that the high mortalities observed frequently in various kinds of
fish larvae receiving yeast-fed rotifers as their sole feed is due to an EFA
deficiency in the fish.
Based upon these results, a new kind of yeast has been developed as a
culture organism for rotifers in order to improve upon the nutritional value
for fish larvae of rotifers cultured on baker’s yeast (Imada et al., 1979).
This new type of yeast (designated as o-yeast) was produced by adding fish
oil or cuttlefish liver oil as a supplement to the culture medium of baker’s
yeast, resulting in a high content of lipid and 03 HUFA, the EFA for marine
fish (Table XIV). The rotifers cultured with w -yeast were high in lipid con-
tent in general, together with w3 HUFA, as a result of the oils added to
the baker’s yeast. The incorporation of w3 HUFA from w-yeast reached a
maximum at around 12 h of feeding (Fig. 4). Furthermore, the dietary
value of the rotifers to fish larvae was significantly improved, compared to
that of rotifers cultured on marine ChZoreZZaas shown in Table XV (Kitajima
130

25-
l&l

i .

20- ",

. 20:5+22:6
ii? .
- 15-
rr
8

E lo-
ym

5- 6
Lipid
L-L L L

II-
0 0.5 1 2 3 7

Culture period in days

Fig. 4. Changes of fatty acid distribution of total lipids from the rotifer,

8 -10
3 -4

1-_ 9 -12

22:5w 3 0.9- 1.4 o-o.4 2 -3

22~6~3 12.8-15.6 7 -9

zw3 HUFA 33.5-35.8 25 -26

Lipid % l.O- 1.6 12.3-15.6 1.4-1.9 3.3- 5.4

 131

TABLE XV

Comparison of growth and survival rates of larval red sea bream fed on rotifers cultured
with respectively yeast or w-yeast.

Rotifer No. of Total length Rate of Survival at

used fish at end survival activity test
(mm) (%I (%I

Expt. I w -Yeast 30 000 9.281tO.77

Yeast 30 000 7.1OkO.78 13.0 12.5

Expt. II w -Yeast 15 000 10.11*0.87 76.2 92.9

Chlorella 15 000 10.21?1.60 57.1 91.7
Y12hC 15 000 9.11k1.24 27.9 93.2

Expt. III w -Yeast 24 000 10.32tr1.26 76.9 92.5

Chloreiia 24 000 9.78* 70.1 91.5
Y 3hC 24 000 8.85+ 1.09 27.6 55.8

Expt. IV’ w -Yeast 10 000 10.91+0.94 95.5

Yeast 10 000 6.24+0.62 3.2 45.9

‘Black sea bream.

et al., 1980a, 1980b; Oka et al., 1980). These results clearly indicate that
rotifers grown on the newly developed yeast have a superior food value to
those grown on the original yeast. Arakawa et aL(1979) also .found that
rotifers grown on w-yeast had a superior food value for larval puffer, Fugu
rubripes rubripes. Recently, Fukusho et al. (1980) mass-cultured Tigriopus
with w-yeast and found that its nutritional quality for mud dab was much
enhanced. This method may be called the indirect method for improving
the dietary value of living feeds.
Another method has also been developed (Watanabe et al., 1982). This
is called the direct method. Lipids containing w3 HUFA were homogenized
with a small amount of raw egg yolk and water and the resulting emulsion
was fed directly to rotifers together with baker’s yeast (Fig. 5). As shown
in Fig. 6, rotifers took up lipids very easily and the concentration of
w3 HUFA reached a maximum between 6 and 12 h of feeding, as observed
in the indirect method. The two methods, direct and indirect, are also found
to be very effective for improving the dietary value of other living feeds. By
using both methods, it was found possible to further improve the dietary
value of living feeds by allowing them to take up from the culture medium
not only w3 HUFA but also fat-soluble vitamins together with lipids
(Watanabe et al., 1982).
132

50
Rotifel
f

10

z!
:
0 3 6 12 24

Culture period in hours

Fig. 5. The direct method for improving the dietary value of living feeds.

Fig. 6. Incorporation of lipids emulsified with various kinds of reagents (A, B, C and D)
in rotifers by the direct method. Reproduced with permission from Bull. Jpn. Sot. Sci.
Fish., Watanabe et al., 1983.

