A study on enrichment of the rotifer Brachionus “Cayman” with iodine and selected vitamins

Aquaculture 319 (2011) 430–438

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

A study on enrichment of the rotifer Brachionus “Cayman” with iodine and selected vitamins Ashutosh Srivastava a, b, Joachim Stoss c, Kristin Hamre a,⁎ a b c

National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida (U.P.), India Stolt Sea Farm Turbot Norway AS, Øyestranda, Norway

a r t i c l e

i n f o

Article history: Received 6 May 2011 Received in revised form 20 July 2011 Accepted 22 July 2011 Available online 2 August 2011 Keywords: Brachionus Vitamin A Vitamin E Vitamin C Thiamine Iodine

a b s t r a c t Brachionus “Cayman” is a widely used live feed organism in fish larvae culture. Our previous studies indicate that these live feeds may not fulfill the nutritional requirement of marine fish larvae. The present study aimed to enrich rotifers with the micronutrients thiamine, vitamins C, A, and E, and iodine up to the levels found in copepods, the feed for fish larvae in the wild. Various levels of these micronutrients were supplemented along with the basal diet: Baker's yeast–pronova oil–live Chlorella (65:25:15 DW). Thiamine was supplemented as thiamine HCl, vitamin A as retinyl palmitate, vitamin C as Stay C or ascorbic acid polyphosphate, vitamin E as ® DL-α-tocopherol and iodine as Lipiodol , ethyl esters of iodized fatty acids from poppy seed oil. Rotifers were cultured with these supplemented diets for 4 days. There was a significant positive relationship between dietary and rotifer concentrations of thiamine, vitamins C and E, and iodine, as indicated by the regression equations: thiamine y = 0.73x + 30, r = 0.88; vitamin C: y = 0.023x + 125, r = 0.91; vitamin E: y = 0.078x− 12, r = 0.96; and iodine: y = 0.88x + 5, r = 0.96, y being the rotifer concentration and x being the concentration of active component in the diet, both in mg kg− 1 DW. Compared to copepod levels, the control diet gave sufficient amounts of thiamine in the rotifers (25 ± 14 mg kg− 1 DW). The highest level of vitamin C enrichment did not give copepod levels of vitamin C (500 mg kg− 1) in rotifers, but by extrapolation the diet should contain 16 g kg− 1 vitamin C from Stay C to obtain this level. This corresponds to 4.6% Stay C in the diet. A dietary concentration of 1.6 g kg− 1 DW of α-tocopherol is needed to enrich rotifers up to copepod levels (110 mg kg− 1), while 52 mg kg− 1 dietary iodide would be needed to obtain the lower range of copepod levels of iodine (50 mg kg− 1). There was no significant relationship between dietary and rotifer vitamin A concentrations (y= 0.0097x + 5.4, r = 0.57). Control rotifers contained 4.9 ± 0.5 mg kg− 1, while rotifers enriched with the highest concentration of 200 mg kg− 1 vitamin A contained 7.0 ± 1.7 mg kg− 1 vitamin A. However, both control and enriched rotifers contained sufficient concentrations of vitamin A to cover the requirement in fish larvae. This study shows that concentrations of thiamine, vitamin C, vitamin E and iodine increase almost linearly in Brachionus in response to simultaneously increasing dietary concentrations. This indicates that rotifers can be enriched up to copepod levels with these nutrients, to satisfy marine fish larvae requirements. Vitamin A was not increased in the rotifers by dietary retinyl palmitate under the conditions used in the present study. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Despite the recent progress in the production of inert diets for fish larvae, feeding of most species of interest for aquaculture still relies on live feeds during the early life stages. Rotifers are the most widely used live feed in intensive aquaculture. Its body size makes this organism an appropriate prey at start feeding. It also has high population growth rate at high densities and it feeds by filtrating

