Aquaculture Research, 2011, 42, 1737^1740 doi:10.1111/j.1365-2109.2010.02752.

SHORT COMMUNICATION
Protocol to enrich rotifers (Brachionus plicatilis)
with iodine and selenium

Ana Rita A Ribeiro1,2, Laura Ribeiro1, Maria T Dinis1 & Mari Moren2
1
CCMAR, Centre of Marine Sciences of Algarve, University of Algarve, Faro, Portugal
2
NIFES, National Institute of Nutrition and Seafood Research, Nordnes, Bergen, Norway

Correspondence: Ana Rita A. Ribeiro, CCMAR, Centre of Marine Sciences of Algarve, University of Algarve, Faro, Portugal or NIFES,
National Institute of Nutrition and Seafood Research, PO Box 2029 Nordnes, N-5817 Bergen, Norway. E-mail: anarita.bio@gmail.com

Fish production is the fastest growing food produc- development of thyroid lesions, e.g. goitre (Bichsel &
tion sector of the world (FAO 2006). Iodine (I) and Von Gunten1999; Sherrill,Whitaker & Wong 2004) in
selenium (Se) are both essential for thyroid hormone particular if the live prey is not enriched with I
production and hence crucial for normal ¢sh devel- (Ribeiro, Ribeiro, Sle, Hamre, Dinis & Moren 2009).
opment (Leatherland 1994; Kohrle, Jakob, Contempre While I de¢ciency has been shown to cause thyroid
& Dumont 2005). Consequently, if one of these ele- lesions in Senegalese sole juveniles (Ribeiro et al.
ments is missing in the water systems, it must be 2009), a de¢ciency in Se can lead to nerve cord and
added to ¢sh diets. Most larval ¢sh species are depen- liver damage in adult rainbow trout (Bell, Pirie,
dent on live prey at ¢rst feeding (Tocher, Mourente & Adron & Cowey 1986) or lethargy; loss of appetite
Sargent 1997), and rotifers are one of the most widely and reduced muscle tone in salmon fry (Poston &
used live preys (Dhert, Rombaut, Suantika & Sorge- Combs Jr 1979). Hence, a minimum of both minerals
loos 2001). Compared with wild copepods, the natur- has to be delivered in a bio-available form to the fast-
al prey for marine ¢sh larvae, rotifers are low in I and developing larvae. The aim of the present study was
Se. Copepods contain between 50 and 350 mg of I to establish a protocol to enrich rotifers with I and
per kg of dry weight (dry wt.) (Moren, Opstad, Van Se up to levels found in copepods, which could be
Der Meeren & Hamre 2006; Hamre, Srivastava, easily applied to the rotifer protocols used in com-
Ronnestad, Mangor-Jensen & Stoss 2008), whereas mercial ¢sh larvae hatcheries.
the I content in rotifers ranges between 0.52 and Experiment1aimed to increase the I concentration
7.9 mg kg 1 dry wt. (Hamre, Mollan, Sle & Erstad in rotifers and was conducted at the University
2008; Hamre, Srivastava et al. 2008). Se levels range of Algarve, Faro, Portugal. In this experiment,
between 2 and 5 mg kg 1 dry wt. in copepods and the rotifers (Brachionus plicatilis) were cultured
between 0.08 and1.4 mg kg 1 dry wt. in rotifers (Sol- in 120 L circular tanks, at a starting density of
bakken, Berntessen, Norberg, Pittman & Hamre 200 rotifers mL 1. They were fed micro-algae on the
2002; Hamre, Mollan et al. 2008; Hamre, Srivastava ¢rst day and baker’s yeast during the following days
et al. 2008). As stated by FAO (FAO 2005), the expan- (2 days). The tanks were continuously aerated (95%
sion and the pro¢tability of aquaculture of marine saturation) with water temperature and salinity at
and ornamental species are likely to be dependent 26 1C and 24 g L 1 respectively. Based on the levels
on the use of recirculation systems. In these systems, of potassium iodide and potassium iodate used to en-
the water is often treated with ozone for quality con- rich Artemia by Moren et al. (2006), it was estimated
trol (Rueter & Johnson 1995; Krumins, Ebeling & that 260 mg of sodium iodide (NaI) had to be added
Wheaton 2001). However, ozonation changes the for each million rotifer in the enrichment tank. The I
physiochemical properties of the water, i.e. bio-avail- salt was mixed with a commercial enrichment pro-
able I is removed and this can in turn lead to the duct (CP) (DC DHA Selco, INVE Aquaculture NV,

r 2011 Blackwell Publishing Ltd 1737

Protocol to enrich rotifers A R A Ribeiro et al. Aquaculture Research, 2011, 42, 1737^1740

