Possibility of enrichment of rotifer (Brachionus rotondiformis) with calcium (Ca) for feeding the fish fry was investigated. Rotifer was kept for 24 h with aeration in normal seawater (Treatment 1), seawater with 400 mg/l supplemental Ca from Ca-lactate (Treatment 2) and seawater with 400 mg/l supplemental Ca from Ca-chloride (Treatment 3). After the experimental period, Ca contents of rotifer were 0.20, 0.29 and 0.39% of dry weight in T-1, T-2 and T-3, respectively. Ca content of media did not affect phosphorus, zinc and manganese contents of rotifer. Results revealed that rotifer can be enriched with Ca for feeding fish fry and Ca-chloride might be a better source for Ca enrichment

Furuichi, M.

Hossain, M.A.

Rahman, M.S.

Aquatic Commons

Bangladesh Fish. Res., 11(1), 2007: 51-56 	 ©BFRI 
Enrichment of rotifer Brachionus rotundiformis with calcium 
M.A. Hossain* 1, M.S. Rahman' and M. Furuichi 3 
Fishery Research Laboratory, Kyushu University, Fukuoka 811-0033, Japan 
`Present address: Department of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University 
Gazipur 1706, Bangladesh 
Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh 
'Present address: Wakamiya 3-3-16, Higashi-Ku, Fukuoka City 813-0036, Japan 
*Corresponding author. Email: m_amzad_hossain@hotmail.com 
 