⁎ Corresponding author at: NIFES, PO Box 2029, 5817 Bergen, Norway. Tel.: + 47 4818 5034; fax: + 47 55905299. E-mail address: [email protected] (K. Hamre). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.07.027

particles in suspension which makes it easy to enrich with potentially lacking nutrients. Copepods, the main prey in the wild, have mostly been used at a pilot scale or in locations where abundant collection of natural (or induced) zooplankton bloom is possible (Conceição et al., 2010). Establishing a cost effective protocol for the mass production of copepods is still a challenge. Several studies have proven that marine fish larvae show high survival and growth, normal pigmentation and low frequencies of skeletal deformities when fed with live natural prey (Busch et al., 2010; Drillet et al., 2006; Evjemo et al., 2003; Finn et al., 2002; Hamre, 2006; Hamre et al., 2002; Næss et al., 1995; Rajkumar and Kumaraguru vasagam, 2006; Seoka et al., 2007; Shields et al., 1999; Støttrup et al., 1998; van der Meeren and Naas, 1997). The data from some of our studies on the nutrient profile of rotifers and

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copepods show that there are numerous differences in nutrient composition between these pray types (Hamre et al., 2008a; Srivastava et al., 2006; van der Meeren et al., 2008). The difference in nutrition is one of the several possible reasons, like environmental factors, bacterial and viral activity, for the poor performance of marine fish larvae in intensive culture systems (Hamre et al., 2008a). Hamre et al. (2008a) found that thiamine and vitamins A, C and E were lower in rotifers fed a yeast based diet than in copepods while levels of the other B-vitamins were similar. There were also differences in most of the trace elements. Different vitamins and minerals play important roles in proper growth and development of the fish larvae. Vitamin A plays a key role in visual function, reproduction, the immune system and skeletogenesis (Blanner and Olson, 1994; Fernández et al., 2009). It is known to be essential for establishing body and organ axes in vertebrate embryos and interact with other nutrients such as vitamin D and fatty acids through the steroid/thyroid nuclear hormone receptor family (Hamre et al., 2010a). The deficiency of vitamin A in fish leads to retarded growth, poor vision, mortality, anemia and anorexia (Goswami and Basmatari, 1988; Goswami and Dutta, 1991; Moren et al., 2004b; Poston et al., 1977; Saleh et al., 1995). Excess vitamin A induced developmental abnormalities including patterning defects, skin hemorrhage, abnormal skin color and bone deformity in fish (Dedi et al., 1997; Haga et al., 2002; Hilton, 1983; Martinez et al., 2007; Mohamed et al., 2003; Suzuki et al., 1999; Takeuchi et al., 1998; Tarui et al., 2006; Vandersea et al., 1998). Vitamin E acts as antioxidant. It reduces peroxyl radicals in lipids and prevents the chain reaction leading to lipid peroxidation (reviewed by Hamre, 2011). Vitamin E is therefore crucial for normal development of the tissues including bone and cartilage (Hamre, 2011; Lall and Lewis-McCrea, 2007). Ascorbic acid, by donating electrons to the α-tocopheroxyl radicals reduces it back to α-tocopherol (Azzi and Stocker, 2000). This has been demonstrated in yellow perch (Lee and Dabrowski, 2003) and Atlantic salmon (Hamre et al., 1997). The ascorbate radical formed may be regenerated by glutathione, a reaction catalyzed by a group of selenium dependent enzymes, glutathione peroxidases. Oxidized glutathione can be reduced by NADPH formed in energy metabolism (Hamre et al., 2010a). Ascorbic acid is the most studied vitamin in fish. Updates in vitamin C research have been recently reviewed by Waagbø (2010). Supplementation of ascorbic acid is essential for a variety of biological and physiological functions including increased disease resistance and wound healing (Halver, 2002), improved tolerance to environmental stressors (Gapasin et al., 1998; Merchie et al., 1996; Wang et al., 2006), regulation of collagen synthesis (Dabrowski, 1992) and proper bone calcification and metabolism (Johnston et al., 1994; Tietz et al., 1983). Thiamine is a co-enzyme in carboxylation and decarboxylation reactions. It is important for energy production, especially from carbohydrates as well as from amino acids (Hamre, 2006). Morito et al. (1986) studied thiamine deficiency symptoms and requirement in rainbow trout, and found that thiamine deficiency symptoms are predominantly neurological: irritability and instability. Other signs include convulsions, feed refusal, dark pigmentation and finally mortalities. Some recent studies show that thiamine deficiency causes general lethargy or weakness in fry, among other signs (Fisher et al., 1998; Fitzsimons et al., 2005; Ketola et al., 2000, 2009). Such lethargy or weakness in thiamine deficient fry and migrating adults may be related to a reduced production of the high-energy metabolite adenosine triphosphate, which requires thiamine diphosphate for its production (Butterworth, 1989). Many reports linked mortality with thiamine (Åkerman et al., 1998; Brown et al., 2005a; Everitt, 2006; Fisher et al., 1996; Fitzsimons et al., 2007; Honeyfield et al., 2005; Jaroszewska et al., 2009; Ketola et al., 2000; Pickova et al., 1998; Wolgamood et al., 2005). In addition to thiamine's well known role in human nutrition as enzyme cofactor; recent studies suggested that

