Embed Size (px)
Citation preview
広塩性ツボワムシ類に対する栄養強化時間が脂肪酸組成及びマダイ仔魚飼育成績に与える効果
誌名誌名 水産増殖 = The aquiculture
ISSNISSN 03714217
巻/号巻/号 652
掲載ページ掲載ページ p. 133-144
発行年月発行年月 2017年6月
農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat
Aquacult. Sci. 65 (2), 133-144 (2017)
Effect of the duration of nutritional enrichment on由efa均racid
composition of commonly used rotifers Brachionus plicαtilis sp. complex and larviculture performance
of red sea bream Pαgrus mαrjor
Tomonari KoTANI1' *, Takumi H却 AGUCHI2,Yuta y,雌 AZAKI¥Tatsuya Dm1,
Hideaki MA’rsur2, Saichiro YOKOYAMA¥ Manabu lsHIKAWA1 and Shunsuke Kosmo1
Abs仕acむ Thisstudy aimed to con宣rmthe dietary value of the ro位fersBrachionus plicatilis sp. complex enriched for the various durations and the rotifer feeding effect of these rotifers on larviculture performance of red sea bream Pagrus major. S and L勿perotifers were used in the enrichment. Rotifers were enriched with Chlorella vulgaris containing n-3 HUFAs for 12 or 24 h (12 h
EN, 24 h EN). Rotifers cultured primarily with fed C. vulga焔 containingn-3 HUFAs (FEN) were also prepared. In larval rearing of red sea bream, the grow仕lin standard body length during the rearing
and the survival rate at 20 days post-hatching (dph) were compared among the feeding conditions of 24 h EN and FEN. EPA and DHA contents of bo出 rotiferspecies between 12 h and 24 h EN did not differ. In polar lipids of L type ro世ers,FEN rotifers included more DHA content than 12 h and 24 h
EN rotifers.τbe survival rate of larvae fed FEN rotifers was significantly higher出an24 h EN rotifer feeding group, and the growth in standard body length of larvae fed FEN group was also faster.
Key words: Rotifer species; Nutritional enrichment; Polar lipid; DHA
Improvement of the nutritional value of the
euryhaline rotifer Brachionus ρlieαtilis sp.
complex has contributed to the development
and performance of fish larviculture tech-
nology (Watanabe 1993; Dhert et al. 2001).
Improvement of fa句racid components in ro世－
fers, especially n・3HUFA, including EPA and
DHA, has increased the survival and grow血rate
of fish larvae (Watanabe 1993; Dhert et al. 2001).
Recently, the contents of arachidonic acid, tau-
rine and minerals have been added to nutritional
materials to enrich live feeds (Koven et al. 2001;
Hamre et al. 2008; Matsunari et al. 2005).
Methodologies of nutritional enrichment
have been discussed since enrichment has
been recognized to be necess訂 yfor improving
the larviculture performance (Watanabe et al.
1978; Imada et al. 1979; Watanabe et al. 1983;
Received 23 August 2016; Accepted 26 April 2017.
Ben-Amotz et al. 1987; S1:0t仕upand Attramadal
1992; Fernandez-Reiriz et al. 1993; Lie et al.
1997; Yukino et al. 2002; Vagner et al. 2014).
Currentlぁtwomethods have been used. In the
first method, the enrichment diet is produced
with mixing required nutrients and rotifers
are directly enriched with that diet (Imada et
al. 1979; Watanabe et al. 1983; St前 回pand
Attramadal 1992; Fernandez-Reiriz et al. 1993;
Lie et al. 1997). In the second method, roti-
fers are enriched with feeding the microalgae
including or added essential fatty acids in cell
(Watanabe et al. 1978; Ben-Amotz et al. 1987;
Lie et al. 1997; Yukino et al. 2002; Vagner et al.
2014). These methods are effective for obtain-
ing enrichment performance. Although bo出
methods can enhance the fa句racid composi”
tion of rotifers, especially n-3 HUFA, DHA and
1 Faculty of Fisheries, Kaogoshima University, Kagoshima, Kagoshima 890-0056, Japan. 2 Graduate School of Fisheries, Kagoshima University, Kagoshima, Kagoshima 89Cト0056,Japan. *Corresponding author: Tel/Fax, (+81) 99-286-4192; Email, kotani＠宣sh.kagoshima-u.ac.σ.Ko句ni).
134 T. Kotani, T. Haraguchi, Y. Yamazaki, T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
EPA, manufacturers of each diet determine
the enrichment method for that diet (Kotani
et al. 2010). However, the effect of nutritional
enrichment can be altered in fa向racid contents
and composition with changing the duration of
enrichment and/ or the amount of enrichment
diet (Kotani et al. 2010).
Current enrichment methods are effective
for increasing the content of n・3HUFA in roti-
fers, as mentioned above. A higher enrichment
effect can be obtained when more n-3 HUFA,
DHA and/or EPA are included in polar lipids
or phospholipid than in non-polar lipids (Bell et
al. 2003; Kj0rsvik et al. 2009; Word et al. 2009;
Olsen et al. 2014). On the other hand, there
have been few studies that have addressed how
to enrich the fa町racid contents of polar lipids
in rotifers (Li and Olsen 2015). In the report
of Watanabe et al. (1978), rotifers always fed
Nannochloropsis oculata, called in their report as
“marine Chlorella”， had high contents of EPA in
polar lipids. This high content likely stemmed
from the fact that N oculαta was fed to rotifers
in the primary culture and rotifers were always
exposed to EPA included in N oculata.
This study aimed to re-consider the method-
ology of nutritional enrichment for rotifers in
order to obtain a higher effect of enrichment. In
this re-consideration, we focused on the enrich-
ment duration. To evaluate the effect of dura開
tion, the fa句racid contents of polar and non-polar
lipids in rotifers were analyzed. Moreover, larvae
were reared using rotifers enriched with three
treatments. Survival and growth were then com-
pared among仕eatments.Because the effect of
enrichment is expected to vary among rotifer
species, S and L type rotifers Briαchionus were
used. Based on these evaluations, we extensively
considered the effect of nu仕itionalenrichment
method tested in this study.
Note α：bout the scientific n，α：me ofγotifeγBτac沿lonus
Mills et al.。016)reported that euryhaline
rotifer Brachionus can be genetically discrim-
inated into 15 species. Although each species
was given scientific name, all already known
strains or populations have not been classified
to any species出atMills et al. (2016) determined.
It is also unclear which rotifer species suggested
by Mills et al. (2016) L type and S type rotifers
used in this study belong to. In this report, L
type and S type rotifers訂 e仕eatedas different
species. However, those descriptions are indi-
cated as“L type rot江er”and“Stype rotifer”h B.
plicatilis sp. complex, respectively, because their
species names have not been determined.
Materials and Methods
Primary rot約rculture
The S type rotifer Okayama strain and the
L type rotifer Obama strain of Brachionus pli-
catilis sp. complex were used. S type rotifers
and L type rotifers were maintained in the incu-
bator of the Facul句rof Fisheries, Kagoshima
University, at 25°C, and were cultured in the lab-
oratory by feeding Nannochloro戸sisoculata.
Each rotifer strain was raised on commercial
concentrated freshwater chlorella ChloreUαvul-
garis (Fresh Chlorella-V周辺 (dry matter weight:
135 g/l), Chlorella Industry Co., Ltd., Tokyo,
Japan). The rotifers were cultivated using the
batch method (Lubzens 1987). Seawater was
diluted at 60% (20 psu) with tap water that was
aerated over night and was used as culture
medium. Batch culture was performed in a
5 l plastic beaker or a 30 l polycarbonate tank.
