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: tkotani@ma.fuma.fukuyama-u.ac.jp

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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