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thiamine could also function to alleviate stress in different organisms like bacteria, fungi, animals and plants (Jung and Kim, 2003; Lukienko et al., 2000; Mehta et al., 2008; Tunc-Ozdemir et al., 2009; Wang et al., 2007). Recent reports implicate thiamine in oxidative stress, protein processing, peroxisomal function, and gene expression (Gibson and Blass, 2007). More recently, Waagbø (2010) reviewed the role of thiamine in marine fish ontogeny. Mineral nutrition of marine fish larvae has been given little attention in research and commercial culture (Hamre et al., 2008a). Iodine is essential part of thyroid hormone which is a key regulator of metamorphosis in fish larvae (Schreiber and Specker, 1998; Yoo et al., 2000). The hormone stimulates gastric gland formation and pepsinogen synthesis in summer flounder larvae (Huang et al., 1998). T3, together with retinoic acid also increases transcription of growth hormone in carp (Stenberg and Moav, 1999). Iodine also acts as antibacterial agent. Hatcheries use several iodine based disinfectants (Smail et al., 2004; Verner-Jeffreys et al., 2008, Ribeiro et al., 2011). Maier et al. (2007) reported activation of antioxidant genes and DNA damage in the thyroid gland of rats and mice by iodine deficiency. Excess iodine has been observed to induce apoptosis in thyrocytes and mammary cells (Upadhyay et al., 2002). Our previous study on rotifer micronutrient profiling (Hamre et al., 2008a) showed that vitamins A, E, and C, thiamine and trace element contents of rotifers fed diets based on raw ingredients were below the levels found in copepods but mainly within the range of requirement of fish. Since there are few direct measurements of fish larval nutrient requirements, the nutrient levels of copepod were considered as target for the enrichment. In the present study, rotifers were cultured on a yeast based diet with graded levels of vitamins A, E, and C, thiamine and iodine, given as Lipiodol®, to identify which concentrations of micronutrients in the culture diet would give copepod levels of micronutrients in the rotifers. The rotifer Brachionus “Cayman” was used in the study. This rotifer strain was identified by Baer et al., (2008), using genetic barcoding techniques. It is a member of the Brachionus plicatilis species complex, the nearest names relative being B. ibericus, and is commonly used in marine fish hatcheries around the world, including Norwegian cod hatcheries (Baer et al., 2008). 2. Materials and methods 2.1. Culture of Brachionus Rotifers, Brachionus “Cayman”, were cultured at Stolt Sea Farm AS, Øyestranda, Norway. Stock rotifers were grown on the standard Øye diet (Baker's yeast–cod liver oil (10:1, weight/volume; Peter Møller, Norway) with vitamin supplement and the live algae, Isochrysis galbana. These rotifers had been held in culture in the hatchery and fed the Øye diet for several years. For the study, the basic diet was Baker's yeast–pronova oil–live Chlorella (65:25:15 DW). The pronova oil was a synthetic fish oil EPAX 5010, from Pronova As, Sandefjord, Norway having EPA and DHA level of 10% and 50% of fatty acids, respectively. Live Chlorella (13.5% DW) was from Chlorella Industry Co. Ltd. Tokyo Japan. Lipidol® Ultra-fluid (Laboratoire Guerbet, France), which contains ethyl esters of the iodized fatty acids of poppy seed oil, was used as iodine source. Vitamin A was supplemented in the form of retinyl palmitate (Hoffmann-LaRoche, Basel, Switzerland) and DL-α-tocopherol (Royal DSM N.V, Netherland) was used as source for vitamin E. Thiamine was added in the pure powder form (Thiamine HCl, Sigma-Aldrich Logistik GmbH, Germany). Stay C (Hoffmann-LaRoche, Basel, Switzerland) was used as vitamin C source in the diet. The water soluble vitamins thiamine and Stay C were given along with casein (Sigma-Aldrich Logistik GmbH, Germany) to make accurate weighing of the enrichment possible. The fat soluble vitamins A and E were supplied as emulsion in Lipidol. Iodine was present in Lipidol. Vitamins and