Dendermonde, Belgium) to simulate the procedures Julshamn, Dahl and Eckho¡ (2001), and those of Se
run daily at ¢sh hatcheries. The I enrichment tests were processed using an acid decomposition as
were conducted at the same water temperature and described by Julshamn, Lundebye, Heggstad, Bernts-
salinity as the culture and at a rotifer density of sen and Boe (2004). All statistical analyses were per-
200^300 ind mL 1 and the amount of CP was formed with STATISTICA software (version 7, Statsoft,
250 mg L 1 water. Tulsa, OK, USA). Data from I enrichment were sub-
Two enrichments containing NaI plus CP (experi- jected to one-way ANOVA to test di¡erences in I con-
mental enrichments) were prepared as well as two tent. Fisher’s LSD was used to test for signi¢cant
containing only CP (controls) by mixing them with di¡erences between group means. Di¡erences were
0.5 L of water for 2 min in a kitchen blender. Two pro- considered signi¢cant at Po0.05. Data from Se
tocols, di¡ering in time used for enrichment, were enrichment were subjected to a linear regression
tested. In protocol A, half of the enrichment was analysis to establish the correlation between the Se
given to the rotifers (two tanks, n 5 2) at 6 h, and the amount in the enrichment and the content in the
other half at 3 h before sample collection. In protocol rotifers.
B, the total amount of enrichment was given to roti- Results from Experiment 1 showed that rotifers
fers (n 5 2) 3 h before sample collection. Correspond- were enriched with I when NaI was added to the en-
ing controls without I addition were run in parallel in richments (Table 1). I content in rotifers from con-
two tanks (n 5 2). trol enrichments were signi¢cantly lower (Po0.01)
Experiment 2 aimed to increase the level of bio- than the levels found in rotifers from the di¡erent
available Se in the rotifers and was conducted at protocols (A and B). In addition, these values were
NIFES in Bergen, Norway. The rotifers, B. plicatilis, six to seven times higher than I levels found in
were obtained from Sagafjord Cod Hatchery (Stord, control rotifers from previous experiments (Lie,
Norway). They were cultured at a density of 500 Haaland, Hemre, Maage, Lied, Rosenlund, Sandnes
ind mL 1 in 200 L tanks with continuous aeration & Olsen 1997; Hamre, Mollan et al. 2008); however,
(95% saturation) with water temperature and sali- the di¡erences observed might be due to seasonal or
nity of 25 1C and 18^22 g L 1 respectively. Rotifers species variation, or due to di¡erent methods used
were fed micro-algae and an amount of 0.021mL mil- for cultivation of rotifers. The I levels in the en-
lion rotifers day 1 of ¢sh oil (Rich S.A., Rich, Greece). riched rotifers were 15 times higher than those of
The Se was given to the rotifers (n 5 3) 3 h before the control rotifers (Table 1). Signi¢cant di¡erences
sampling, without a commercial enrichment pro- in I levels were observed between rotifers from the
duct. The Se source tested was a selenized yeast di¡erent protocols (Po0.01). Rotifers from protocol
(Selplex, ParanaŁ, Brazil), where most of the Se exists B contained 1.3 times more I compared with rotifers
in the form of seleno-methionine (Encinar, Sliwka- from protocol A (Table 1), suggesting that when
Kaszynska, Polatajko, Vacchina & Szpunar 2003). using protocol A, the I ingested by rotifers had time
Based on the levels of sodium selenite used in a pre- to be evacuated by gut evacuation, and conse-
vious study (Hamre, Mollan et al., 2008), a range of Se quently less I was retained in the rotifers. In
concentrations was tested (0.01, 0.02, 0.025, 0.04 and addition, this level was within the range of con-
0.08 g million rotifers 1) to determine the optimal centrations found in copepods (Moren et al. 2006),
level of enrichment. Rotifers were enriched in 15 L
tanks continuously aerated (95% saturation) with
water temperature and salinity of 26 1C and
24 g L 1, respectively, and at a density of 200 Table 1 Iodine (I) concentration in rotifers, Brachionus
ind mL 1. plicatilis (mg kg 1 dry weight  SD), from protocols A, B
In both experiments, all sampled rotifers were col- compared with control rotifers
lected by ¢ltering and thoroughly washed in distilled
1
water. Excess water was removed by a hygroscopic Protocol mg kg dry wt.
material and the rotifers were stored in 1.5 mL tubes A 58  3.2b
at 20 1C before analysis. Both I and Se levels were B 74.2  7.9c
determined using inductively coupled plasma mass Control 5.35  0.9a
spectroscopy. Samples of rotifers of 100 mg dry wt. Means  SD (n 5 2).
were processed for I analyses using alkaline decom- Di¡erent superscripts within the same column represent signi¢-
position according to the protocol described by cant di¡erences among treatments.