Abstract 
Possibility of enrichment of rotifer (Brachionus rotondiformis) with calcium (Ca) for 
feeding the fish fry was investigated. Rotifer was kept for 24 h with aeration in normal 
seawater (Treatment 1), seawater with 400 mg/1 supplemental Ca from Ca-lactate 
(Treatment 2) and seawater with 400 mg/I supplemental Ca from Ca-chloride 
(Treatment 3). After the experimental period, Ca contents of rotifer were 0.20, 0.29 and 
0.39% of dry weight in T-1, T-2 and T-3, respectively. Ca content of media did not 
affect phosphorus, zinc and manganese contents of rotifer. Results revealed that rotifer 
can be enriched with Ca for feeding -fish fry and Ca-chloride might be a better source 
for Ca enrichment. 
Key words : Rotifer, Calcium, enrichment 
Introduction 
Rotifer, Brachionus spp., have a number of features that make them useful for 
aquaculture, including rapid reproduction, slow swimming speed, high nutritional 
quality and easy culture method. The biological composition of rotifer closely reflects 
what they eat. Therefore, rotifer can be fed specific nutrients and antibiotics that in 
turn can be transferred to larval fish. Gastesoupe (1982) observed that in fish hatcheries 
rotifers are considered living food that transmit macro and micronutrients, vitamins and 
even antibodies to fish larvae. Recently, rearing procedures for various kinds of fish and 
method for the mass production of live feeds have been markedly progressed (Stottrup 
and Attramdal 1992). As a consequence, the number of cultured fish species is 
increasing year by year. Among zooplanktons, the rotifers have been extensively used 
for the feeding of larva of marine and freshwater fishes. Larval rearing of marine fishes 
would be virtually impossible without the mass culture of rotifers (Watanabe et al. 
1983a). It is a widely accepted opinion in Japan that the number of fish and crustacean 
larvae produced is limited by the amount of rotifers available (Hirata 1979, Hirata et al. 
1983). However, the nutritional values of live feeds have not been widely reported. 
M.A. Hossain et al. 
Watanabe et al. (1978) reported the proximate and mineral composition of rotifers, 
where culture media such as backer's yeast and freshwater and marine chlorella greatly 
affected the proximate but not the mineral composition. The digestibility of protein in 
rotifers was high as 89-94%, irrespective of culture organism used. They observed it 
important to 'enrich' or 'pack' rotifers with essential fats, vitamins and other nutrients 
just before feeding to larval fish. 
The nutritional quality of rotifers as a live food for fish larvae has been investigated 
mainly from the viewpoints of essential fatty acid (EFA) required for fish (Watanabe et 
al. 1983b,c). The fatty acid composition of rotifer is readily affected by the fatty acid 
composition of the organisms in culture media. So, direct and indirect methods have 
been developed for enrichment of rotifers with n3-HUFA for feeding fish larvae 
(Yoshimura et al. 2003). 
A high incidence of lordosis (bone deformity) has been reported in several cultured 
marine fishes and many authors have suggested possible counter measures (Kitajima et 
al. 1977 8z 1994, Isheda et al. 1982, Hayashida et at 1984), which mainly considered the 
shortage of EFA as the main cause for lordosis. Inadequacy of Ca as a possible cause 
for lordosis is yet to be investigated. There is a general consideration that as seawater 
contains adequate Ca, which is available to marine fish through absorption, marine fish 
may not need a dietary Ca supplement (Love 1980). However, dietary Ca supplement 
has been found necessary for some marine fishes (Hossain and Furuichi 1998, 2000a,b). 
At the growing stage of fish, Ca is needed for building the hard tissues and for other 
physiological functions. Lordosis in fish may be due to shortage of dietary Ca, as it is a 
major component of bone. Therefore, the supplement of an increased level of Ca to 
larval diets might fulfill the Ca requirement of larval fish and might prevent lordosis. 
Rotifer is the widely used live food for larval stage of fish and could be an important 
source of feeding Ca to fish larvae. In order to feed Ca to larval fish through rotifer, it is 
necessary to enrich rotifer with Ca. However, reports on the enrichment of rotifer with 
Ca and on the feeding of Ca-enriched rotifers to fish larvae are scarcely available. In the 
present study, the possibility of enrichment of rotifers with Ca has been investigated. 
Materials and methods 
The experiment was conducted in 9 round, polycarbonate tanks of 50L size. Each 
tank was filled with 40L sand-filtered seawater. There were 3 treatments each with 3 
replications. Treatment 1 was without supplemental calcium (Ca), treatment 2 was with 
400 mg/1 Ca supplement from Ca-lactate and treatment 3 was with 400 mg/1 Ca 
supplement from Ca-chloride. Necessary amount of chemicals was measured and 
dissolved in the tank water to obtain required level of supplemental Ca. Tanks were 
monitored every 4 hours to observe salinity, dissolved oxygen, pH, ammonia, rotifer 
density and Ca-content. Marine rotifer (B. rotundiformis) were cultured in a 500-1 tank 
following the method of Yoshimura et al. (1996). Rotifer were cultured at around 20,000 
individuals/ml by feeding them exclusively on condensed marine chlorella 
(Nannochloropsis sp.) Culture media were supplied with constant oxygen, pH was 
52 
Enrichment of rotifer with calcium 
adjusted to 7 by addition of hydrochloric acid to avoid an increase of free ammonia in 
the culture system. The excess suspended organic matter was removed by a nylon 
filtration mat. Rotifer cultured in the 500-1 tank were harvested with a plankton net and 
distributed to the experimental tanks to obtain a concentration of 5000 rotifer/ml 
seawater. Tanks were provided with aeration. After a 24 h experimental period, rotifers 
were harvested and three samples (each sample contained approximately 10 g of wet 
rotifers) from each tank were collected for Ca determination. Samples were washed 