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iodine were supplemented at graded levels in the enrichment diet according to Table 1. Rotifers were fed three meals daily. The amount of diet fed was based on daily culture condition observations and amounted to between 0.2 and 0.6 g dry diet per million rotifers per day. The rotifers were not fed above the satiation level to avoid water quality deterioration. The rotifers were grown in 1000 L tanks inoculated at 400 ind mL − 1. No water exchange was performed during the 4-day culture cycles. Particles were removed daily by sedimentation. Water temperature varied between 21 °C and 24 °C and pH between 7.18 and 8.16 during the culture period, measured with Metrohm 632 pHMeter, Switzerland. Oxygen was measured using a DO meter, model 550A from Yellow Springs Instruments (YSI), USA. All rotifers were harvested after 4 days. At the time of harvest, the rotifer cultures were filtered (65 μm, mesh size), and rinsed in tap water. To compensate the lack of replicates, the experiment was repeated three times. The concentrated rotifers were packed and stored in liquid nitrogen for vitamin analysis and at −20 °C for iodine analysis before being transported to NIFES, Bergen, Norway, for analysis. At NIFES, vitamin samples were kept at − 80 °C and iodine samples at − 20 °C, until analyzed. The rotifers were supplemented with known amount of vitamins and iodine, but in addition to calculating total amount of nutrients in the diet, samples of diet with supplement were also taken for analysis.

tetrahydrofuran in n-hexane as mobile phase as described in Hamre et al. (2010b). Ascorbic acid in B. plicatilis was analyzed by HPLC with amperometric electrochemical detector as described in Mæland and Waagbø (1998). Thiamine analysis was carried out according to CEN (2002). Iodine was analyzed in freeze-dried samples of diet ingredients and rotifers. The samples were wet digested by use of a microwave technique in nitric acid with 30% hydrogen peroxide and diluted to 25 ml (Julshamn et al., 2000). The samples were then analyzed for the iodine by an Agilent 7500 inductively coupled plasma-mass spectroscopy (ICP-MS) instrument with HP computer and Chem Station (Julshamn et al., 2001).

2.3. Statistics Statistical analyses were performed using Statistica (StatSoft inc., Tulsa, OK, USA, version 9). Data were checked for homogenicity of variances using Levene's test before ANOVA analysis. Differences between treatments were analyzed by one-way ANOVA and Scheffe post hoc test. Significance level was set at 5% (p b 0.05). Multiple regression analysis was performed for the correlation of the data on dietary and rotifer nutrient levels. Significance level was set at 5% (p b 0.05).

3. Results 2.2. Analytical methods 3.1. Culture parameters

Table 1 Supplementary concentration (mg active substance kg− 1 DW feed) of different vitamins and iodine in diets for rotifers. The basic diet was Baker's yeast–pronova oil–live Chlorella (65:25:15 DW). The pronova oil was the synthetic fish oil EPAX 5010, from Pronova AS, Sandefjord, Norway. Live Chlorella was from Chlorella Industry Co. Ltd. Tokyo Japan. Lipidol® Ultra-fluid (Laboratoire Guerbet, France), which contains ethyl esters of the iodized fatty acids of poppy seed oil, was used as an iodine source. Vitamin A was supplemented in the form of retinyl palmitate (Hoffmann-LaRoche, Basel, Switzerland) and DL-α-tocopherol (Royal DSM N.V., Netherlands) was used as source of vitamin E. Thiamine was added in the pure powder form (Thiamine HCl, Sigma-Aldrich Logistik GmbH, Germany). Stay C (Hoffmann-LaRoche, Basel, Switzerland) was used as vitamin C source in the diet. The water soluble vitamins thiamine and Stay C were given along with casein (Sigma-Aldrich Logistik GmbH, Germany), while vitamins A and E were supplied as emulsion in Lipidol. Iodine was present in Lipidol.

