ORIGINAL ARTICLE Aquaculture
Effect of cultivation methods on nutritional enrichmentof euryhaline rotifer Brachionus plicatilis
Tomonari Kotani Æ Teruhisa Genka ÆHiroshi Fushimi Æ Masahiro Hayashi ÆKristof Dierckens Æ Patrick Sorgeloos
Received: 8 September 2008 / Accepted: 19 March 2009 / Published online: 12 May 2009
� The Japanese Society of Fisheries Science 2009
Abstract This study aimed at comparing fatty acid con-
tents of rotifers cultured with different methods after
nutritional enrichment in order to evaluate the rotifer
quality produced by these methods. Rotifers were cultured
using either a batch or a continuous culture. From the batch
culture, three experimental subpopulations were used,
sampled from the culture at 1, 24, and 48 h after rotifer
inoculation. The continuous culture was performed with
two tanks; one was for cultivation with continuous feeding
and water supply (cultivation tank), and another was for
harvesting from the cultivation tank by overflow (harvest
tank). From the continuous culture, two subpopulations
were used: one from the cultivation and one from the
harvest tank. Nutritional enrichment was performed after
each culture. Each population was enriched with Nanno-
chloropsis oculata or a commercial enrichment diet. When
the enrichment was performed with N. oculata on popu-
lations at 24 h after inoculation originating from either of
the two tanks of continuous culture or the batch culture
tank, a higher quantity of arachidonic acid (ARA) and
eicosapentaenoic acid (EPA) was obtained from the two
tanks of continuous culture. The same results were
obtained when enrichment diet was used, this time
including docosahexaenoic acid (DHA).
Keywords Batch culture � Continuous culture �Fatty acid � Nutritional enrichment � Rotifer
Introduction
Euryhaline rotifers Brachionus plicatilis complex are the
zooplankton used mostly as the first feed for larviculture.
Since Ito [1] introduced rotifers as live feed, methods for
mass culture of rotifers have been developed [2–6]. After
the mass culture of rotifers had been established, mass
production of fish larvae was put into practice [7–10]. On
the other hand, some problems with fish larvae in relation
to rotifers became prevalent. Many of them were caused by
the inappropriate nutritional content of rotifers, e.g., mass
mortality [11], malpigmentation [12], and deformity [13].
Others were caused by instability of the rotifer culture [14].
So far, the causes of nutritional issues have been clarified
[11–13], and some of them have been solved by the
development of nutritional enrichment diets for rotifers
[15, 16]. n-3 highly unsaturated fatty acid (HUFA)
including eicosapentaenoic acid (EPA) and docosahexae-
noic acid (DHA) is important for health and activities of
fish larvae [17], and therefore the studies on the enrichment
of rotifers have focused on n-3 HUFA, especially EPA and
DHA.
The stabilization of rotifer cultures was achieved by the
improvement of culture methods and equipment, e.g., the
introduction of filters to remove waste [18], the stable
supply of freshwater Chlorella as feed [19], and the
development of chemostat culture [20]. On the other hand,
T. Kotani (&) � T. Genka � H. Fushimi
Institute of Marine Bio-resources, Faculty of Life Science
and Biotechnology, Fukuyama University, 452-10
Innoshima-Ohama, Onomichi, Hiroshima 722-2101, Japan
e-mail: [email protected]
M. Hayashi
Department of Biological Production and Environmental
Science, Faculty of Agriculture, University of Miyazaki,
Gakuen-kibanadai-nishi-1-1, Miyazaki 889-2192, Japan
K. Dierckens � P. Sorgeloos
Laboratory of Aquaculture and Artemia Reference Center,
Faculty of Bioscience Engineering, Ghent University, Rozier 44,
9000 Ghent, Belgium
123
Fish Sci (2009) 75:975–984
DOI 10.1007/s12562-009-0105-1
Tomoda et al. [21, 22] reported that the population growth
phase of the rotifers affects the growth of finfish larvae.
This is due to the environmental factors affecting the
physiological status of the rotifers during the culture [21–
23]. The success of finfish larviculture depends on the
nutritional value of live feed. In continuous culture, the
water quality is stable because of continuous supply of
water and phytoplankton (freshwater Chlorella) and
therefore we can obtain a stable quality of the rotifers,
theoretically. However, the level and stability of the rotifer
quality cultivated with continuous culture is unknown.
There is little knowledge on the correlation of rotifer
population growth in batch cultures compared with con-
tinuous cultures. Furthermore, no comparison has been
made between the nutritional compositions of rotifers
originating from a batch or from a continuous culture after
nutritional enrichment.
In this study, we aimed to evaluate the effect of the
rotifer culture method, continuous or batch, on nutritional
enrichment. In order to evaluate these effects, we analyzed
the fatty acid contents of the rotifer population from each
culture before and after nutritional enrichment.