several times with distilled water and dried at 105 °C for 12 h. Dried rotifer samples were 
digested with wet digestion method with a nitric acid-perchloric acid mixture. Ca, 
phosphorus (P), zinc (Zn) and manganese (Mn) in the digested sample (insert Ref for 
analytical methods followed (Hossain and Furuichi 2000a)) were determined with an 
Atomic Absorption Spectrophotometer (Perkin-Elmer 3300, Perkin Elmer, USA). Data 
were analyzed for significant difference with Fisher PLSD (full Protected Least 
Significant Difference) test. 
Results 
The average salinity, DO, pH, rotifer density, ammonia and Ca-content of the 
culture media are shown in Table 1. The average values of salinity, dissolved oxygen, 
pH, rotifer density, ammonia, were similar in all the treatments. The ranges of all those 
water quality parameters were optimum for rotifer culture (Yoshimura et al. 1996). Ca 
contents of culture media of rotifer were 396 mg/1, 780 mg/1 and 770 mg/1 in treatment-
1, treatment-2 and treatment-3, respectively. High calcium contents in treatment 2 and 
treatment 3 were due to the addition of Ca-lactate and Ca-chloride, respectively. Ca 
content of seawater was 396 mg/l. The Ca contents of rotifer after the experimental 
period with different treatments are presented in Fig. 1. The Ca content of rotifer was 
0.23% of dry matter in the control group (Treatment 1). Calcium content of rotifer 
increased when reared in Ca supplemented seawater for 24 hours, which were 0.33% in 
Treatment 2 and 0.42% in Treatment 3. The P, Zn and Mn contents of rotifers are 
shown in Fig. 2. Concentrations of these minerals in rotifers were independent of Ca 
supplement to the rearing media. 
Table 1. Water quality parameters of different culture media of rotifer (Brachionus plicatilis) 
Parameters 
T-1 
Treatments* 
T-2 T-3 
Salinity (ppt) 35.4±0.3 34.5±0.5 36.3±34.9 
DO (mg/I) 5.6±0.4 5.8±0.2 5.6±0.3 
pH 7.8±0.2 7.8±0.3 8.0±0.4 
Rotifer density (incl./nil) 5000±50 4900±60 5100±65 
Ammonia (mg/1) 45.4±3.2 33.3±2.5 37.4±7.0 
Ca contents (mg/1) 396±32 780±34 770±20 
* T-1, Control; T-2, Ca-lactate; T-3, Ca-chloride. 
53 
M.A. Hossain et al. 
0.5 
0.4 
^cn 
0.3 
t 0.2 
O 
r 	 0.1 
0 
a 
b 
C 
Control 	 Ca- actate (400 Ca-chloride (400 
ppm) 	 ppm) 
Ca source 
Fig. 1. Ca contents of rotifer after 24 hours enrichment with Ca. Analyses were made of three 
samples from each of three replicates (N=9 samples per treatment). Different letters indicate 
significant differences among each other (p<0.05, Fisher PLSD test). 
P (mg/g) 	 Zn (Ag/g) 	 0 Mn (x10 nig) 
Control 	 Ca-lactate (400 	 Ca-chloride (400 
ppm) 	 ppm) 
Calcium source 
Fig. 2. P, Zn and Mn contents of rotifer after 24 hours enrichment with Ca. Analysis was made of three 
samples from each three replicate groups (N=9 samples per treatment). No significant difference for a 
mineral within 3 treatments (p>0.01, Fisher PLSD test). 
54 
Enrichment of rotifer with calcium 
Discussion 
The Ca content of rotifer was 0.23% of dry matter in the control group (Treatment 1). 
The Ca content of rotifers in a dry matter basis was 0.12-0.22%, 0.09-0.24%, and 0.08-
0.17% when cultured on backer's yeast, backer's yeast and marine chlorella, and marine 
chlorella, respectively (Watanabe et al. 1978). Calcium content of rotifer increased 
when reared in Ca supplemented seawater for 24 hours, which were 0.33% in treatment 
2 and 0.42% in treatment 3 (Fig. 1). Although dietary Ca sometimes affect the P, Zn and 
Mn contents in fish (Hossain and Furuichi 2000a), Ca in media did not affect the 
contents of these minerals in rotifers in the present study (Fig 2). It indicated that 
rotifer can be enriched with Ca just before feeding to larval fish to fulfill the dietary Ca 
requirement in the larval stage of fishes without affecting other important minerals in 
rotifer. Reports on the enrichment of rotifer with Ca are scarcely available. However, it 
has been reported that rotifer can take HUFA and vitamins directly from medium. 
Rotifer are harvested, concentrated and then soaked in HUFA rich emulsion for 2-3 
hours, thus becoming enriched with all the HUFAs required by larval fish (Watanabe et 
al. 1983c). Marine fishes need Ca for growth and bone mineralization. In the initial 
stage of growth, the process of bone mineralization is very rapid, hence the requirement 
of Ca may be even high. Generally, marine fish absorb calcium from seawater. However, 
Ca absorption from seawater is not sufficient for some of the marine fishes and they 
require dietary Ca supplementation (Hossain and Furuichi 1998, 2000a,b). Besides the 
investigations that considered the shortage of n3 HUFA is the possible cause of lordosis 
(Kitajima et al. 1977 & 1994, Isheda et al. 1982, Hayashida et al. 1984), the lordosis 
problem in larval marine fishes may be due to the shortage of Ca, because the major 
fraction of the body Ca (99%) are present in skeleton in the form of ca-hydroxyapatite. 
From the present study, it is evident that rotifer can be enriched with Ca. As rotifer is 
the major food of larval fishes, the enrichment of rotifer with Ca before feeding fish 
larvae may be effective when dietary Ca supplementation become necessary. Calcium 
contents of rotifer was higher in Treatment 3 than in Treatment 2, where Ca contents of 
the media were the same but sources were Ca-chloride and Ca-lactate, respectively. It is 
evident from the present study that rotifers may be enriched with Ca addition to culture 
media, and that Ca-chloride seems better as a source. Therefore, enrichment of rotifer 
with Ca may be an appropriate way to supply Ca to larval fish. Further studies are 
necessary to find out the effects of feeding Ca-enriched rotifers to fish larvae. 
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(Manuscript received 3 September 2006) 
56 


Enrichment of rotifer Brachionus rotundiformis with calcium

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Enrichment of rotifer Brachionus rotundiformis with calcium

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