Artemiu

The nauplii of Artemiu salina have been widely used as a food in the pro-
duction of juvenile marine fish (Morris, 1956; Kurata, 1959; Fujita, 1962;
Hirano and Ohshima, 1963; Shelboume, 1964; Riley, 1966). However,
feeding with Artemia alone frequently resulted in high mortalities in various
marine fish (Fushimi, 1971; Fujita, 1973; Kitajima, 1978), although this
phenomenon depended upon the fish species as well as the site of production
of Artemia. Some species of flounder, one species of mullet (Liza haemoto-
cheilu), one salmonid (Plecoglossus altiuelis) and some gobiid fish are not
easily affected, but the larvae of yellowtail (Seriola quinquerudiata) are very
susceptible to this phenomenon (Fujita, 1972, 1973). In addition, many
investigators have reported heavy losses of larval prawns, crabs, and marine
fish fed on Artemiu nauplii from Utah (Slobodkin, 1968; Little, 1969;
Reeve, 1969; Bookhout and Costlow, 1970; Wickins, 1972). The dietary
value of Artemia nauplii was found to be improved when the nauplii were
fed to fish with marine copepods such as Tigriopus and AcurtM (Fukusho,
1974).
Recently, in Japan, a large number of red sea bream fry have been pro-
duced by using rotifers cultured with marine Chlorellu. It was found that
feeding the fish with yeast-fed rotifers resulted in various pathological syn-
dromes, such as a dark body color, lack of appetite, lethargy and heavy
mortalities as described above (Kitajima and Koda, 1976; Kitajima, 1978;
Kitajima et al., 1979; Fujita, 1979). The chief cause of these syndromes was
found to be an EFA deficiency in the fish (Watanabe et al., 1978a, 1979).
 133

The symptoms observed in these red sea bream were very similar to those
induced by Artemia nauplii. At first Watanabe et al. analyzed fatty acid
compositions of Artemia eggs and nauplii from different locations and found
that Artemia could be classified into two types according to the fatty acid
composition; one (the freshwater type) contained a high concentration of
18:3w3, which is an EFA for freshwater fish, and the other (the marine
type) was high in 20:503, which is one of the EFA for marine fish (Tables
XVI-XVIII) (Watanabe et al., 1978d). Furthermore, Artemia of the marine
type were found to be a satisfactory food for juvenile red sea bream, and the
dietary value of the nauplii was improved by feeding them marine Chlorella
and u-yeast, both containing substantial amounts of the EFA required by
marine fish. These results suggest that the class of EFA contained in Artemia
is the principal factor in the variation in food value of Artemia to fish, as
demonstrated in the case of rotifers. Wickens (1972) reported that the food
value of Artemia from Utah was improved by allowing them to feed on
Isochrysis galbana. This may be due to the incorporation of w 3 HUFA from
I. galbana, which generally contained high amounts of 20:5w3 and 22:603
(Watanabe and Ackman, 1974). Moreover, the food value of Artemia is ef-
fectively improved when they are fed to fish together with Tigriopus or

TABLEXVI

CertainfattyacidsoftotallipidsinArtemia eggs and naupliifrom San Francisco

Fatty acid 1979 1980 1981

A A B C D E F' G' H' A B C
14:o 2.3 3.5 2.9 3.6 1.3 2.1 2.2 0.6 0.7 1.6 1.1 0.7
16:0 13.3 26.6 25.3 25.9 14.9 23.7 9.2 11.0 12.2 15.2 13.6 10.6
16:1~7~ 16.4 16.3 15.7 12.9 5.5 7.4 14.8 3.8 10.4 10.5 4.3 5.4

18:O 2.4 5.1 5.1 3.7 3.5 4.1 2.0 3.3 3.2 2.9 3.2 3.0
18:lw9' 28.2 25.8 27.6 19.8 28.0 23.7 19.1 26.7 34.9 28.5 27.1 26.3
18:2w6 8.3 2.6 2.9 2.5 6.3 5.4 8.3 8.9 6.6 7.1 6.1 7.6
18:3w6 0.9 0.5 0.4 0.6 - - 1.2 - 0.4 0.6 - 0.8
18:3w3 2.3 3.3 4.2 4.8 22.4 14.7 5.4 27.6 17.2 17.2 28.1 27.0
18:4w3 0.2 0.3 0.9 1.1 3.2 3.4 0.6 6.0 2.5 2.7 3.6 5.2