Materials and methods
In each experiment, the Obama strain of euryhaline rotifer
Brachionus plicatilis was used. This high-fecundity strain
originated from a rotifer population maintained at low tem-
perature (10–15�C) in the Obama Station of The Japan Sea
Farming Association (presently the National Center for Stock
Enhancement, Fisheries Research Agency) for 20 years.
Two methods of rotifer cultivation, batch and continu-
ous culture, were applied. Concentrated freshwater Chlo-
rella vulgaris (Nihon Chlorella Co. Ltd, Kunitachi, Japan)
was used as feed in both methods. Water temperature was
adjusted and controlled at 25 ± 1�C. Seawater for culture
medium was pumped up from sea around the Institute of
Marine Bio-resources, Fukuyama University, Onomichi,
Japan and sterilized with ultraviolet (UV) light after sand-
filtering. Seawater was diluted with tapwater such that
salinity was adjusted to 20 psu confirmed with a refrac-
tometer. In batch and continuous cultures, pH and con-
centrations of dissolved oxygen (DO) and ammoniac
nitrogen were measured to assess the water quality. DO
was measured using a Tox-90 meter (Toko-Kagaku Co.
Ltd., Tokyo, Japan), and pH was measured using a pH
meter (HM-30V, TOA Electronics Ltd., Tokyo, Japan).
Total ammoniac nitrogen was measured by the sodium
salicylic acid method [24]. The ammonia cyanurate reagent
powder (Cat. 23955-66, HACH Co., Loveland, CO, USA)
and the ammonia salicylate reagent powder (Cat. 23955-
66, HACH Co.) were added to the sampled culture water
diluted 100 times with 60% diluted seawater, and absor-
bance was measured after 3 min with a DR/4000 spectro-
photometer (0–0.80 mg/l NH3–N, HACH Co.). Based on
the pH, water temperature, salinity, and total ammoniac
nitrogen concentration, the unionized ammonia fraction of
the total ammoniac nitrogen measured was calculated from
the following formula of Bower and Bidwell [25];
%unionized ammonia¼100ð1þantilog10 pKasðTÞ�pHð Þ�1
in which
pKasðTÞ ¼ pKa
sðT ¼ 298Þ þ 0:0324ð298� TÞ;
T = degrees Kelvin, and the value for pKas in 18–22 psu
seawater at 25�C was assumed to be 9.29 [25].
Population density was measured daily in each culture
and the growth ratio from the previous day in the batch
culture was calculated. The growth ratio was defined as
follows:
growth ratio (%) ¼ ððDp � Dp�1Þ=Dp�1Þ � 100;
where Dp is the population density on a day and Dp-1 is the
population density on the previous day.
Batch culture
Batch culture was performed in a 500-l cylindro-conical
tank. Initial stocking density was adjusted to 700 rotifers/ml.
C. vulgaris inoculation was carried out daily at the rate of
20 9 103 cells/rotifer. Aeration was provided by a ceramic
air-stone suspended 5 cm from the centre of tank bottom. To
remove particulate waste, four nylon filter nets (Tanaka
Sanjiro Co. Ltd., Ogori, Japan, 60 cm 9 40 cm 9 2 cm)
were placed vertically in the culture tank and changed daily.
Batch culture was stopped at 2 days after rotifer inoculation
and a portion of the culture was used as the initial population
of new batch culture. The rotifer populations were harvested
at 1, 24, and 48 h after the inoculation of rotifers and these
were used for the nutritional enrichment experiments.
Continuous culture
The continuous culture method we used was a modification
of method described by Kuwada [26]. Three 500-l cylin-
dro-conical tanks, one refrigerator, one 18-l plastic tank,
and two pumps were used (Fig. 1). Initially, two cylindro-
conical tanks were filled with 60% diluted seawater
(20 psu). Nothing further was added to one tank (supply
tank), while rotifers were inoculated at a rate of approxi-
mately 750 rotifers/ml in the other tank (cultivation tank).
Twenty million C. vulgaris cells/ml was added to the
rotifer cultivation tank. A vinyl chloride pipe, adjusted to
the height and water volume of the 500-l cultivation tank,
976 Fish Sci (2009) 75:975–984
123
was placed in the centre of the tank. The cultivation tank
was connected to the third tank (harvest tank) with a hose.
A quantitative pump (EBN-B20, Iwaki Co. Ltd., Japan)
was used to supply 60% diluted seawater from the supply
tank to the cultivation tank at a rate of 300 l/day (208 ml/
min). Concentrated C. vulgaris (3.3 l; 15 9 109 cells/ml)
and 13.7 l freshwater were mixed and supplied at a rate of
11.8 ml/min to the cultivation tank. Freshwater was added
in order to avoid C. vulgaris cells piling up in the pump
and vinyl tubes and in order to keep fluidity. The rotifer
and C. vulgaris-containing culture water overflowed from
the cultivation tank through the vinyl chloride pipe and
hose to the harvest tank. In both cultivation and harvest
tanks, aeration was supplied by one ceramic air-stone
suspended 5 cm from the centre of tank bottom. To remove
particulate waste, four nylon filter nets (Tanaka Sanjiro Co.