Control Diet 1 Diet 2 Diet 3

Basic diet

Vitamin C

Thiamine

Vitamin E

Vitamin A

Iodine

Basic Basic Basic Basic

0 1600 3200 6400

0 200 400 800

0 1000 2000 4000

0 50 100 200

0 100 200 400

diet diet diet diet

No significant difference (P b 0.05) was observed in egg ratio between rotifers grown on the different diets (Fig. 1), but growth rate was significantly higher in the rotifers fed diet 1 than in rotifers fed the other diets (Fig. 2). The egg ratio varied between 0.22 ± 0.06 and 0.43 ± 0.05 on day 0 and 3, respectively. Dissolved oxygen level (DO) decreased from day 0 to 2, from 7.41 ± 0.13 to 4.55 ± 0.66 mg L − 1 (103 ± 2 to 63 ± 9% saturation). After day 2, DO increased gradually and reached 5.50 ± 0.59 mg L − 1 (76 ± 8% saturation) on day 4 just before sampling. After feeding, there was a sudden drop in oxygen concentration due to the nutrient load in the tank.

0.60

Control diet Diet 1

Diet 2 Diet 3

0.55

Egg ratio (rotifers with egg/total rotifers)

Dry weight was determined gravimetrically after drying the samples at 105 °C over night. Ash content was measured gravimetrically after combustion at 500 °C for 6 h. Nitrogen was analyzed after total combustion using a nitrogen analyser (PE 2410 SERIES II, Perkin Elmer, Oslo, Norway) and protein was calculated as N × 4.46 (see Srivastava et al., 2006 for determination of the factor). Lipid was determined gravimetrically after extraction with ethylacetate and isopropanol (Lie et al., 1988) and glycogen was measured colorimetrically after enzymatic digestion (Hemre et al., 1989). The fatty acid composition of total lipids was analyzed according to Lie and Lambertsen (1991) and individual fatty acids were identified by known purified standards. Vitamin A was determined according to a modified HPLC method by Nöll (1996) as described in Moren et al. (2004a). Vitamin E was also extracted as vitamin A. After extraction vitamin E was quantified by HPLC equipped with a Si column and a florescent detector with 5%

0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10

Day-1

Day-2

Day-3

Day-4

Fig. 1. Rotifer egg ratio (rotifer with egg/total rotifers, mean ± SD, n = 3) on various days of culture.

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120 Control diet Diet 1

Diet 2 Diet 3

100

Table 3 Analyzed dietary concentration (mg kg− 1 DW, mean ± SD, n = 3) of different vitamins and iodine in the feed for the rotifers. Control diet is Baker's yeast–pronova oil–live Chlorella (65:25:15 DW), nd — not detected.

Growth rate (% /day)

80 Control diet Diet 1 Diet 2 Diet 3

60

40

433

Vitamin C (mg kg− 1 DW)

Thiamine (mg kg− 1 DW)

nd

14.0 ± 0.3

860 ± 109

1966 ± 198 3904 ± 402 6215 ± 491

225 ± 7 405 ± 28 706 ± 23

2210 ± 206 3230 ± 213 5090 ± 238

Vitamin E (mg kg− 1 DW)

Vitamin A (mg kg− 1 DW)

Iodine (mg kg− 1 DW)

nd

2.53 ± 0.37

65 ± 5 107 ± 11 183 ± 19

23 ± 2 36 ± 1 69 ± 5

20

0

-20

-40

Day-1

Day-2

Day-3

Day-4

Fig. 2. Growth rate (% per day, mean ± SD, n = 3) of the rotifers fed with different feeds.

3.2. Proximate composition Proximate composition and fatty acid profile of Brachionus “Cayman” cultured with Baker's yeast–pronova oil–live Chlorella (65:25:15 DW, control) is presented in Table 2. Crude protein content was 39.5 ± 0.9% of DW, which is in the range (34–41%) of previous studies (Lie et al., 1997; Srivastava et al., 2006). This result further confirms that stable levels of proteins are found in fed rotifers. The fat content of these rotifers was 15.7% DW in which, total fatty acids contributed 12.0%. DHA, EPA and ARA levels were 21.4, 9.2 and 1.5% of total fatty acids, respectively. Glycogen content was 7.83% DW. 3.3. Vitamins and iodine Analyzed dietary concentration of vitamins and iodine are presented in Table 3. Vitamins C and A were not detected in the control diet. All vitamins were more than the added concentration due to presence of vitamins in the control diet. Analyzed iodine concentration in diet was less than the supplement. Thiamine in the rotifers (25 ± 14 mg kg − 1 DW) fed with the control diet (14.0 ± 0.3 mg kg − 1 DW) was in the upper range of