Initial stocking density was adjusted to 1,000
rotifers/ml in S type rotifers and 500 rotifers/
ml in L句rperotifers. Aeration was provided to
each tank via a ceramic air-stone suspended
2-3 cm from the center of tank bottom. To
remove particulate wastes, 1 nylon filter net
(Vilene mat, Tanaka Sanjiro Co. Ltd., Fukuoka,
Japan, 20×10×1 cm in a 5 l plastic beaker and
25×15×1 cm in a 30 l polycarbonate tank) was
placed in the culture tank and changed daily. S
句rperotifers were harvested on day 3 from the
start of cultivation and a portion of the stock
was used as the initial stock for the next cultiva-
tion after rinsing.仁 vulgariswas supplied to the
S type rotifer culture at a ratio of 10×103 cells/
rotifer I day. The culture temperature of S句rperotifer was 28°C. L type rotifers were harvested
on day 2 from the start of cultivation.仁 vulg，αris
was supplied to the L type rotifer culture at a
Effect of length of nu tr悩onalenrichment 135
ratio of 20×103 cells/rotifer/day. The culture
temperature of L type rotifers was 25°C.
Nutritionαl enrichment
To enrich白erotifers nu凶tionally,commercial
concen仕ated企eshwaterchlorella C. vulgaris
containing n・3HUFA (Super Fresh Chlorella-V12
(SFC, dry matter weight: 135 g/l), Chlorella
Industry Co., Ltd.) was used. After the harvest
from primary cultures, rotifers were transferred
to fresh culture medium and fed SFC. The
rotifers were enriched for 12 or 24 h (12 h EN,
24 h EN). In 12 h EN, the quantity of SFC was
adjusted to be 5×103 cells/rotifer. The rotifers
were fed SFC just after inoculation to a new
culture medium. They were then harvested at
12 h after enrichment began. In 24 h EN, nutri-
tional enrichment was performed for 24 h. The
quantity of SFC was adjusted to be 5×103 cells/
rotifer /feeding. Rotifers were fed SFC just after
inoculation to new culture medium and were
fed SFC again at 12 h after the first feeding.
They were harvested at 24 h after the first feed-
ing. As in the other experimental仕eatment,
rotifers were enriched during full time (FEN).
FEN ro世ferswere cultured using the batch
method and were fed SFC.τbis batch method
was the same as the method mentioned above
for S and L type rotifers.τbe FEN cultures
were started from one week before the rearing
experiment. FEN cultures were established
as仕ieindependent culture from the primary
culture of 12 h EN and 24 h EN. FEN cultures
were maintained during the rearing experiment.
For the feeding, FEN rotifers were harvested
from出eculture which more than one week
had passed since the beginning.τbe tempera-
加reof nu仕itionalenrichment was the same as
the primary culture for each type of rotifer.
Rotifers enriched wi出 threetreatments were
analyzed for protein contents, lipid content and
fatty acid composition in polar and non-polar
lipids. For仕ieseanalyses, ro世ferswere frozen
just after the harvest and the removal of extra water, and were stored at -80。Cuntil analyses.
Five populations were collected企omeach treat-
ment in different batches.
In larval rearing of red sea bream larvae, 24 h
EN and FEN rotifers were supplied to larvae
after the harvest.
Chemicalαnalysis To investigate the chemical contents of roti-
fers, crude protein, crude lipid and fa向racid
composition in polar and non-polar lipid were
analyzed. The contents of crude protein were
analyzed with a Kjeldahl distilling apparatus
（悶eltecsystem 1002, Tecator, Sweden). Crude
lipids were extracted using the method of
Folch et al. (195ηand were then separated
into non-polar lipid (NL) and polar lipid (PL)
fractions by column chromatography on Sep-
Pak Silica Car仕idges(Waters, S. A, U.S.A.)
σuaneda and Rocquelin 1985), with chloro-
form同methanol (98:2) for NL and methanol
for PL. Methyl-esterificated PL and NL were
prepared prior to gas chromatography (GC・
17, Shimadzu Co. Ltd., Kyoto, Japan) analysis.
The extracted lipids were resuspended in 1 ml
of chloroform containing 2 mg/ml of fat町acid
standard, C19:0. The suspension was仕ans司
ferred to a 10-ml centrifuge tube and 1 ml of 5%
hydrogen chloride methanol solution (Wako
Pure Chemical Indus廿ies,Ltd., Osaka, Japan)
was added to the tube.τbe tube was then
heated at 80°C for 3 h. A立ercooling, 1 ml of
hexane and 5.5 ml of distilled water were added,
and the mixture was vortexed. The tube was
centrifuged at 2,000 rpm for 5 min. The hexane
layer was transferred to a screwed bo仕leand
stored at -80°C until GC analysis.τbe hexane
layer, including fa向racids, was subject to GC
analysis. Data were analyzed based on the
C19:0 standard.
Laγ匂α1γeαγ'ing
Broodstocks of red sea bream were main-
tained at the Fish Farming Center established
by Nagashima-cho, Kagoshima Prefecture. Eggs
were spawned and fertilized naturally on 9出
May, 2012. These fertilized eggs were trans-
ported to Kagoshima University on the 10白 of
May. Initial stocking densities were set at 1,500
eggs per tank. The hatching rate of these fertil-
ized eggs was 67.8 ± 18.5%.
Larval rearing was performed for 22 days
136 T. Kotani, T. Haraguchi, Y. Yamazaki, T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
from the 10th to 31st May, 2012. Two expe子
imental conditions, feeding 24 h EN or FEN
rotifers, were established in triplicate. Six 100 l
black polyethylene tanks were used as exper-
imental rearing tanks. In each tank, aeration
was provided via a ceramic air-stones, which
was increased with larval growth. To ensure
circulation of water within the rearing tank, an
air-stone was suspended approximately 2-3 cm
above the tank bottom. Water was drained via
siphoning through a 15 mm diameter vinyl hose
and screened through a polyethylene mesh on
a metal frame (14×15×55 cm) in the center of
tank. The mesh size was changed from 0.2 to
0.5 mm with larval growth. The water tempera-
加rewas maintained at 21°C using a 1 kw tita-
nium heater applied to each tank. Photoperiod
was controlled at 12L12D with a light intensity
of 600 Ix during the daytime. The rearing water was exchanged at a ratio of 100 l/day from the
stocking of fertilized eggs to 15 days post-hatch
(dph) and at a ratio of 300 l/ day from 16 to 20 dph. The removal of waste on the tank bottom
was not performed during the period of single
feeding of rotifer, but it was started from 15 dph
via siphoning through a 7 mm diameter plastic
pipe with a 9 mm diameter vinyl tube.
S句rperotifers were fed to the larvae from 2 to
9 dph, and L type rotifers were fed to the larvae
from 10 to 19 dph. Artemia nauplii, hatched
from the commercial eggs (Great Salt Lake
Artemia, Ogden, UT, USA) and enriched with
Super Capsule Powder (Chlorella Industry Co.,
Ltd.) at 25°C and a ratio of 70 mg/l for 24 h,
were fed from 14 to 19 dph. Rotifer density was
checked at 8: 00 am and 3 : 00 pm, and Density
of S type rotifers was adjusted to ten rotifers/
ml, while L type rotifer density was adjusted
to five rotifers/ml. Artemia nauplii density
was checked at 9: 00 am and 4: 00 pm and was
replenished to 12 nauplii/larva.