Ltd., Ogori, Japan, 60 cm 9 40 cm 9 2 cm) were placed
vertically in the cultivation and harvest tanks. These filters
were changed daily. Rotifer harvest from the cultivation
and harvest tanks was performed in the morning on random
days as per the requirement for the nutritional enrichment
experiments. The population remaining in the harvest tank
was discarded after the harvest.
Nutritional enrichments
Two nutritional enrichment treatments were compared. In
one treatment, Nannochloropsis oculata was fed at
60 9 106 cells/ml in 20 g/l diluted seawater at 25 ± 1�C
for 24 h. In the other treatment, commercial enrichment diet
(DHA Protein SELCO; INVE Technologies NV, Dender-
monde, Belgium) was fed at 0.25 g/l of concentration at
25 ± 1�C for 8 h. The initial population density in each
treatment was 1 9 103 rotifers/ml. After the enrichment,
each population was washed on a 45-lm-mesh net. After
the removal of extra moisture, these populations were
stored at -80�C until analysis of fatty acid content. Three
populations were sampled from each culture (batch or
continuous) on different days. As controls, populations
were also sampled before the nutritional enrichment.
Fatty acid contents
The extraction of lipids was based on the method of Folch
et al. [27]. Lipids were extracted from 1 g of each frozen
sample. A lipid methyl ester was prepared prior to gas
chromatography (GC; G-3000, Hitachi Industry Co. Ltd.,
Tokyo, Japan) analysis. The extracted lipid was resus-
pended in 1 ml chloroform containing 2 mg/ml fatty acid
standard, C19:0. The suspension was transferred to a 10-ml
centrifuge tube and 1 ml 5% hydrogen chloride methanol
solution (Wako Pure Chemical Industries, Ltd., Osaka,
Japan) was added to it. The tube was then heated at 80�C
for 3 h. After cooling, 1 ml hexane and 5.5 ml 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 bottle and stored at -80�C
until GC analysis. The hexane layer including fatty acid
was subject to GC analysis. The data detected were ana-
lyzed based on the C19:0 standard.
Statistical analysis
Kruskal–Wallis tests were performed to compare the
amount of lipid and each fatty acid among conditions before
enrichment treatments, and Mann–Whitney’s U test also
Quantitative pump
60 %dilutedseawater
ConcentratedfreshwaterChlorella
Refrigerator
Filter
Supply tank Cultivation tank Harvest tank
Filter
Pipe
18l tank
Air pump
Quantitative pump
Bulb
Drain
Fig. 1 Schematic overview of
the continuous culture system
modified from the system of
Kuwada [24]
Fish Sci (2009) 75:975–984 977
123
was performed in order to compare multiply. These analy-
ses were performed with the statistical analysis software
Statview version 5.0 (SAS Institute Inc.) for Windows.
Results
Rotifer density in each culture
Eight consecutive batch cultures were performed in
17 days and the continuous culture was maintained for
27 days (Figs. 2, 3). In batch cultures, rotifers were inoc-
ulated at an average density of 676 rotifers/ml and
increased to 1,066 rotifers/ml after 24 h and to 1,204 rot-
ifers/ml after 48 h (Table 1). All batch cultures, except the
fifth one (9th to 11th days), maintained the increase from
inoculation to harvest (Fig. 2). The average of the growth
ratio from the first to second day was higher than that from
second to third day (Table 1; Mann–Whitney’s U test,
U value = 5.0, z value = -2.8, P \ 0.01).
In the continuous culture, rotifers were inoculated at
about 750 rotifers/ml. Population density in the cultivation
tank reached a maximum density (1,296 ± 43 rotifers/ml)
on the fourth day and then kept fluctuating between 500
and 1,000 rotifers/ml (Fig. 3). The density in the harvest
tank reached a maximum (1,332 ± 21 rotifers/ml) on the
fifth day and then kept fluctuating between 500 and 1,000
rotifers/ml (Fig. 3).
Water quality
pH value of the culture water in the batch culture was 7.1 at
1 h after inoculation. Although it rose to 7.4 the next day
(24 h after inoculation), there was no significant change
after that (Table 2). The concentration of dissolved oxygen
decreased from 7.12 to 2.62 mg/l during the batch culture,
while the concentration of free ammonia kept increasing
(Table 2).
pH value and concentration of dissolved oxygen of the
culture water in the continuous culture did not differ
between the two tanks and they tended to be higher than in
the batch culture at 24 and 48 h after inoculation (Table 2;
Mann–Whitney’s U test, P \ 0.05). The unionized
ammonia concentration was significantly higher in the
continuous culture than in the batch culture (Table 2;
Mann–Whitney’s U test, P \ 0.05). The concentration of
total ammoniac nitrogen and unionized ammonia increased
during the batch culture (Table 2; Mann–Whitney’s U test,
P \ 0.05).