2O:l 0.1 0.9 1.2 1.1 0.3 1.0 tr 0.5 0.5 0.4 0.4 0.5
20:2w6 0.1 1.2 1.2 1.0 0.3 0.6 tr 0.1 0.1 - - 0.1
20:4w6 7.7 2.7 0.7 0.6 0.8 0.8 3.6 1.1 1.4 1.4 0.7 1.3
20:4w3 - 0.1 tr tr 0.2 0.5 0.3 0.9 0.3 0.4 - 0.9
20:5w3 7.5 3.9 1.7 0.9 2.7 0.6 6.8 0.3 3.5 3.6 2.4 2.1

22:l 0.7 0.5 0.3 0.6 0.3 tr - 0.3 - 0.2 tr

22:4w6 - 0.3 0.2 0.4- - 0.2 0.1 - - - 0.2
22:6w3 0.1 0.4 0.1 0.2 0.1 0.1 0.2 - tr - - -

Zw3 HUFA3 7.6 4.4 1.8 1.1 3.0 1.2 7.3 1.2 3.8 4.0 2.4 3.0

Lipid % 2.g_ - - - - 1.9 3.9 2.3- - 3.5

'Naupliijust after hatching.

*Smallamounts of the other monoenes wereincluded.
'C20:3 < w3 fattyacids.
134

TABLE XVII

Certain fatty acids of total lipids in Artemia eggs from Brazil and Australia in 1980

Fatty acid Brazil Australia

A B C D E E’ F
14:o 3.3 3.4 2.1 3.6 2.4 1.7 2.0 1.6
16:0 16.0 18.2 13.7 18.0 14.7 12.2 13.7 13.9
16:lw7’ 18.6 14.4 13.8 14.6 14.7 12.8 14.1 9.9

18:O 1.9 2.9 3.2 2.8 2.7 4.1 2.6 2.8

18:1wg2 21.8 23.7 28.9 16.2 26.6 30.7 28.3 33.3
18:2w6 7.2 6.4 8.5 3.1 7.7 9.3 11.8 5.2
18:3w6 1.9 3.2 0.8 5.1 - - - 0.1
18:3w3 3.3 1.1 3.2 0.9 3.6 3.3 2.7 10.1
18 :4w 3 0.3 tr 0.6 tr 0.7 0.4 0.9 3.8

20: 1 0.9 1.2 0.4 1.9 0.5 0.5 0.3 0.4

20:2w 6 0.5 0.3 0.1 0.2 0.1 - -- 0.2
20:4w6 2.7 3.2 4.5 3.2 4.0 4.6 3.6 1.1
20: 4w 3 0.1 tr 0.4 tr 0.3 0.4 0.2 1.0
20:5w3 3.9 3.5 5.9 4.6 5.8 6.5 5.8 8.6

22:l 0.7 1.0 0.4 1.8 0.5 -.- - tr

22:4w6 0.4 0.5 0.1 0.9 - - - tr
22~6~3 0.4 0.6 tr 1.6 0.1 - 0.2 0.2

c w 3 HUFA” 4.4 4.1 6.3 6.2 7.2 6.9 6.2 9.8

Lipid % - 5.4 - - - - 7.9

‘Nauplii from egg E.

‘Small amounts of the other monoenes were included.
‘Czc:s < w 3 fatty acids.

Acartiu (Fukusho, 1974), both rich in 20:5w3 and 22:6w3 (Watanabe et al.,
1980).
Watanabe et al. (1980, 1981b) conducted further experiments in order to
improve the dietary value of Artemiu nauplii of the freshwater type by the
direct and indirect methods, both used for improving the dietary value of
rotifers. As shown in Fig. 7, Artemia nauplii also took up lipids very easily
by the direct method, and the concentration of 03 HUFA reached a maxi-
mum between 6 and 12 h of feeding as observed in the indirect method. The
dietary value of the nauplii to fish larvae was found to be improved by
incorporating w3 HUFA from emulsified lipids, and was proportional to the
w 3 HUFA content in the nauplii (Tables XIX-XXI). On the other hand, the
dietary value of the nauplii fed on baker’s yeast alone or on corn oil, con-
taining few 03 HUFA, was very low for marine fish. Although any type of
 135

(%)
25
Artemia
w3HUFA
B
20 E

2 D
;; 15
u.

?
s 10

oa
e
.P 5

2 E

0 3 6 12 24

Culture period in hours

Fig. 7. Incorporation of lipids emulsified with various kinds of reagents (A, B, C and D)
in Artemia nauplii by the direct method. Reproduced with permission from Bull. Jpn.
Sot. Sci. Fish.; Watanabe et al., 1982.