Twenty larvae were collected at 0, 2 dph
and every 5 days from 5 dph and fixed in 10%
formalin-based sea water for growth mea-
surements of standard length (i.e., notochord
length）.百iesefixed samples were stored in a
refrigerator until measurements. The measure-
ment was performed under microscope based
on the micro-ruler. The stocking number in
each tank was estimated on the hatch day by
the volumetric method.官官 PVCpipe (40 mm仇100 cmL) with ball valve was inserted into the
rearing water from the side without the valve.
After the valve was closed, the pipe was pulled
out of the water. The water column included in
that pipe was received in a plastic beaker. The
number of red sea bream larvae included in the
sampled water was counted. Also, the volume of
that sampled water was measured with the grad-
uated plastic beaker.τbe stocking density was
estimated based on the counted宣shnumber
and the measured water volume. This water
sampling was replicated five times in each tank.
Survival in each tank was estimated by direct
counting at 20 dph.
Cαle叫αtionof coefficient of nαtural悦 0γtαlit'ヲ
The coefficient of natural mortality during
the larval rearing was difficult to determine
because the final survival count includes
fish removed for sampling. Therefore, the
coefficient of natural mortality was calcu-
lated using a model of natural mortality that
includes removed samples (Kotani et al. 2011).
Generally, the decrease of a natural population
is indicated by the following formula,
dN/dt=-mN
where N is population size and m is the mor-
tality coefficient. When N0 signifies the initial
value of Natt = 0, the above formula becomes:
Ni ＝~おmt.
Taking the sample size in each sampling into
consideration, the population size 包） after
all samplings is indicated by the following
equation:
N1=e一mT(No一エNsnen').
where T represents the total rearing period, dn
represents the rearing period until the nth sam-
pling event and N sn indicates the sampled size in the nth sampling event.
Sample sizes and final survival counts are
known values; thus, we substituted values for m
Effect of length of nutritional enrichment 137
Table 1. Crude protein and crude lipid contents of Land S type rotifers enriched for various durations
Ro出erspecies Ltype S type
Enrichment condition 12h 24h Full time 12h 24h Full time
Crude protein (%dw) 38.7 ± 0.6 39.3 ± 0.6 38.7 ± 0.2 38.3 ± 0.7 38.3 ± 0.5 38.7 ± 0.6 Crude lipid (%dw) 15.7 ± 1.9 16.2 ± 1.4 15.3 ± 2.7 15.0 ± 1.6 15.9 ± 2.7 13.0 ± 0.8 Crude lipid (%ww) 2.5 ± 0.4 2.5± 0.3 2.3± 0.2 2.6 ± 0.5 2.7± 0.3 2.2 ± 0.2
in the above formula with successive approxi-
mations and estimated the coefficient of natural
decrease, from which the actual survival num-
bers were calculated with the substitution.
Stαtisticαl anαlysis Fa町racid profiles of rotifers among treat-
ments were tested using Kraskal-Wallis test
and then compared using Steel-Dwass test.
Wilcoxon test was used to compare the coeffi・
cient of natural mortality of larvae, calculated
under the experimental conditions. We used a
chi-square test to compare the estimated su子
vival rate at 20 dph. Standard length of larvae
was compared using Wilcoxon test. The growth
coefficient in each treatment was calculated
from the regression formula of an exponential
curve自社edto total length data. These coef-
ficients between treatments were compared
using two-way ANOVA. Statistical analyses were
performed using JMP 11.0.0 for Mac OS X (SAS
Institute Incよ
Results
Chemical analysis
Rotifers contained on average 38-39% crude
protein and 13-16% crude lipid on a dry weight
basis. Crude protein and lipid contents did not
differ among rotifer species and enrichment
durations (Table 1).
The ratio of PL of total lipid of S type rotifer
was approximately 45% in all treatments. L type
rotifers tended to increase from 36.7% (12 h
EN) to 49.9% (FEN) with the passage of enrich-
menttime σig. 1; Kruskal-Wallis test, Pく 0.05).
The percent components of some fa町racids
in both lipids of S type rotifers did not change
among treatments (Table 2 and 3）.官iepercenι
age component of eicosapentaenioic acid (C20:5
Rotifer Enrichment
species condition
印
印
叩
ι
H
L
H
ル昨
吋，
ι
a
斗
U
唱
i
、，ιr
r
「lili－－
L
e
－，
nv uv
Eτつ＇：＇C:τー「？＝？でーー了？？句...,-7-
一 一一 ’
印
印
四
ι
H
L
H
仙川即
、，ιa
u
守
U
1
2
F
「Illi－－
－
L
e
nv uv t
p
、d
。 so 100
Percentage in crude lrpid
Fig. 1. The ratio of non-polar and polar lipids of L and S type rotifers in three enrichment durations. Blank columns show the mean percentage of non-polar lipids (NL) and gray columns show the mean percentage of polar lipids (PL). Each horizontal line shows the standard deviation of mean (n = 5). An asterisk indicates that th巴Krusl王altest found a significant difference among the enrichment treatments (Pく0.05).
n・3,EPA) in NL of S type rotifers increased
with the passage of enrichment timeぐTable2;
Kruskal-Wallis test, Pく 0.05;Steel-Dwass test,
p < 0.05）.百iepercentage component of arachi-
donic acid (C20:4 n-6, ArA) in PL of S type roti-
fers decreased with the passage of enrichment
timeぐTable3; Kruskal-Wallis test, P < 0.05; Steel-
Dwass test, Pく 0.05）.百iepercentage compo-
nents of some fa町racids of both lipids of L type
rotifers changed among仕切佃ientsぐTable2 and
3; Kruskal-Wallis test, Pく 0.05;Steel-Dwass test,
Pく0.05）.百iepercentage of C16:3 and C18:4
n-3 decreased, and the percent component of
EPA and docosapentaenoic acid (C22:5 n-6)
increased in both lipidsぐTable2 and 3; Kruskal-
Wallis test, Pく0.05;Steel-Dwass test, P < 0.05).