1738 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740

Aquaculture Research, 2011, 42, 1737^1740 Protocol to enrich rotifers A R A Ribeiro et al.

showing that protocol B is the best to enrich rotifers (Wang, Lovell & Klesius 1997), and an Se supplemen-
and to later feed them to ¢sh larvae. tation can improve ¢sh immune response (Cotter,
Results from Experiment 2 showed that rotifers Craig & McLean 2008), growth (Abdel-Tawwab, Mou-
were enriched with Se when an organic source of Se sa, & Abbass 2007) and survival (Hamre, Mollan et al.
was added to the enrichment tanks. The rotifer con- 2008). Therefore, the use of this yeast in the enrich-
centrations of Se ranged from 35.9 ( 0.4 SD) to104.0 ment of rotifers is bene¢cial to marine ¢sh larvae in
( 0.4 SD) mg kg 1 dry wt., when enriched with intensive culture. Unlike I, Se is known to be toxic
0.01^0.08 g selenized yeast per million rotifers re- (NRC 1993), and consequently new experiments
spectively. These values were more than 10 times should be conducted in order to determine the re-
higher than levels found in copepods (Hamre, Srivas- quirement level of Se in ¢sh larvae.
tava et al. 2008). Nevertheless, the regression ana- In summary, rotifers can be enriched with I and Se,
lyses showed a linear (R2 50.7438) relation between by using a 3 h enrichment protocol with sodium (I)
Se retention in rotifers and the amount of yeast used and a selenized yeast respectively. Applying the same
in the enrichment (Fig. 1), indicating that this was a enrichment protocol in the culture of di¡erent ¢sh
good predictor to determine the amount of Se that species allows the standardization of enrichment
should be given to rotifers. The Se levels obtained protocols and simpli¢es the evaluation of the e¡ects
were higher than what has been observed in cope- of nutrients. This type of study is therefore essential,
pods (Hamre, Srivastava et al. 2008). Hence, less Se because it is an easy and practical protocol, applic-
should be given to rotifers in order to achieve a cope- able to any hatchery.
pod level. According to the linear regression, an
amount of 0.003 g of selenized yeast should be given
per million rotifers. This amount was used in a ¢sh
experiment byA. R. A. Ribeiro, L. Ribeiro, . Sle, K. Acknowledgments
Hamre, M. T. Dinis and M. Moren (unpubl. data) and
We are indebted to the highly skilled technicians at
the rotifers reached concentrations between 3 and
the Centre of Marine Sciences of Algarve and at the
5 mg of Se g 1 dry wt. This, together with the results
National Institute of Nutrition and Seafood Research.
of the current experiment, shows that rotifers can be
This work was supported by the DIGFISH project,
e¡ectively enriched with high levels of Se by using an
POCI/CVT/58790/2004 (Fundacao para a CieŒncia e
organic source of Se. The yeast used in the enrich-
Tecnologia ^ FCT, Portugal). Ribeiro A R A and Ri-
ment contains (Se) in the organic form of seleno-
beiro L bene¢t from grants SFRH/BD/24803/2005
methionine (Encinar et al. 2003). Organic forms of Se
and SFRH/BPD/7148/2001 (Fundacao para a CieŒncia
have been shown to be more bio-available for ¢sh
e Tecnologia ^ FCT, Portugal) respectively.

References

Abdel-Tawwab M., Mousa M.A.A. & Abbass F.E. (2007)

Growth performance and physiological response of Afri-
can cat¢sh, Clarias gariepinus (B.) fed organic (Se) prior to
the exposure to environmental copper toxicity. Aquacul-
ture 272, 335^345.
Bell J.G., Pirie B.J.S., Adron J.W. & Cowey C.B. (1986) Some ef-
fects of selenium de¢ciency on glutathione peroxidase
(EC 1.11.1.9) activity and tissue pathology in rainbow trout
(Salmo gairdneri). BritishJournal of Nutrition 55, 305^311.
Figure 1 Selenium (Se) concentration (mg kg 1 dry wt.) Bichsel Y. & Von Gunten U. (1999) Determination of iodide
in rotifers Brachionus plicatilis sp. as a function of level of and iodate by Ion chromatography with postcolumn re-
selenized yeast given in the enrichment. Five di¡erent le- action and UV/visible detection. Analytical Chemistry 71,
vels of selenized yeast were tested: 0.01, 0.02, 0.025, 0.04 34^38.
and 0.08 g million rotifers 1) given to rotifers 3 h before Cotter P.A, Craig S.R. & McLean E. (2008) Hyperaccumula-
sample collection. The regression equation was y 51530, tion of selenium in hybrid striped bass: a functional food
3x; r2 50.7438. for aquaculture? Aquaculture Nutrition 14, 215^222.

r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740 1739

Protocol to enrich rotifers A R A Ribeiro et al. Aquaculture Research, 2011, 42, 1737^1740