Fatty acids contents
The total lipid amount, four fatty acids (16:0, 16:2, 16:3,
and 18:2 n-6), and total n-3 HUFA in the rotifers from the
different culture systems were significantly different
(Table 3). There was no difference among total lipid
amount after the nutritional enrichment. However, we
found significant differences in more kinds of fatty acids
after nutritional enrichment than just after the culture: for
N. oculata: nine fatty acids, total n-3 and n-6 HUFA
(Table 4; Kruskal–Wallis test, P \ 0.05); for the enrich-
ment diet: seven fatty acids, total n-3 and n-6 HUFA
(Table 5; Kruskal–Wallis test, P \ 0.05). For all fatty
500
750
1000
1250
1500
1750
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time (Day)
Den
sity
(ro
tifer
s /m
l)
Fig. 2 Rotifer population density in eight consecutive batch cultures.
Mean ± SE (n = 5)
500
750
1000
1250
1500
1 3 5 7 9 11 13 15 17 19 21 23 25 27
500
750
1000
1250
1500
(a)
(b)
Time (Day)
Time (Day)
1 3 5 7 9 11 13 15 17 19 21 23 25 27D
ensi
ty (
rotif
ers
/ml)
Den
sity
(ro
tifer
s /m
l)
Fig. 3 Rotifer population density in the culture and harvest tanks of
the continuous culture system for a period of 27 days. Mean ± SE
(n = 5). a Density in the culture tank. b Density in the harvest tank
978 Fish Sci (2009) 75:975–984
123
acids with significant different concentrations in the roti-
fers after the culture, the level in the rotifers from the
harvest tank was the highest (Tables 3, 4, 5; U test,
P \ 0.05) and that value was approximately two times
higher than that from the sample taken from the batch
culture at 48 h after inoculation (Tables 3, 4, 5).
After the nutritional enrichment, of all fatty acids with
more than 20 carbons, significant differences were only
detected in arachidonic acid (20:4 n-6, ARA), EPA (20:5
n-3), and/or DHA (22:6 n-3). However, these trends differed
between enrichment diets (Tables 4, 5). When N. oculata
was used, the samples from the continuous culture contained
more ARA and EPA than in the batch culture (Table 4;
Kruskal–Wallis test and U test, P \ 0.05). On the other
hand, when the artificial enrichment diet was used, the
sample from 24 h after inoculation in batch culture and the
one from the cultivation tank of the continuous culture
contained higher amounts of ARA, EPA, and DHA than
the ones from the other treatments (Table 5; Kruskal–Wallis
test and U test, P \ 0.05).
The levels of the fatty acids in the samples taken from
the batch culture at 24 h after inoculation tended to be the
highest of the batch culture (Table 3; U test, P \ 0.05). We
found a similar trend of nutritional enrichment with the
artificial enrichment diet of batch-cultured rotifers
(Table 5; U test, P \ 0.05). Moreover, the values of the
samples from 24 h after inoculation reached a similar level
to those from the harvest tank (Tables 3, 5). Such trends
were not seen in the results from the nutritional enrichment
with N. oculata after the batch culture. Those values were
lower than those from the continuous culture (Table 4; U
test, P \ 0.05). On the other hand, there was no significant
difference between the amounts of each fatty acid detected
in the samples from the two tanks of the continuous culture
(Tables 3, 4, 5).