TABLE XVIII

Certain fatty acids of total lipids in Artemio eggs from Tien-tsin during 1979 to 1981

Fatty 1979 1980 1981

acid
A’ B’ A B C A B C A
14:o 0.9 0.8 3.0 2.8 2.0 5.0 5.5 2.1 2.0
16:0 9.7 9.3 12.1 12.7 12.7 23.0 21.2 12.5 13.1
16:lw7’ 13.6 13.4 22.6 24.0 22.4 24.7 22.8 20.1 19.1

18:0 6.0 6.0 3.5 2.9 3.3 4.4 3.8 3.2 3.3
18:1wg2 33.5 33.8 26.2 20.2 28.3 22.1 17.4 24.9 25.3
18:2w6 4.4 4.4 4.1 3.8 4.3 1.6 2.2 4.2 5.0
18:3w3 5.3 5.1 5.5 6.0 5.1 0.4 0.6 6.4 6.6
18:4~; 3 0.6 0.6 0.9 1.0 0.7 0.4 0.9 1.0 1.3

2O:l 0.7 0.7 0.2 0.4 - 1.7 1.9 0.4 0.4

20:4w6 2.8 3.0 1.2 1.1 1.5 0.8 0.7 1.8 1.4
20~4~3 0.7 0.7 - - - - - 0.2 0.1
20:5w3 13.0 13.2 9.2 10.2 11.3 1.9 1.3 10.9 9.3

22:l - -. -- 0.6 0.6 tr 0.7

22:6w 3 - - - 0.2 0.3 tr -

xw3 HUFA 13.7 13.9 9.2 10.2 11.3 2.1 2.2 11.1 9.4

Lipid % 4.2 4.4 3.7 3.9 5.2 - 9.3 2.5

‘Nauplii from egg A and B.

‘Small amounts of the other monoenes were included.
136

TABLE XIX

Improvement of dietary value of Artemia for fIatfiih by the direct method (Expt. I)’

Lipid ~3 HUFA Total body Average Survival Normal fish

given to in length (mm) body wt. rate in activity
Artemia Artemia (%) test (%)
Initial Final (mg)
(%)

Cuttlefish 0.40 7.36 12.36 13.7 67.6 80.0

liver oil
Control2 0.05 7.30 11.15 9.8 35.6 13.3
Corn oil 0.05 7.16 9.86 5.8 27.1 0

‘Feeding period was 19 days.

‘Nauplii just after hatching (48 h).

TABLE XX

Improvement of dietary value of Artemia for rock sea bream by the direct method (Expt.
II)’

Feed given w 3 HUFA Total body3 Average body3 Survival Normal fish
to in length (mm) weight (mg) rate in activity
Artemia Artemia (%) test (%)
Initial Final Initial Final
(%)
w-yeast 0.30 9.7 20.4 9.0 145.1 78.3 86.7
Cuttle fish 0.31 9.7 20.3 9.0 142.9 81.4 100
liver oil
Baker’s 0.08 9.7 19.3 9.0 117.2 41.4 3.4
yeast
Control’ 0.10 9.7 19.5 9.0 124.8 59.2 10.0
Trigriopus 0.40 9.7 19.4 9.0 123.0 77.1 100

‘Feeding period was 10 days.

*Nauplii just after hatching.
“Average values of 30 fish.

Artemia may be satisfactory for freshwater fish, judging from their EFA
requirement (CasteIl et al., 1972a; Watanabe et al., 1974a, 1974b, 1975b;
Takeuchi et al., 1979, 1980), it is necessary to check the fatty acid composi-
tion of Artemia for use as a food for marine fish. If its fatty acid composi-
tion is not known, the Artemia should be fed to fish together with other
marine copepods or should be fed on lipids containing w3 HUFA to prevent
heavy fish losses from various syndromes.