In PL, the percen臼geof ArA decreased, and
the percentage of docosahexaenoic acid (C22:6
n-3, DHA) increased with the passage of enrich-
ment timeぐTable3; Kruskal
138 T. Kotani, T. Haraguchi, Y. Yamaz品d,T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
Table 2. Fatty acid profiles in non-polar lipids (NL) of Land S type ro位fersenriched for various durations
Rotifer species Ltype Stype
Enrichment condition 12h 24h Full time 12h 24h Full time
Fa町 acid(%)14:0 1.95 ± 0.18 1.90 ± 0.10 2.90 ±2.27 2.68 ± 1.76 2.04 ± 0.71 1.81 ±0.17 16:0 13.03 ± 0.93 11.75 ± 1.34 11.77 ±0.71 13.79 士1.75 13.34 ± 1.28 16.26 ± 1.96 16:1 キ 2.71b±0.24 4.48 ab ± 3.54 11.32 a ± 0.47 4.25 ± 3.95 3.26 ± 1.73 2.38 ± 0.14 16:2 * * 12.49 a ± 1.12 12.13 a ± 1.57 3.02 b ±0.25 7.90 ± 4.37 9.41 ±4.62 7.78 ±4.12 16:3 ** 3.73 a ± 0.46 3.52 a ± 0.36 0.11 b ±0.07 2.75 ± 0.96 2.92 士0.67 0.74 ± 1.24 18:0 6.39 ± 1.48 5.33 ± 1.80 4.10 ±0.83 5.63 ± 1.39 7.22 ±2.35 6.25 ± 0.71 18:1 n-9 ホ 1.43b ± 1.75 3.49 ab± 1.75 4.65a土0.40 2.91 ± 2.40 1.24 ± 1.56 0.00 ± 0.00 18:2 n-6 本 29.71a ± 2.41 27.59 ab ± 4.27 24.58 b ± 0.85 25.81 ± 1.30 24.96 ± 1.26 26.35 ± 2.05 18:3 n-3 5.67 ± 4.80 7.51 土3.75 8.30 ±0.64 7.15 ± 2.99 4.71 ±3.80 3.45 土4.0418:4n”3 ** 0.16 a ± 0.04 0.10 ab土0.04 0.03 b ±0.03 0.15 ± 0.08 0.14 ± 0.06 0.12 ± 0.06 20:4n司6 1.10 ± 0.26 1.27 ± 0.20 1.21 ±0.40 1.02 ± 0.08 1.03 ±0.05 0.82 ± 0.36 20:4 n-3 0.72 ± 0.27 0.83 ± 0.13 0.61 ±0.16 0.46 ± 0.18 0.42 ±0.12 0.35 ± 0.15 20:5 n-3 * 3.26 b土 1.05 3.87 ab ± 1.42 4.97 a ±0.39 * 4.69 b土0.30 4.98 ab ± 0.35 5.62 a± 0.36
22:0 1.04 ± 1.55 0.28 ± 0.00 0.26 ±0.01 0.17 ± 0.05 0.23 ±0.07 0.20 ± 0.04 22:5 n-6 * 1.32 b ± 0.66 1.80 ab± 0.72 2.46a ±0.35 2.54 士0.49 2.80 ± 0.33 3.38 ± 0.47 22:6 n-3 * 5.46 ± 2.84 7.26 ± 2.85 9.52 ± 1.17 8.64 ± 0.85 9.81 ±0.93 9.73 ± 0.92
Contents (mg/ g dw) ArA 0.45 ± 0.22 0.67 ± 0.17 0.65 ±0.41 0.60 ± 0.30 0.42 ± 0.12 0.26 ± 0.16 EPA 1.31 ± 0.75 2.37 ± 1.25 2.52 ±0.79 2.69 ± 0.98 2.05 ± 0.63 1.74 ± 0.69 DHA 2.16 ± 1.73 4.44 ± 2.32 4.83 ± 1.52 5.18 土2.60 3.94 ± 0.83 3.02 ± 1.26 In-3HUFA 3.75 ± 2.63 7.26 ± 3.70 7.66 ±2.40 8.13 土3.69 6.17 ± 1.52 4.88 土2.01
Imonoene 9.14 ± 3.58 10.64 ± 3.68 9.39 ± 1.95 12.31 ± 3.06 9.48 ±3.81 7.63 ± 3.13 In-3 6.88 土4.83 12.16 ± 6.69 11.93 ±3.76 12.61 ± 6.50 8.33 士3.17 6.40 ± 4.20 In-6 13.11 土4.98 16.67 ± 5.33 14.38 ±4.41 17.36 ± 8.04 11.77 ± 3.27 9.42 ± 3.67
DHAノEPA 1.45 ± 0.80 1.83 ± 0.17 1.91 ±0.17 1.86 ± 0.31 1.98 ± 0.28 1.73 ± 0.18 ArA/EPA 0.36 ± 0.08 0.50 ± 0.54 0.24 ±0.07 0.22 ± 0.03 0.21 ±0.02 0.15 ± 0.08 ル3/n-6 0.49 ± 0.20 0.67 ± 0.32 0.83 ±0.06 0.72 ± 0.12 0.70 ±0.14 0.64 士0.22
Values indicate me叩± SD in that田 atment.Asterisks indicate that there are significant differences among悦 atmentsh仕rntfatty acid (Kruskal-Wallis匂st, *: Pく0.05,**: pく0.01).Each alphabetical subscript indicates the result of a Steel-Dwass test among仕田町ients(Pく0.05,a> b).
Steel-Dwass test, Pく0.05).
There was no significant change in fatty acid
contents (mg/ g dry weight) and ratio in NL for
both rotifer species among enrichment dura-
tions (Table 2). In PL, the content (mg/ g dry
weight) of ArA and DHA in L type rotifers and
the content of ArA in S type ro柱ferssignificantly
changed among enrichment durationsぐTable3;
Kruskal開Wallistest, Pく 0.05).The ratio of ArA
加 dEPA in PL of L旬pero世ferssigni宣cantly
decreased with出epassage of enrichment time
ぐfable3; Krusl王al
Dwass test, Pく 0.05).
Sur匂初αlofγedseαbγω悦 lαγ切巴
Initially, larvae were stocked at approximately
800-1,500 in出vidualsin each tankぐTable4）.百ie
coefficient of natural mor旬lityin 24 h EN feed-
ing group was 0.09620 ± 0.05374 (mean± SD).
The coefficient of natural mortality in FEN
feeding group was 0.04184 ± 0.02118ぐfable4).
τbere were no significant differences in these
values among rotifer treatments.τbe survival
rates in 24 h EN and FEN groups, estimated
by those coefficients, were 20.54 ± 18.53% and
45.99 ± 19.52%, respectivelぁ andthere was
a significant difference between treatments
(fable 4, chi-square test, Pく 0.05).
Gγowth ofγedseαbγeαmlα刊 αe
Changes in the standard length of red sea
bream larvae in both trea出ientsare shown in
Fig. 2. Initial standard leng仕1of newly hatched
larvae was 1.36 ± 0.10 mm (O dph, mean± SD,
n = 50). At 5 dph, 24 h EN group was larger
血anFEN groupσig. 2, Wilcoxon test, Pく 0.01).