Discussion
In general, the batch culture method is performed for 3–
5 days. During that period, the population growth of the
rotifers changes from lag phase to logarithmic and finally
to stationary phase. These differences of population
growth phases reflect the physiological activities of the
rotifers [28]. In this study, the growth ratio was higher
during the first 24 h of the batch culture than from the
second to the third day (Table 1). Therefore, we can
determine that the second day of batch culture in this
study corresponds to the logarithmic growth phase. Lipid
and fatty acid content of the rotifers from the batch cul-
ture at 24 h after inoculation, before the nutritional
enrichment, tended to be higher than of rotifers from the
other sampling moments (Table 3). Since the same trend
was shown after the nutritional enrichment with the arti-
ficial enrichment diet (Table 5), it seems that the physi-
ological activity of the rotifers in the logarithmic growth
Table 1 Statistics of rotifer populations of eight replicates of the batch culture
Time after
inoculation (h)
Average density
(rotifers/ml)
Minimum density
(rotifers/ml)
Maximum density
(rotifers/ml)
Average growth ratio
from previous day (%)
1 676.0 ± 88.8 582 852 –
24 1,065.8 ± 183.8 808 1,368 61.3 ± 42.3
48 1,203.8 ± 243.5 928 1,662 13.0 ± 11.6
Values indicate mean ± standard deviation (SD) of three to five replicates
Table 2 pH, dissolved oxygen, and total and unionized ammoniac nitrogen in the culture water of batch and continuous culture
Rotifer culture pH Dissolved
oxygen (mg/l)
Total ammonia-N
(mg/l)
Estimated unionized
ammonia (mg/l)a
Batch culture (after inoculation)
1 h 7.2c ± 0.1 7.12a ± 1.0 1.0e ± 1.0 0.01e ± 0.01
24 h 7.4b ± 0.1 3.80a,b ± 3.20 19.7d ± 3.2 0.25d ± 0.08
48 h 7.7b ± 0.3 2.62b ± 2.20 42.0b ± 5.0 1.00c ± 0.43
Continuous culture
Cultivation tank 8.2a ± 0.0 7.23a ± 0.78 62.7a ± 7.6 4.18a ± 0.49
Harvest tank 8.2a ± 0.0 7.35a ± 1.03 30.0c ± 3.5 2.04b ± 0.41
Values indicate mean ± SD during the culture period
Values with different letters indicate the result of Mann–Whitney’s U test among rotifer cultures (P \ 0.05; a [ b [ c [ d [ e)a Values were calculated from the formula of Bower and Bidwell [25]
Fish Sci (2009) 75:975–984 979
123
phase of the batch culture increases and rotifers absorb
higher quantities of fatty acids. Moreover, rotifer popu-
lations under logarithmic or exponential growth phase
include many individuals bearing eggs [29, 30]; rotifer
eggs contain relatively high amount of lipid [31]. There-
fore, we inferred that higher lipid content of rotifer pop-
ulations from batch culture at 24 h after inoculation also
could reflect the number of eggs. On the other hand, after
enrichment treatments, lipid contents did not differ among
initial states, though some fatty acid contents differed
significantly (Tables 3, 4). It is not clarified how many
eggs rotifers bear after enrichment treatment. In order to
confirm this hypothesis, it is necessary to count the
number of eggs rotifers bore after enrichment treatment.
In previous studies, chemostat continuous cultures were
developed [4, 5, 32, 33]. Recently, extensive continuous
culture was improved and enabled the continuous method
to be performed in a tank larger than 10 m3 [20, 26]. The
continuous culture method in this study was modified from
the methodology of Kuwada [26] and the population den-
sity in this study (500–1,000 rotifers/ml) was higher than in
the cases of Kuwada [26] (100–400 rotifers/ml) (Fig. 2).
Previous studies reported that chemostat rotifer culture has
greater stability and productivity than any conventional
rotifer culture method [5, 20, 26, 32]. However, the rotifer
population density fluctuated during the cultivation period,
though the population did not crash [24, 32]. In this study,
the rotifer population densities in both tanks of continuous
culture fluctuated as well (Fig. 3). Fluctuation of rotifer
population density is an essential feature of chemostat
continuous culture [34, 35]. When rotifer population
growth leads to shortage of phytoplankton as feed, the
frequency of rotifer females bearing eggs decreases, which
results in the decline of population growth. After that
decline, the amount of phytoplankton per individual rotifer
increases and the population growth rises. Accordingly, the
fluctuation of the rotifer density in the continuous culture
did not influence the physiological activity of the rotifers,
at least not the absorbance of fatty acids by rotifers. On the
other hand, we were not able to evaluate the physiological
activities by the fatty acid composition only. Therefore, in
order to evaluate the details of physiological activities, it is
necessary to use other methods, e.g., percentage of feeding
individuals, extent of digestive organs in the body, fre-
quency distribution of lorica length, tolerance to hypersa-
line environment [28], and enzyme activity [36].