Other living feeds

The marine copepods Tigriopus and Acartia and the freshwater Moina
and LIaphnia are also well known to be suitable as foods for rearing juvenile
 137

TABLE XXI

Improvement of dietary value of Artemia for red sea bream by the direct method (Expt.
III)’

Feed given w 3 HUFA Total body’ Average body’ Survival Normal fish
to in length (mm) weight (mg) rate in activity
Artemia Artemia
Initial Final Initial Final (%) test (%)
(%)
Baker’s 0.12 14.7 22.0 35.2 151.9 58.9 23.0
yeast
Corn oil 0.03 14.7 22.6 35.2 158.0 52.3 31.5
Pollock 0.21 14.7 23.7 35.2 188.9 76.3 86.5
liver oil
Cuttlefish 0.77 14.7 23.6 35.2 182.5 83.1 99.6
liver oil
Methyl 0.71 14,7 23.4 35.2 178.7 72.0 99.3
w3 HUFA
Tigriopus 0.50 14.7 22.6 35.2 181.0 89.1 100

‘Feeding period was 9 days.

‘Average values of 50 fish.

50-
A
B
C

0’
3 6 12 24
Culture period in hours

Fig. 8. Incorporation of lipids emulsified with various kinds of reagents (A, B and C) in
nfoina by the direct method. Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.;
Watanabe et al,, 1983.

fish of about 7 mm in body length. Watanabe et al. (1978e) analyzed fatty

acids of these zooplankters. As shown in Table XXII, Tigriopus was found to
contain relatively high amounts of 20:5w3 and 22:6w3, irrespective of
culture media and organisms, such as baker’s yeast and soy sauce cake; this
result suggests a high nutritional value for fish. On the other hand, the
content of w3 HUFA in the lipid of Moina (Table XXIII) was significantly
affected by the culture organisms, as observed in rotifers. Moina cultured
TABLE XXII
z
cc
Certain fatty acids of total lipids from Tigriopue cultured with marine chlorella (Chlorello minutissimc) soy sauce cake. baker’s
yeast (Sccchromyces cereuisice) and the yeast supplemented with cuttlefish liver oil (S yeast), respectively

Fatty acid Cultured under Tigriopue japonicus cultured with

natural
Yeast Yeast + Chlorella Yeast + formula S yeast SOY sauce intake
conditions
diet of prawn Nov.
Nov.-Feb. Dec. Nov. 1977’ Jun. 19762 Nov. Dec. Nov.
1977
197+1976 1976 I II I II 1977 Nov. 1977 1976 1977
-
14:o 1.7 1.2 1.1 1.6 0.9 1.1 1.4 1.3 2.4 0.6 0.9
14:l 0.2 4.5 6.0 6.5 3.9 3.5 6.6 5.1 2.0 2.3 2.9
16:O 2.1 2.0 2.2 1.6 2.0 1.9 3.0 2.2 1.6 1.3 1.9
16:0 14.0 10.4 8.6 13.3 10.6 10.6 10.3 11.5 12.1 20.1 16.5
16:1w7s 6.3 10.9 14.2 11.1 11.6 10.7 10.9 8.7 8.1 5.1 4.1

18:0 2.3 6.0 5.0 4.6 4.5 3.8 3.1 3.1 3.6 4.3 3.6
18:1w9’ 20.1 11.2 23.0 15.3 19.4 18.6 16.6 20.1 21.3 15.0 19.9
18:2w6 2.5 3.6 2.1 2.7 3.5 3.8 3.1 5.2 1.7 7.0 18.1
18:3w3 8.2 6.0 1.8 5.6 6.1 7.8 7.4 6.1 1.3 1.8 2.7

18:4w3
3.2 1.9 4.3 2.0 2.2 3.7 1.4 0.8 2.0 0.3
20:o 1

2O:l 1.0 2.1 0.9 1.8 1.0 0.4 1.1 4.8 0.5 0.8
20:3w3
1 1.7 2.1 0.7 2.0 2.5 1.4 1.8 2.6 1.6 2.2
20:4w6
2014~3 2.6 2.5 1.3 2.9 3.1 1.3 2.2 1.0 1.0 1.2
20:5w3 8.2 7.2 4.2 5.8 5.9 6.8 6.8 10.8 9.3 4.4

22:6w3 0.7 1.6 0.6 2.0 2.2 0.6 0.6 1.2 0.5 0.6
22:6w3 6.6 12.0 8.6 9.6 9.7 6.4 7.8 13.6 16.7 7.9

X:w3 HUFA 17.9 23.3 14.8 20.3 20.9 16.1 17.4 26.6 27.5 14.1

Lipid 96 2.6 3.2 2.4 3.6 2.8 3.1 2.6 2.6 1.7 3.0

’ Tigriopus cultured using a 1 ton (I) or a 200 ton tank (II).