copepod level (13–23 mg kg − 1 DW; Hamre et al., 2008a) but high standard deviation (±14) was observed in the replicates. There was a significant increase in rotifer thiamine concentration with increased dietary levels, as shown by the regression equation y = 0.73x + 30, r = 0.88 (Fig. 3). Rotifers fed with diet 3 (706 ± 23 mg kg − 1 DW) retained 81 ± 5 mg kg − 1 DW thiamine. There was no significant difference in rotifers fed with diets 1 and 2 (p N 0.05) while rotifers fed with diet 3 were significantly higher than the other groups. A significant positive relationship was also observed between dietary vitamin C and rotifer vitamin C content (Fig. 4; y=0.023x+125, r = 0.91). In the control rotifers, the vitamin C level was 133 ± 13 mg kg− 1 DW, which significantly (pb 0.05) increased up to 263± 53 mg kg− 1 DW in rotifers fed with diet 3. This concentration of vitamin C was below the copepod level (500 mg kg− 1 DW, Hamre et al., 2008a). Assuming a continuous linear relationship, the regression equation shows that 16,295 mg kg− 1 DW vitamin C, in the form of Stay C, will be needed to get copepod vitamin C content in rotifers. No significant difference was observed between rotifers fed with diets 2 and 3. Rotifers fed with diet 1 had significantly lower vitamin C concentration than the rotifers fed with diet 3 but no significant difference were observed in between rotifers fed with the control diet, diet 1 and diet 2. There was also a significant positive relationship between dietary and rotifer concentration of α-tocopherol; regression equation, y = 0.078x− 12.4; r = 0.96 (Fig. 5). The maximum concentration was 406 ± 27 mg kg− 1 DW in rotifers fed with diet 3, was significantly (pb 0.05) higher than in rotifers fed the other diets and is approximately 4 times higher than the copepod level (110 mg kg− 1 DW, Hamre et al., 2008a). On the basis of the regression equation, 1569 mg kg− 1 DW α-tocopherol will be needed in the diet, to enrich rotifers up to the copepod level. Traces of β and δ tocopherol were present in a few samples (data not shown). There was no significant difference (pN 0.05)

Table 2 Biochemical compositions of rotifers cultured with the control diet — Baker's yeast– pronova oil–live Chlorella (65:25:15 DW). The pronova oil was a synthetic fish oil EPAX 5010, from Pronova AS Sandefjord, Norway. Live Chlorella from Chlorella Industry Co. Ltd. Tokyo Japan. DHA — docosahexanoic acid (22:6n−3), EPA — eicosapentanoic acid (20:5n−3), ARA — arachidonic acid (20:4n−6). Concentration

Dry wt (% wet wt) Ash (% dry wt) Crude protein (% dry wt) Fat (% dry wt) Glycogen (% dry wt) Total fatty acids (% dry wt) Saturated fatty acids (% total fatty acids) Monounsaturated fatty acids (% total fatty acids) Total n−3 (% total fatty acids) Total n−6 (% total fatty acids) DHA (% total fatty acids) EPA (% total fatty acids) ARA (% total fatty acids) n−3/n6 DHA/EPA EPA/ARA

13.9 ± 0.6 2.3 ± 0.2 39.5 ± 0.9 15.7 ± 1.1 7.8 ± 0.4 12.0 ± 2.7 11.1 ± 0.5 31.4 ± 2.4 39.3 ± 3.3 7.9 ± 0.4 21.4 ± 1.7 9.2 ± 1.7 1.5 ± 0.4 5.5 ± 0.7 2.4 ± 0.5 6.9 ± 2.7

90 80 70 -1

Parameter

R- thiamine (mg kg -1 DW) = 30.4 + 0.073 * Dietary thiamine (mg kg-1 DW) Correlation: r = 0.88

60 50 40 30 20 10 0 -100

0

100

200

300

400

500

600

700

800

Dietary thiamine (mg kg-1 DW) Fig. 3. Relationship between dietary and rotifer thiamine content (mg kg− 1 DW). Rthiamine — rotifer thiamine content.