Table 3 Fatty acid contents of cultured rotifers before enrichment
Batch culture (after inoculation) Continuous culture
1 h 24 h 48 h Cultivation tank Harvest tank
Total lipid contents (%) 13.36c ± 1.03 16.06a,b ± 1.52 14.12b,c ± 0.44 16.60a ± 1.38 17.06a ± 0.35
Fatty acids contents (mg/g DW)
14:0 0.99 ± 0.30 1.52 ± 0.86 0.94 ± 0.20 1.25 ± 0.24 1.00 ± 0.13
16:0 6.94b ± 1.74 11.79a ± 1.15 6.68b ± 1.06 13.14a ± 0.57 12.91a ± 1.10
16:1 0.68 ± 0.22 0.94 ± 0.32 0.67 ± 0.21 0.99 ± 0.18 0.83 ± 0.22
16:2 3.68b ± 0.74 7.49a ± 2.18 3.88b ± 0.81 5.91a ± 0.36 5.93a ± 0.56
16:3 0.71b ± 0.08 0.49b,c ± 0.38 0.61c ± 0.02 1.12a ± 0.08 1.15a ± 0.04
18:0 1.65 ± 0.34 2.01 ± 0.99 1.49 ± 0.29 2.82 ± 0.08 2.75 ± 0.19
18:1 n-9 0.63 ± 0.56 1.01 ± 0.53 0.48 ± 0.09 0.81 ± 0.08 0.85 ± 0.13
18:1 n-7 0.58 ± 0.05 0.53 ± 0.47 0.57 ± 0.04 0.99 ± 0.07 0.66 ± 0.57
18:2 n-6 14.80b ± 2.69 24.31a ± 3.99 14.09b ± 1.37 26.14a ± 1.95 26.43a ± 1.70
18:3 n-3 2.14 ± 1.17 3.49 ± 1.47 1.46 ± 0.04 3.86 ± 2.51 5.31 ± 0.29
20:4 n-6 0.06 ± 0.10 0.31 ± 0.36 0.30 ± 0.26 0.21 ± 0.19 0.23 ± 0.21
20:4 n-3 0.16 ± 0.28 0.33 ± 0.32 0.02 ± 0.04 0.31 ± 0.27 0.09 ± 0.16
20:5 n-3 ND 0.02 ± 0.03 ND ND ND
22:5 n-6 ND ND ND ND ND
22:5 n-3 ND ND ND ND ND
22:6 n-3 ND ND 0.01 ± 0.01 0.04 ± 0.07 0.39 ± 0.36
Rn-3 HUFA 0.16b,c ± 0.28 0.35a,b ± 0.35 0.03c ± 0.05 0.35a,b ± 0.33 0.48a ± 0.41
Rn-6 HUFA 0.06 ± 0.10 0.31 ± 0.36 0.30 ± 0.26 0.21 ± 0.19 0.23 ± 0.21
Values indicate mean ± SD of three replicates
Values with different letters indicate the result of Mann–Whitney’s U test among rotifer cultures (P \ 0.05; a [ b [ c)
ND not detected
980 Fish Sci (2009) 75:975–984
123
In the batch method, the physiological activities of
rotifer were reduced due to the increase of unionized
ammonia concentration [23, 36–38] and the decrease of
dissolved oxygen [39]. In the batch culture of this study,
the unionized ammonia concentration increased and DO
decreased with the progress of batch culture (Table 2). The
fatty acid content after the enrichment with N. oculata or
the artificial enrichment diet using the rotifers from the
batch culture at 1 and 48 h after inoculation was lower than
24 h after inoculation (Tables 4, 5). We inferred that the
low physiological activities of rotifer in the batch culture at
1 h after inoculation were because of handling in the
inoculation, and that those at 48 h were because of the
increase of unionized ammoniac nitrogen and low DO
concentrations. Compared with the result of Yu and Hi-
rayama [37], the unionized ammonia concentrations in this
study were not so high (Table 2). Therefore, we deduced
that rotifers influenced by low DO concentration lowered
their physiological activities, and this resulted in the
decrease of lipid and some fatty acid contents even if
unionized ammonia concentration increased slightly.
Moreover, the DO concentrations were higher in both tanks
of the continuous culture than in batch culture, and lipid
and some fatty acid contents of rotifers from continuous
culture were also higher even if the unionized ammonia
concentrations in continuous culture were higher than in
batch culture (Tables 2, 3, 4, 5). Taking these factors into
consideration, we can hypothesize that, if the unionized
ammoniac nitrogen concentration increases slightly, it does
not affect the physiological activities of rotifers so much
under high DO concentration. In order to confirm this
hypothesis, it is necessary to observe the physiological
activities of rotifers under high or low DO and high
unionized ammonia concentrations.
In the continuous culture, if the exchange rate of culture
water is fixed and the population density of rotifer is at a
steady state, the population growth rate of the rotifers is at
a steady state as well. Although the population densities in
this continuous culture were fluctuated, the culture did not
collapse and the population number did not increase
extremely (Fig. 2). As the exchange rate of culture water in
the continuous culture was 60%/day, it seems that the
population growth rate was around 60%/day as well.