After 10 dph, the pattern reversed in direction:
the standard body length of FEN group was
F同 1
Effect of length of nu仕itionalenrichment 139
Table 3. Fatty acid profiles in polar lipids (PL) of Land S type rotifers enriched for various durations
Ro世erspecies Ltype Stype
Enrichment condition 12h 24h Full time 12h 24h Full time
Fatty acid(%) 14:0 1.04 ±0.19 1.03 ± 0.15 1.02 ± 0.07 1.46 ±0.36 1.44 ± 0.29 1.66 ± 0.35 16:0 * 21.49 ab土 0.88 20.98 b ± 0.62 23.14 a土0.93 20.93 ± 2.14 22.11 ± 0.88 24.05 ± 1.54 16:1 1.58 士0.27 1.81 土 0.29 1.77 ± 0.40 2.46 ±0.56 2.43 ± 0.32 2.11 ± 0.27 16:2 * 1.69 土 0.69 1.39 土 0.80 0.34 ±0.08 1.65 ± 1.08 0.75 ± 0.77 1.84 ± 0.72 16:3 ** 0.37 a士0.04 1.20 a土 1.67 0.01 b ±0.02 1.98 ± 1.95 1.76 ± 1.83 1.02 ± 1.82 18:0 5.21 ±0.50 4.71 ±0.64 4.75 ± 0.37 11.88 ±7.85 8.75 ± 4.85 8.73 ± 2.02 18:1 n-9 3.56 ±0.28 2.79 ± 1.40 3.42 ± 0.20 0.47 土0.95 0.88 ± 1.76 0.00 ± 0.00 18:2 n-6 キ33.86 ± 2.80 31.55 ± 2.49 28.80 ± 1.61 23.39 土4.35 24.21 ± 3.32 22.81 ± 4.19 18:3 n-3 キ 8.55 ±4.28 9.71 ±0.40 6.92 ± 3.51 5.43 ±3.09 6.70 ± 1.24 4.84 ± 2.41 18:4 n-3 ** 0.19 a ± 0.04 0.18 a ± 0.03 0.05b ±0.06 0.19 ±0.09 0.20 ± 0.08 0.23 ± 0.13 20:4 n-6 ** 1.25 a ± 0.21 l.08a ± 0.30 0.37b ± 0.05 * 0.53 ab ± 0.10 0.59a土 0.08 0.39b土 0.0520:4 n-3 2.50 土 1.03 2.81 ±0.17 2.04 土0.60 指 0.85 ±0.17 0.87 ± 0.14 0.58 ± 0.10 20:5 n-3 * 3.66 b土 0.39 3.88 ab ± 1.32 5.63a土0.65 3.95 ±0.68 4.48 ± 0.64 4.70 士0.6222:0 0.49 ±0.13 0.42 ±0.06 0.47 ± 0.08 0.44 ±0.21 0.65 ± 0.37 0.51 ± 0.30 22:5 n-6 ** l.65b士0.80 2.42 ab ± 0.93 4.27 a± 0.40 3.16 ±0.63 4.05 ± 0.59 4.03 ± 0.92 22:6 n-3 ** 2.24 b ± 1.02 3.02 ab土 1.06 4.51a±0.47 4.14 ± 1.00 4.88 ± 0.81 4.54 ± 1.47
Contents (mg/g dw) ArA * 0.28 ±0.12 0.29 ±0.13 0.12 ± 0.09 キ 0.18 ±0.11 0.13 ± 0.06 0.04 土O.Ql
EPA 0.81 土 0.29 1.05 ±0.50 1.91 土 1.68 1.55 ± 1.12 1.01 ± 0.58 0.57 ± 0.29 DHA * 0.50 ±0.30 0.80 ± 0.35 1.54 ± 1.37 1.68 ± 1.26 1.11 ± 0.65 0.59 ± 0.42 In-3HUFA 1.89 ±0.91 2.59 ±0.98 4.19 ± 3.78 3.56 ±2.61 2.32 ± 1.35 1.23 ± 0.74
Imonoene 6.14 ± 1.71 7.06 ± 1.20 9.53 ± 7.28 10.43 ±6.62 6.87 土3.11 4.00 ± 1.10 In-3 3.99 ± 1.96 5.17 ± 1.44 5.67 ± 2.98 5.15 ±2.80 3.93 ± 2.33 1.80 土 0.94In-6 7.97 ±2.07 9.09 ± 1.38 10.83 土8.31 10.26 ±6.81 6.66 ± 3.99 3.36 士1.83
DHA/EPA 0.59 ±0.28 0.77 ±0.12 0.81 ± 0.09 1.03 土 0.13 1.09 ± 0.08 0.94 ± 0.22 ArA/EPA 判 0.34a ± 0.06 0.33a士0.19 0.07 b ± 0.02 0.14 ±0.06 0.14 ± 0.05 0.09 ± 0.02 n-3/n-6 0.47 ±0.15 0.56 ±0.10 0.57 ± 0.09 0.53 ±0.09 0.59 土0.04 0.56 ± 0.12
Values indicate mean ± SD h白attreatment. Asterisks indicate that there are significant differences among廿eatmentsin白紙fattyacid 侭ruskal-Wallistest, * : Pく0.05，叫：Pく0.01).Each alphabetical subscript indicates the result of a Steel Dwass test among treatments (P < 0.05, a> b).
Table 4. Comparison of survival rates of r吋 seabream larvae between two rotiferたedinggroups.
Group Tank Stock疋d Survival Coefficient mean ± SD Survival mean ± SD number fish number number at of of rate(%) survival rate(%)
at hatching 20 days post natural coefficient of hatch mortality natural mortality
24h EN 1 857 283 0.0446 0.09620 ± 0.05374 41.0 20.54 ± 18.53 b 2 1525 216 0.0921 15.9 3 1060 42 0.1519 4.8
FEN 123
1064 798 794
376 421 182
0.0436 0.0198 0.0621
0.04184 ± 0.02118 41.8 67.3 28.9
45.99 ± 19.52 a
Each alphabetical subscript indicates the result of a chi-square test among仕eatments(P < 0.05, a> b).
larger (Fig. 2, Wilcoxon test, P < 0.05).
The growth coef宣dentof FEN group during
the whole period was higher than that of 24 h
EN group (Table 5, two-way ANOVA, P < 0.05).
Although the growth coefficient of FEN group
during 0-10 dph was higher, the growth coe壬
:ficient of 24 h EN group during 10-20 dph was
higher (Table 5, two-way ANOVA, Pく 0.01).
Discussion
Although euryhaline rotifers of the genus
Brach ion us訂 eimportant live feed h白rfishlar-
vi culture，吐ieydo not have n-3 HUFA, especially
r
T. Kotani, T. H訂aguchi,Y. Yamazaki, T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
enrichment methods used for rotifer (Kotani et
al. 2010）.百ierehave been no studies也athave
examined the effects of nutritional enrichment
on rotifers using methods that have not been
developed by manufacturers, except for the
study of Kotani et al. (2010). When enrichment
methods exceeded dose and/ or duration rec-
ommendations, the dose-exceeded diets have
been shown to be more effective for raising
fatty acid content, especially for DHA content,
than duration-exceeded diets (Kotani et al.
2010). In this study, we used DHA-enriched C.
vulgaris (SFC) for the enrichment diet. Kotani et al.。010)used DHA Protein SELCOR (INVE,
BV), the product that they used was powdered
and included vitamins, fa句racids and proteins.
The manufacturer of由atproduct does not
assume that the dose or the duration of enrich-
ment will be in excess. The duration for general
usage is between 6 to 12 h (Moretti et al. 1999).
On仕ieother hand, SFC is not processed in
appearance and just incorporates DHA in the
cell. Although the Chlorella industry does not
determine the exact enrichment dura柱。n，出e
company conventionally assume long enrich-
ment duration using SFC. Taking the difference
in usage between the two products into consid-
eration, the effects of exceeding dose or -dura-
tion recommendations訂 edifferent.
Watanabe et al. (1978) reported the fatty acid
compositions of rotifers fed Nannochloropsis oculata or yeast. In their report, rotifers fed N oculata full time had higher values of EPA由m
rotifers fed yeast and enriched wi白 N oculata.
20
Fig. 2. Growth in standard length of red sea bream larvae between two enrichment durations. Solid squares indi駒
cate the average standard length in FEN group (n = 60) and open circles indicate that in 24 h EN group (n = 60). Each vertical line shows the standard deviation of mean (n = 60). Each alphabetical subscript indicates the result of a Wilcoxon test among仕eatments伊く0.05,a>b).