*Culture was conducted in duplicate 200 ton tanks (I and II).
’ Smaii amounts of the other monoenes were included.
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al.. 1978e.
TABLE XXIII

Certain fatty acids of total lipids from Moino and Acartia (area 96)

Moina sp. cultured with Acadia clausi Natural Diatom

plankton for prawn
Fatty acid Yeast’ Yeast + Poultry manure Natural
for ayu’ Sept.
poultry May Dec. Sept.
Gifu Hiroshima 5 days 10 days 19,6 1977
manure 1976 1977
14:o 2.6 2.4 0.8 1.3 3.2 4.2 4.4 12.1 1.1 23.1
16:0 6.6 6.5 5.8 10.7 9.2 16.9 16.5 18.3 16.2 9.9
16:1w7s 37.9 33.6 19.9 13.8 18.1 2.4 1.8 10.7 3.2 14.5
18:0 1.4 2.6 3.0 5.6 2.3 5.4 3.8 6.0 3.9 0.2
18:1w9’ 25.1 24.1 26.2 10.4 3.1 4.1 3.6 5.4 7.4 2.2
18:2w6 4.9 4.2 6.6 7.3 2.1 1.1 0.6 1.1 8.6 1.7
18:3w3 0.6 0.8 0.8 10.1 2.3 1.1 0.7 1.0 34.5 0.2

18:4w3 -
1 0.1 - 1.1 2.0 2.3 3.2 2.5 1.6 0.1
20:o

20:1 0.2 0.3 0.2 0.1 - 0.7 0.7 2.3 1.6 1.6
20:3w3
1 0.3 4.4 8.9 5.5 1.7 1.1 0.4 1.0 2.1 1.7
20:4w6
20~4~3 - 0.1 0.2 0.2 0.2 0.6 0.6 0.4 1.0 0.2
20:5w3 0.2 1.5 7.0 14.5 20.8 20.1 29.2 16.6 4.6 12.9

22:6w3 - - 0.2 0.2 0.5 0.5 1.0 0.8 - -

22:6w3 0.3 tr tr 28.6 27.2 12.3 - -

Cw3 HUFA 0.2 1.6 7.7 14.9 21.5 49.8 58.0 30.1 5.6 13.1

Lipid % 2.9 - 1.3 1.3 3.3 1.1 1.6 1.3 - 1.8

’ Moina cultured with baker’s yeast Saccharomyces cereuisiae at Gifu and Hiroshima Prefectural Fisheries Experimental
Stations, raspectively.
‘Mainly consisted of Moina and Bmchionuspltcotilis.
‘Small amounts of the other monoenes were included.
Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978e.
140

with yeast contained high levels of monoethylenic fatty acids and low levels
of w3 HUFA, while those cultured with poultry manure had high contents
of 20:5w3, indicating that the former is inferior to the latter as a living feed
for fish. Moina was also found to take up emulsified lipids very easily by the
direct method, as shown in Fig. 8. Acartiu collected in the sea was found to
be a very good food for marine fish; it contained both 20:5w3 and 22:603
at fairly high levels, making a total w3 HUFA of 30-60%, although some
seasonal variation in 03 HUFA was apparent. The fatty acid spectrum of
Daphniu (Table XXIV) also makes it a suitable food for fish from the view-
point of EFA.

TABLE XXIV

Certain fatty acid of total lipid, polar lipid and triglyceride fractions from Daphnio sp.

Fatty acid Total lipid Polar lipid Tricglyceride

12:o 0.8 0.1 2.4
14:o 2.1 1.5 3.6
15:o 2.2 1.2 1.8
16:0 15.3 14.3 17.1
16:lw7’ 12.4 9.6 12.2
18:O 4.8 6.6 6.3
18:lw9’ 12.8 17.7 16.7
18:2w6 4.4 4.7 3.9
18:3w 3 11.0 11.0 6.4

18:4w 3
1 2.9 2.0 1.8
20:o

20:3w 3
1 3.6 4.0 5.2
20:4w 6
20~4~ 3 0.6 0.4 0.4
20:5w 3 16.5 16.2 7.5

22~6~3 0.2 0.7 -

‘Small amounts of the other monoenes were included.

Reproduced with permission from Bull. Jpn. Sot. Sci. Fish.; Watanabe et al., 1978e.

ACKNOWLEDGEMENT

We express here our sincere thanks to Dr. C.B. Cowey, Institute of Marine
Biochemistry, Aberdeen, Great Britain, who kindly read the manuscript and
gave valuable suggestions.

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