The content of different fatty acids of the samples from the
continuous culture was similar to those related to the
samples from the batch culture at 24 h after inoculation
Table 4 Fatty acid contents of cultured rotifer enriched with Nannochloropsis oculata
Batch culture (after inoculation) Continuous culture
1 h 24 h 48 h Cultivation tank Harvest tank
Total lipid contents (%) 13.41 ± 1.46 14.25 ± 0.47 13.74 ± 1.43 15.35 ± 1.60 15.87 ± 1.22
Fatty acids contents (mg/g DW)
14:0 1.17b ± 0.32 1.24b ± 0.16 1.16b ± 0.10 1.96a ± 0.14 2.34a ± 0.84
16:0 6.87b ± 0.44 5.94c ± 0.25 5.83b,c ± 0.91 9.88a ± 1.66 10.37a ± 2.38
16:1 2.62d ± 0.19 3.54b ± 0.06 3.25c ± 0.24 5.81a ± 0.49 6.21a ± 1.35
16:2 1.88b,c ± 0.25 1.81c ± 0.06 1.37d ± 0.38 2.70a,b ± 0.59 3.40a ± 0.78
16:3 0.98 ± 0.13 0.10 ± 0.09 0.33 ± 0.57 0.65 ± 0.45 0.33 ± 0.17
18:0 1.77 ± 0.22 0.74 ± 0.11 1.12 ± 0.45 1.92 ± 0.55 1.60 ± 0.14
18:1 n-9 1.11b ± 0.34 1.02b ± 0.08 1.01b ± 0.03 1.67a ± 0.07 1.76a ± 0.22
18:1 n-7 1.02 ± 0.05 0.85 ± 0.06 1.08 ± 0.51 1.58 ± 0.39 1.54 ± 0.13
18:2 n-6 10.08a ± 1.49 6.66b ± 0.19 6.47b ± 0.94 10.79a ± 1.33 11.16a ± 1.83
18:3 n-3 1.22a ± 0.39 0.77b ± 0.07 0.60c ± 0.05 1.30a ± 0.17 1.39a ± 0.17
20:4 n-6 1.29b ± 0.18 1.01b ± 0.14 1.19a,b ± 0.36 1.91a ± 0.42 1.81a ± 0.24
20:4 n-3 0.32 ± 0.15 0.00 ± 0.00 0.08 ± 0.14 0.18 ± 0.19 0.05 ± 0.08
20:5 n-3 2.91d ± 0.22 5.38b ± 0.48 4.62c ± 0.21 8.03a ± 0.57 9.21a ± 1.20
22:5 n-6 ND ND ND 0.04 ± 0.07 0.18 ± 0.32
22:5 n-3 0.01 ± 0.02 ND 0.06 ± 0.10 0.07 ± 0.11 ND
22:6 n-3 0.29 ± 0.27 ND 0.05 ± 0.09 0.22 ± 0.18 0.26 ± 0.22
Rn-3 HUFA 3.54d ± 0.41 5.38b ± 0.48 4.81c ± 0.13 8.50a ± 0.36 9.51a ± 1.17
Rn-6 HUFA 1.29b ± 0.18 1.01b ± 0.14 1.19a,b ± 0.36 1.95a ± 0.40 2.00a ± 0.48
Values indicate mean ± SD of three replicates
Values with different letters indicate the result of Mann–Whitney’s U test among rotifer cultures (P \ 0.05, a [ b [ c)
ND not detected
Fish Sci (2009) 75:975–984 981
123
(Tables 3, 4, 5). Considering the growth rate, it is possible
that the population growth in the continuous culture was
consistent with logarithmic phase, and therefore those
rotifers could include higher contents in some fatty acids
because they had higher physiological activities. On the
other hand, the water quality of the batch culture at 24 h
after inoculation and that in the continuous culture was
quite different (Table 2). Moreover, although Tomoda
et al. [40] did not use the population in the middle loga-
rithmic phase, they reported that the DHA and EPA con-
tents of rotifers from batch culture in the logarithmic phase
were significantly lower than those of rotifers from con-
tinuous culture. Accordingly, we cannot conclude that the
physiological activities of rotifers in the continuous culture
were similar to or the same as the ones under logarithmic
phase in the batch culture.
Most hatcheries use the batch culture method and
rotifers are harvested at the end of the culture period in
order to feed finfish larvae. In the batch culture of this
study, the population of batch culture at 48 h after inocu-
lation corresponded to this situation. In the case of the
continuous culture, rotifers in the harvest tank are utilized
as the feed for finfish larvae. After the nutritional enrich-
ment using N. oculata and commercial enrichment diet,
rotifers from the harvest tank of the continuous culture
contained 1.5–2 times as much fatty acids as the ones from
the batch culture at 48 h after inoculation (Tables 4, 5). In
the result of Tomoda et al. [40], rotifers from the contin-
uous culture contained approximately 1.5 times as much
fatty acids as the ones from the batch culture in later log-
arithmic phase. Stationary phase is subsequent to later
logarithmic phase and the activities of rotifers in later
logarithmic phase are similar to stationary phase. Accord-
ingly these results are not contradictory to the result of
Tomoda et al. [40]. The quantity of EPA, DHA or total n-3
HUFA has various effects on growth, morphology, and
survival of finfish larvae [11, 41–45]. Previous studies on
the nutritional enrichment used batch-cultured rotifers.
Therefore, nutritional enrichment diets are likely to be
designed for rotifers cultivated with the batch method.