EPA and DHA, in sufficient quantities for finfish
larvae. Thus, feeds of this type require nutri-
tional enrichment (Watanabe et al. 1978). When
the nutri柱。nalenrichment practices for rotifers
had just begun, enrichment was purely quanti-
tative (Imada et al. 1979; Watanabe et al. 1983),
and aquaculturists related to larval rearing have
had no problem for those me仕10ds(Kitaiima et
al. 1979; Takeuchi et al. 1994).
Generally, the manufacturers
diets have determined the
of enrich-
nutritional
。 15
Days post hatching
10 5
ー．
4
2
。
8
6
140
ment
（
EE）
FZωcω一〉℃
0222EE的
Table 5. Comparison of exponential regression curve of standard leng白 ofred sea bream larvae between two rotifer たedinggroups
Group
10-20 days post hatching
0.0658 0.7963
く0.0001
Rearing period
0-10 days post hatching
0.0806 0.7657
く0.0001
Whole period
0.0684 0.9193
く0.0001
Coefficient R2 p
24hEN
0.0524 0.7987 <0.0001
0.0970 0.8987
く0.0001
0.0717 0.9375 <0.0001
Coefficient R2 p
FEN
0.3354 25.6353 <0.0001
0.2857 10.5197 0.0013
0.1030 4.5948 0.0324
Sum of square Fvalue p
Two-way ANOVA
Effect of length of nu仕itionalenrichment 141
This phenomenon is similar to the DHA content
and fa均racid composition of rotifers fed SFC
in this study. In the report of Watanabe et al.
(1978), EPA content increased up to 22% of all
fa均racids in PL. However, EPA content in this
study increased up to 5.6%. Consequent！）ろ the
enrichment effect of FEN was still insuf宣cient.
Although the cause of this increase is not clear,
enrichment of DHA in PL, especially phos-
pholipids is important for the development or
growth of larval宣shes(Bell et al. 2003; Kj0rsvik
et al. 2009; Word et al. 2009; Olsen et al. 2014).
Because the effect on the enrichment method
used in this study was restricted, the improve-
ment of enrichment diet or method should be
considered in future studies.
The increment of DHA in PL was observed
in only L type rotifer. And the decearse of
ArA content in FEN rotifers than in other
仕ea出ientswas remarkable (Table 3). ArA is
present at low levels in the cells of仁 vulgaris
(Maruyama et al. 199ηand we could expect
that the ArA content or component decrease.
Nevertheless, the ArA content of NL in L type
rotifers was included slightly more in 24 h EN
and FEN than 12 h EN (Table 2）.官iispa仕ern
was observed even when rotifers were enriched
with SFC, and ArA was not an added nutrient
(Kotani et al. 2013). Kotani et al. (2013) sug-
gested由atincreases in ArA of L type rotifers
stem for the fact that L type rotifers (as well as
any species of its co-flora) may synthesize ArA
from linolenic acid (C18:3 n・3,LA). The synthe-
sis of ArA means that the content of ArA should
not decrease as long as rotifers fed C. vulgaris.
ArA content may have decreased because S
句rperotifers metabolize ArA. In this study, we
performed nu凶tionalenrichment for rotifer
species to accommodate the conditions in each
primary culture. Because the culture tempera-
ture varied among rotifer species we cannot
determine whether the different characteristics
among rotifer species caused the differences in
fa向racid content or composition. Because both
S and L type rotifers are widely used in fish lar-
viculture, required nutritional values and issues
to be solved are common.τherefore, enrich-
ment of DHA to PL is also important for S type
rotifers. S type rotifers are different species
from L type rotifers (Segers 1995); therefore,
their physiological characteristics may also
be different, considering that they differed in
the magnitude of the fecundity characteristics
(Hirayama and Rumengan 1993; Hagiwara et
al. 1995). And the optimum water temperature
for increasing is different among rotifer species
(Hirayama and Rumengan 1993; Hagiwara et al.
1995). Based on these reports, S type rotifers
have the optimum temperature for increase at
more than 25°C and L type ones have between
20 and 25°C. Accordingly，社canbe inferred that
the physiological characteristics, (e.g., en勾rme
activity) are different among rotifer species. In
future studies, the influence of enrichment of
DHA in PL should be investigated when nutri-
tional enrichment is conducted under the same
environmental conditions among rotifer species.
Generally, when fish larvae can ingest rotifers
that can be enriched effectively with DHA and
EPA, larviculture performance can be improved
(Mourente et al. 1993; Park et al. 2006; Satoh
et al. 2005; Takeuchi et al. 1994). However,
improved larviculture performance is related
not only to the contents of DHA and EPA but
also to the ratio of DHA, EPA and/or ArA.
When the DHA/EPA ratio in diet is around 2,
the larval survival is improved (Rodriguez et al.
1997; Bessonart et al. 1997). Even if the DHA/
EPA ratio in the diet is optimum, approximate
2, the growth and survival reduce when EPA/
ArA ratio is over 1 (Bessonart et al. 1999). In
this study, the DHA/EPA ratios were not differ-
ent both in ro世ferspecies and under仕eatments.
Especially，出eDHA/EPA ratios in NL were
around 2. Moreover, we could not observe over 1
of EPN ArA ratio both in species and under any
trea伽ients.Despite these similarities, the sur-
vival rate was higher in FEN group h血isstudy.
If DHA or n-3 HUFA content is increased in roti-
fers excessively, larval survival or growth may
be reduced in some fish and shellfish (Suprayudi
et al. 2002; Takeuchi et al. 1994). However,
the DHA contents in this study （く 5mg/gdw)
were lower than that of Takeuchi et al. (1994)
(15 mg/ g dw）.百ierefore,we can conclude血at
the enrichment method of this study does not
142 T. Kotani, T. Haraguchi, Y. Yamazaki, T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
lead to excesses in DHA content because full-
time enrichment has positive effects on larval
survival and growth. On the other hand, there
were li仕leremarkable differences in fa向racid
composition between 24 h EN and FEN of S type
rotifers (Tables 2 and 3). Nevertheless, their
standard length at 5 dph already significantly
differed (Fig. 2). On fa同racid compositions,
only ArA content of FEN rotifer was less than
24 h EN rotifer (Table 3). In previous studies,
although the increase of ArA composition or content improved the larval survival or growth,
this induced the negative effect, such as
mal-pigmentation (Koven et al. 2001 and 2003;
Estevez et al. 1999; Bessonart et al. 1999). In
this study, no malpigmentiation was observed
and the growth of 24 h EN rotifer feeding group
was better than FEN rotifer group until 5 dph
(Fig. 2). On the other hand, the standard length
of FEN group was larger than 24 h EN group
at 10 dph (Fig. 2). The growth in standard
length until 5 dph might have been influenced
by the difference of ArA contents between
24 h EN and FEN rotifers. However, the reve子
sal of growth at 10 dph could not explain with
the ArA contents. Therefore, we should take
factors other than fa句racid composition into
consideration. From 3 to 5 dph, many larval
fishes should have had their first feeding that
is critical for larviculture performance (Fyhn
1989; Mourente et al. 1993; Bell et al. 2003). The
positive effects of enrichment of free amino acid
and minerals have already been reported (Chen
et al. 2005; Hamre et al. 2008). However, these
materials are included at zero or lower quanti-
ties in仁 vulgaris(Huxtable 1992; Maruyama
et al. 1997). Because these materials need to be
enriched to rotifers or C. vul増alisin other ways
(Maruyama et al. 1997; Simmons and Emery
2011), we presently cannot enrich them more.