Dhert P., Rombaut G., Suantika G. & Sorgeloos P. (2001) Ad- emulsi¢ed oil to microalgae. Aquaculture International 5,
vancement of rotifer culture and manipulation techni- 427^438.
ques in Europe. Aquaculture 200,129^146. Moren M., Opstad I., Van Der Meeren T. & Hamre K. (2006)
Encinar J.R., Sliwka-Kaszynska M., Polatajko A., Vacchina V. Iodine enrichment of Artemia and enhanced levels of io-
& Szpunar J. (2003) Methodological advances for sele- dine in Atlantic halibut larvae (Hippoglossus hippoglossus
nium speciation analysis in yeast. Analytica Chimica Acta L.) fed the enriched Artemia. Aquaculture Nutrition 12,
500,171^183. 97^102.
FAO (2005) Regional Review on Aquaculture Development, 6. NRC (1993) Nutrient Requirements of Fish, pp.1^116. National
Western European Region, FAO, Rome, Italy. Research Council, National Academy Press, Washington,
FAO (2006) The State ofWorld Fisheries and Aquaculture. FAO, DC, USA.
Rome, Italy. Poston H.A. & Combs G.F Jr (1979) Interrelationships
Hamre K., Mollan T.A., Sle . & Erstad B. (2008) Rotifers between requirements for dietary selenium, vitamin E
enriched with iodine and selenium increase survival in and L-ascorbic acid byAtlantic salmon (Salmo salar) fed a
Atlantic cod (Gadus morhua) larvae. Aquaculture 284, semipuri¢ed diet. Fish Health News 84, 6^7.
190^195. Ribeiro A.R.A., Ribeiro L., Sle ., Hamre K., Dinis M.T. &
Hamre K., Srivastava A., Ronnestad I., Mangor-Jensen A. & Moren M. (2009) Iodine enriched rotifers and Artemia
Stoss J. (2008) Several micronutrients in the rotifer prevents goitre in Senegalese sole (Solea senegalensis) lar-
Brachionus sp may not ful¢l the nutritional requirements vae reared in a recirculating system. Aquaculture Nutri-
of marine ¢sh larvae. Aquaculture Nutrition 14, 51^60. tion (in press), doi:10.1111/j.1365-2095.2009.00740.x.
Julshamn K., Dahl L. & Eckho¡ K. (2001) Determination of Rueter J. & Johnson R. (1995) The use of ozone to improve
iodine in seafood by inductively coupled plasma/ solids removal during disinfection. Aquacultural Engineer-
mass spectrometry. Journal of AOAC International 84, ing 14,123^141.
1976^1982. Sherrill J., Whitaker B.R. & Wong G.T.F. (2004) E¡ects of
Julshamn K., Lundebye A.K., Heggstad K., Berntssen M.H.G. ozonation on the speciation of dissolved iodine in arti¢cial
& Boe B. (2004) Norwegian monitoring programme on seawater. Journal of Zoo andWildlife Medicine 35, 347^355.
the inorganic and organic contaminants in ¢sh caught Solbakken J.S., Berntessen M.H.G., Norberg B., Pittman K. &
in the Barents Sea, Norwegian Sea and North Sea, 1994 Hamre K. (2002) Di¡erent iodine and thyroid hormone le-
to 2001. Food Additives and Contaminants: Part A 21, vels between Atlantic halibut larvae fed wild zooplankton
365^376. or Artemia from ¢rst exogenous feeding until post meta-
Kohrle J., Jakob F., Contempre B. & Dumont J.E. (2005) Sele- morphosis. Journal of Fish Biology 61,1345^1362.
nium, the thyroid, and the endocrine system. Endocrine Tocher D.R., Mourente G. & Sargent J.R. (1997) The use of
Reviews 26, 944^984. silages prepared from ¢sh neural tissue as enrichers for
Krumins V., Ebeling J. & Wheaton F. (2001) Part-day ozona- rotifers (Brachionus plicatilis) and Artemia in the nutrition
tion for nitrogen and organic carbon control in recirculat- of larval marine ¢sh. Aquaculture 148, 213^231.
ing aquaculture systems. Aquacultural Engineering 24, Wang C., Lovell R.T. & Klesius P.H. (1997) Response to
231^241. Edwardsiella ictaluri challenge by channel Cat¢sh fed
Leatherland J.F. (1994) Re£ections on the thyroidology of organic and inorganic sources of selenium. Journal of
¢shes from molecules to humankind. Guelph Ichthyology Aquatic Animal Health 9, 172^179.
Reviews 2, 1^67.
Lie O., Haaland H., Hemre G.-I., Maage A., Lied E., Rosen-
lund G., Sandnes K. & OlsenY. (1997) Nutritional composi-
tion of rotifers following a change in diet from yeast and Keywords: rotifers, enrichment, iodine, selenium

1740 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740