When rotifers cultivated with the continuous culture are
enriched nutritionally, they have higher quantities of fatty
acids than in the case of batch culture, and therefore some
nutritional characteristics will be improved. On the other
hand, we cannot deny that the excess of fatty acid may
have negative effects for larvae [43, 45]. Moreover,
although this study focused on fatty acid content, it is
possible that other kinds of nutrients show the same trend,
Table 5 Fatty acid contents of cultured rotifer enriched with the artificial enrichment diet (DHA Protein SELCO, INVE)
Batch culture (after inoculation) Continuous culture
1 h 24 h 48 h Cultivation tank Harvest tank
Total lipid contents (%) 17.30 ± 2.40 21.24 ± 3.03 18.86 ± 1.61 19.43 ± 1.27 20.44 ± 1.30
Fatty acids contents (mg/g DW)
14:0 1.22b ± 0.20 1.86a ± 0.27 1.23b ± 0.18 1.65a ± 0.11 2.01a ± 0.41
16:0 15.72 ± 7.01 17.67 ± 2.33 11.18 ± 1.67 13.73 ± 1.69 17.94 ± 3.34
16:1 2.96 ± 1.46 4.14 ± 0.24 2.46 ± 0.41 3.15 ± 0.19 3.84 ± 0.62
16:2 2.04 ± 0.91 2.34 ± 0.31 1.44 ± 0.23 1.81 ± 0.35 2.40 ± 0.48
16:3 0.44c ± 0.00 0.94a ± 0.15 0.61b ± 0.13 0.70b ± 0.13 1.15a,b ± 0.49
18:0 3.85 ± 1.19 5.43 ± 0.72 2.63 ± 0.44 3.48 ± 0.84 4.28 ± 0.82
18:1 n-9 6.77b,c ± 1.73 10.33a ± 0.66 5.30c ± 0.78 7.71b ± 0.68 8.20a,b ± 1.47
18:1 n-7 0.68 ± 1.18 0.89 ± 1.31 1.22 ± 0.22 0.52 ± 0.91 1.87 ± 0.34
18:2 n-6 10.85c ± 2.14 18.22a ± 1.56 10.75c ± 1.53 13.75b,c ± 1.45 17.23a,b ± 3.15
18:3 n-3 1.10 ± 0.59 1.47 ± 0.06 0.78 ± 0.12 1.08 ± 0.18 1.28 ± 0.18
20:4 n-6 0.84c ± 0.19 1.45a ± 0.11 0.79c ± 0.23 1.13b,c ± 0.19 1.33a,b ± 0.19
20:4 n-3 0.18 ± 0.18 0.52 ± 0.04 0.59 ± 0.32 0.50 ± 0.28 0.98 ± 0.49
20:5 n-3 2.73c ± 0.48 4.46a ± 0.18 2.49c ± 0.41 3.38b ± 0.34 4.00a,b ± 0.72
22:5 n-6 0.69 ± 0.27 1.06 ± 0.09 0.55 ± 0.24 0.85 ± 0.11 0.90 ± 0.17
22:5 n-3 0.22 ± 0.20 0.33 ± 0.06 0.24 ± 0.05 0.29 ± 0.06 0.39 ± 0.13
22:6 n-3 4.66c ± 0.57 8.52a ± 0.44 4.50c ± 0.91 6.31b ± 0.64 7.31a,b ± 1.15
Rn-3 HUFA 7.80c ± 1.05 13.83a ± 0.69 7.82c ± 1.61 10.47b,c ± 1.02 12.68a,b ± 1.97
Rn-6 HUFA 1.52c ± 0.40 2.52a ± 0.17 1.34c ± 0.38 1.98b,c ± 0.30 2.24a,b ± 0.27
Values indicate mean ± SD of three replicates
Values with different letters indicate the result of Mann–Whitney’s U test among rotifer cultures (P \ 0.05; a [ b [ c)
982 Fish Sci (2009) 75:975–984
123
e.g., protein or vitamins. If finfish larvae obtain excess
amount of vitamin A through rotifers, they will have some
malformation [13, 46]. The introduction of the continuous
culture makes it possible to reduce cost and labor. Besides,
the effect of continuous culture on enrichment of other
kinds of nutrition should be investigated in order to avoid
the negative effect caused by the excess of nutrients.
Depending on the results, it is necessary to improve the
composition of the enrichment diet.
In larval rearing, the nutritional value of live feed at the
larval stage is important for the growth and health of larvae
[47, 48]. Considering the results of this study, it is possible
that the nutritional value of rotifers cultivated with the
continuous method is higher than that with batch method.
On the other hand, it is necessary to clarify the chemical
composition and physiological activities after nutritional
enrichment of rotifers originating from continuous culture.
Acknowledgments We thank Mr. Hiroshi Kuwada, National Center
for Stock Enhancement, Fisheries Research Agency, for his advice on
the continuous culture method and offering the rotifer strain. We also
thank the anonymous reviewers for their useful advice to enhance the
content of this report. We also appreciate Mr. Nobumitsu Sato,
Nagase Biochemicals Sales Co., Ltd., for his valuable assistance.
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