In further studies, we should take these materi-
als into consideration after the method to enrich
them to仁 vulgariswill be developed.
Consequently, full-time nutritional enrichment
for rotifers, using D HA-enriched C. vulgaris
(SFC), was effective for improving larvicul-
ture performance. This improvement likely
stemmed from the fact that because the fa同r
acid contents increased in PL, especially for L
句rperotifers. Based on previous studies (Park
et al. 2006; Kotani et al. 2013), increases in DHA
content have contributed to larviculture perfor-
mance like this study. Howeve乙consideringthat
there were few differences in the effect of enrich-
ment of S type rotifers between treatments,
主irtherstudies must be required to investigate
the relationships between improvement of lar”
viculture performance and changes in fa句racid
composition in PL of rotifers. Moreoveにimprove-
ment in enrichment methods about rotifer spe-
cies should be continued to explore.
Acknowledgemen白
This study was supported by JSPS KAKENHI
Grant Number 24580275.
References
Bell, J. G., L. A. McEvoy, A. Estevez, R. J. Shields and J. R. Sargent (2003) Optimising lipid nutrition in first-feeding flaぜishlarvae. Aquaculture, 227, 211-220.
Ben-Arno包， A,R. Fishier and A. Schneller (198ηChemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fa町racids. Mar. Biol., 95, 31-36.
Bessonart, M., M. S. Izquierdo, M. Salhi, C. M. Hern釘idez-Cruz, M. M. Gonzalez, and H. Fernandez-Palacios (1999) Effect of dietary arachidonic acid levels on growth and survival of gilthead sea bream (Sparus aurata L.) larvae. Aquaculture, 179, 265-275.
Chen, J.-N., T. Takeuchi, T. Takahashi，主 Tomoda,M. Koiso and H. Kuwada (2005) Effect of rotifers enriched with taurine on growth in larvae of Japanese flounder Paralichthys olivaceus. Nippon Suisan
Gakkαishi, 71, 342-347. Dhert, P, G. Rombaut, G. Suantika and P Sorgeloos (2001)
Advancement of rotifer culture and manipulation tech-niques in Europe. Aquaculture, 200, 129-146.
Estevez, A., L. A. McEvoy, J. G. Bell and J. R. Sargent (1999) Growth, survival, lipid composition and pig-mentation of turbot (Scophthalmus mαximus) larvae fed live-prey enriched in arachidonic and eicosapen-taenoic acids. Aquaculture, 180, 321-343.
Fernandez-Reiriz, M. ]., U. Labarta and M. J. Ferreiro (1993) Effects of commercial enrichment diets on the nutritional value of the rotifer (Brachionusρlieαtilis). Aquaculture, 112, 195-206.
Folch, ]., M. Lees and G. H. S. Stanley (1957) A simple method for the isolation and purification of total lip-ides from animal tissues.! Biol. Chem., 226, 497-509.
Fyhn, H. ]. (1989) First feeding of marine fish larvae: Are
""
Effect of length of nutritional enrichment 143
企eeamino acids the source of energy? Aquaculture, 80, 111-120.
Hagiwara, A, T. Kotani, T. W Snell, M. Assava-Aree and K.
Hirayama (1995) Morphology, reproduction, genet-
ics, and mating behavior of small, tropical marine
Brachionus strains (Rotifera). J Exp. Mar. Biol. Ecol., 194, 25-37.
Hamre, K., T. A Mollan，の.Scele and B. Erstad (2008) Rotifers enriched with iodine and selenium increase
survival in Atlantic cod (Gadus morhua) larvae.
Aquaculture, 284, 190-195.
Hirayama, K. and I. M. Rumengann (1993）官1efecundity
patterns of S and L type rotifers of Brachionusρlicati-lis. Hydrobiologia, 255-256, 153-157.
Huxtable, R. J. (1992) Physiological ac世onsof taurine.
Physiol. Rev., 72, 101-163.
Imada, 0., Y. Kageyama, T. Watanabe, C. Kitajima, S. Fujita and Y. Yone (1979) Development of a new yeast as a
culture medium for living feeds used in the produc司
tion of fish seed. Ni仲onSuisan Gakkaishi, 45, 955-
959 (in Japanese with English abstract).
Juaneda, P. and G. Rocquelin (1985) Rapid and convenient
separation of phospholipids and non phosphorus
lipids from rat heart using silica cartridges. Lかids,20, 40-41.
阻同ima,C., S. F吋ita,F. Ohwa, Y. Yone and T. Watanabe (1979) Improvement of dietary value for red sea
bream larvae of rotifers Brachionusρlieαtilis cultured with baker’s yeast Saccharomyces cerevisiae. Nippon Suisan Gakkaishi, 45, 469-471 (in Japanese with
English abstract).
Kj0rsvik, E., C. Olsen, P.-A Wold, K. Hoehne-Reitan, C. L. Cahu, J. Rainuzzo, A I. Olsen, G.のieand Y. Olsen
(2009) Comparison of dietary phospholipids and neu-
tral lipids on skeletal development and fatty acid com-
position in Atlantic cod (Gadus morhua). Aquaculture,
294, 246-255.
Kotani, T., T. Genka, M. Tanabe, A Miyashima, H. Fushimi
and M. Hayashi (2010) Effect of nutritional enrichment
method on fa向racid contents of rotifer Brachionusρlι catilis.J WorldAquacult. Soc., 41, 884-892.
Kotani, T., M. Yokota, H. Fushimi and S. Watanabe (2011) How to determine the appropriate mortali匂rin experi-
mental larval rearing? Fish. Sci., 77, 255-261. Kotani, T., H. Fushimi, Y. Ohta, A Miyashima, K. Sudo,
M. Hayashi, N. Satoh and S. Satoh (2013) Effect of
graded levels of dietary DHA included in rotifers
Brachionus Plicatilis on larviculture performance
of red sea bream Pagrus major. Aquαcult. Sci., 61,
321-330.
Koven, W, Y. Barr, S. Lu包ky,I. Ben-Atia, R. Weiss, M.
Harel, P. Behrens and A Tandler (2001）官官 effect
of dietary arachidonic acid (20:4n -6) on grow出，
survival and resistance to handling stress in gilthead
seabream (Sparus aurata) larvae. Aquaculture, 193, 107-122.
Koven, W, R. van Anholt, S. Lutzky, I. Ben Atia, 0. Nixon,
B. Ron and A Tandler (2003) The effect of dietary
arachidonic acid on growth, survival, and cortisol
levels in different-age gilthead seabream larvae
(Sparus aurat悶：） exposed to handling or daily salinity
change. Aquaculture, 228, 307-320.
Li, K. and Y. Olsen (2015) Effect of enrichment time and
dietary DHA and non-highly unsaturated fa町 acid
composition on the efficiency of DHA enrichment
in phospholipid of rotifer (Brachionus Cayman).
Aquaculture, 446, 31Cト317.
Lie, 0., H. Haaland, G.-1. Hemre, A Maage, E. Lied,
G. Rosenlund, K. Sandnes and Y. Olsen (1997)
Nutritional composition of rotifers following a change
in diet仕omyeast and emulsified oil to microalgae.
Aquacult. Int., 5, 427-438.
Lubzens, E. (198ηRaising rotifers for use in aquaculture.
Hydrobiologia, 147, 245-255.
Maruyama, I., T. Nakao, I. Shigeno, Y. Ando and K.
Hirayama (199ηApplication of unicellular algae
Chlorella vulgaris for the mass-culture of marine roti-fer Brachionus. Hydrobiologia, 358, 133-138.
Matsunari, H., D. Arai, M. Koiso, H. Kuwada, T. Takahashi and T. Takeuchi (2005) Effect of feeding rotifers
enriched with taur担eon growth performance and
body composition of Pacific cod Larvae Gadus macro-cephαlus. Aquacult. Sci., 53, 297-304.
Mills, S., A J. Alc如句ra-Rodriguez,J. Ciros-Perez, A
Gomez, A Hagiwara, K. H. Galindo, C. D. Jersabek,
R. Malekzadeh-Viayeh, F. Leasi, J.-S. I」~e, D. B.
Mark Welch, S. Papakostas, S. Riss, H. Segers, M.
Serra, R. Shiel, R. Smol北， T.W Snell, C.-P. Stelzer, C. Q. Tang, R. L. Wallace, D. Fontaneto組 dE. J.
Walsh (2016) Fr仕eenspecies in one: deciphering
仕1eBrachionusρlicatilis species complex (Rotifera,
Monogononta）血roughDNA taxonomy.均drobiologia,
doi:l0.1007 /s10750-016-272ら7
Morretti, A, M. Pedini Fernandez-Criado, G. Cittolin and
R. Guidastri (1999) Manual on hatchery production of
seabass and gilthead seabream, FAO, Rome, pp. 194.
Mourente, G., A Rodriguez, D. R. Tocher and J. R. Sargent
(1993) Effects of dietary docosahexaenoic acid
(DHA; 22:6n-3) on lipid and fatty acid compositions
and growth in gilthead sea bream (Sparus aurata L.) larvae during first feeding. Aquaculture, 112, 79-98.
Olsen, Y., J. 0. Evjemo, E. Kj0rsvik, H. Larssen, K. Li,
I. Overrein and J. Rainuzzo (2014) DHA content in
dietary phospholipids affects DHA content in phos-
pholipids of cod larvae and larval performance.
Aquaculture, 428-429, 203-214.
Park, H. G., V. Puvanendran, A Kellett, C. C. Parrish and J. A Brown (2006) Effect of enriched rotifers on
growth, survival, and composition of larval Atlantic
cod (Gadus morhua). ICES] Mar. Sci., 63, 285-295.
Rodriguez, C., J. A Perez, M. Diaz, M. S. Izquierdo, H. Fernandez-Palacios and A Lore回 ο（1997)Influence of
the EPA/DHA ratio in rotifers on gilthead seabream
(Sparus aurata) larval development. Aquaculture, 150, 77-89.
Satoh, N., T. Fujioka and Y. Shimizu (2005) Feeding effects of enriched rotifers on the survival, growth and star-
vation resistance of brown sole (Pleuronectes herzen-steinz) larvae. Sci. Rep. Hokkaido Fish. Exp. Stn., 69,
139-144 (in Japanese with English abstract).
144 T. Kotani, T. Haraguchi, Y. Yamazaki, T. Doi, H. Matsui, S. Yokoyama, M. Ishikawa and S. Koshio
Segers, H. (1995) Nomenclatural consequences of some
recent studies on Brachionus plicatilis (Rotifera,
Brachionidae). Hydrobiologia, 313-314, 121-122.
Simmons, D. B. D. and R. J. N. Emery (2011) Phytochelatin
induction by selenate in Chiarella vulgaris, and reg-
ulation of effect by sulfate levels. Environ. Toxicol.
Chem., 30, 469-476.
S初仕rup,J. G. and Y. Attramadal (1992）百leInfluence
of different rotifer and Artemia enrichment diets
on growth, survival and pigmentation in turbot
(Scophthalmus maximus L.) larvae. ］.防匂ridAquacult.
Soc., 23, 307-316.
Suprayudi, M. A, T. Takeuchi, K. Hamasaki and J. Hirokawa (2002) The effect of n-3HUFA content in
rotifers on the development and survival of mud crab, Scylla serrata, larvae. Aquacult. Sci., 50, 205-212.
Takeuchi, T., F. Zheng, K. Yoseda, J. Hirokawa and T. Watanabe (1994) Nutritive value of DHA-enriched
rotifer for larval cod. Nippon Suisan Gakkaishi, 60,
641-652 (in Japanese with English abstract).
Vagner, M., B. de Montgolfier, J. and C. Audet (2014) Effects of algae-enriched rotifers
on winter flounder (Pseudopleuronectes americαnus ）
gene expression during metamorphosis. Mar. Biol.,
161, 985-999.
Watanabe, T, C. Kit司ima,T Arakawa, K. Fukusho and S.
Fujita (1978) Nutritional quality of rotifer Brachionus
ρlicatilis as a living feed from the veiwpoint of essen-
tial fatty acids for fish. Nippon Suis仰 Gakkaishi,44,
1109-1114 (in Japanese with English abstract).
Watanabe, T, T Tamiya, A Oka, M. Hirata, C. Kitajima
and S. F吋ita(1983) Improvement of dietary value
of live foods for fish larvae by feeding them on w 3
highly unsaturated fa町racids and fat-soluble vitamins.
Nippon Suisan Gakkaishi, 49, 471-479 (in Japanese
with English abstract).
Watanabe, T 1993. Importance of docosahexaenoic acid in
marine larval fish.]. World Aquacult. Soc., 24, 152-161.
Wold, P. A, K. Hoehne-Reitan, C. L. Cahu, J. Z. Infante,
J. Rainuzzo and E悶のrsvik(2009) Comparison of
dietary phospholipids and neutral lipids: effects
on gut, liver and pancreas histology in Atlantic cod
(Gadus morhαL.) larvae. Aquacult. Nutr., 15, 73-84.
Yukino，主， K.Tanaka, I. Maruyama, F. Konishi, T
Kumagai, T Hada and M. Hayashi (2002) Lipid and
fa町racid compositions of DHA-fortified Chlorella vul-
garis strain CK22.]. Jpn. Soc. Nutr. Food Sci., 55, 331-
337 (in Japanese with English abstract).
広塩性ツボワムシ類に対する栄養強化時間が脂肪酸組成及び
マダイ仔魚飼育成績に与える効果
小谷知也・原口拓己・山崎悠太・土井達也・松井英明
横山佐一郎・石川 学・越塩俊介
本研究は栄養強化時間の異なる広塩性ツボワムシ類（以下ワムシ）の餌料価値とマダイ仔魚、への給
与効果を明らかにすることを目的とした。強化実験には S型及び L型ワムシを使用した。ワムシは
n-3HUFA含有クロレラで12時間または24時間強化した。さらに， n-3HUFA含有クロレラを一次培養
過程から給餌し続けるワムシ（常時強化）も準備した。マダイはふ化後20日齢までの体長を測定し，
20日齢時点の生残尾数を計数， 24時間強化ワムシと常時強化ワムシの給餌群を比較した。 S型及び L
型ワムシの EPAとDHA含量は12時間強化区と24時間強化区で、は差が無かった。しかし， L型ワムシ
の極性脂質中の EPA及び DHA含量は常時強化区で高くなった。マダイの生残率は24時間強化ワム
シ区給餌群より常時強化区ワムシ給餌群で高く（Pく0.05），体長の成長速度も常時強化ワムシ給餌群
でi車くなった。
,..