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A. Srivastava et al. / Aquaculture 319 (2011) 430–438 R-vitamin A (mg kg-1 DW) = 5.4 + 0.010 * Dietary vitamin A (mg kg-1 DW)

-1

-1

R- vitamin C (mg kg DW) = 125 + 0.023 * Dietary vitamin C (mg kg DW) Correlation: r = .91

320

8,0

280

R-vitamin A (mg kg -1 DW)

R- vitamin C (mg kg -1 DW)

300

260 240 220 200 180 160 140 120 0

1000

2000

3000

4000

5000

6000

7,0 6,5 6,0 5,5 5,0

4,0 -20

7000

Dietary vitamin C (mg kg-1 DW)

0

20

40

60

80

100 120 140 160 180 200 220 240

Dietary vitamin A (mg kg-1 DW)

Fig. 4. Relationship between dietary and rotifer vitamin C content (mg kg− 1 DW). Rvitamin C — rotifer vitamin C content.

in α-tocopherol concentration between rotifers fed the control diet and diet 1. The concentration in rotifers fed diet 2 was significantly (pb 0.05) different from rotifers fed with the control diet and diet 3. No relationship was observed between dietary vitamin A and rotifer content (regression equation: y = 0.0097x+ 5.4143; r = 0.57; Fig. 6). Enrichment of rotifers with vitamin A seems difficult, as maximum retinol content was only 6.98 ± 1.66 mg kg− 1 DW in rotifers fed diet 3, with 200 mg kg − 1 vitamin A. Control rotifers contained 4.9 ± 0.5 mg kg− 1 DW retinol. No significant difference (p b 0.05) was observed in rotifers fed with different doses of vitamin A. There was a significant positive relationship between dietary iodine and rotifer iodine concentration as described by the regression equation y = 0.88x + 4.62, r = 0.96 (Fig. 7). The iodine level increased more than 12 times, compared to control, in rotifers fed with diet 3. In control rotifers, iodine was 5.1 ± 3.0 mg kg − 1 DW. Iodine content in rotifers fed with diet 3 was 64 ± 9 mg kg − 1 DW which is within the range found in copepods (50–350 mg kg − 1 DW, Hamre et al., 2008a). According to the regression equation, 52–392 mg kg − 1 DW iodine will be needed to enrich rotifers up to copepod levels. 4. Discussion Cod larvae reared in semi-extensive systems generally show very high specific growth rate in the range of 25% per day but in intensive

Fig. 6. Relationship between dietary and rotifer vitamin A content (mg kg− 1 DW). Rvitamin A — rotifer vitamin A content.

systems specific growth rate is normally lower than this (Busch et al., 2010). Nutrition is probably a major factor for this difference, since larvae reared intensively on rotifers and copepods also show differences in growth (Koedijk et al., 2010). Our previous studies (Hamre et al., 2008a; Srivastava et al., 2006) show that rotifers have lower levels than copepods of several macro and micronutrients. Proximate analysis shows that the rotifers used in the present study had similar protein content as in the previous study (Srivastava et al., 2006). Glycogen content was also in the range of other reports (Lie et al., 1997; Srivastava et al., unpublished data). Lipids are very important for the normal development of fast growing larvae (Izquirdo and Koven, 2011). For the enrichment of live feed with nutrients other than the fatty acids, it is very important to consider larval lipid and fatty acid requirement. In the present study, the lipid level and fatty acid composition was within the ranges found in copepods (van der Meeren et al., 2008). We used a synthetic oil which was enriched in EPA and DHA, up to 10 and 50% of fatty acids, respectively. Despite the important role in metabolism, micronutrients have been given less emphasis in larval fish nutrition. Our previous report (Hamre et al., 2008a) suggests that rotifers fed with yeast based diets may be low in thiamine. In the present study thiamine in the rotifers (24.86 mg kg − 1 DW) fed with the control diet was up to copepod thiamine content (13–23 mg kg − 1 DW; Hamre et al., 2008a) and would not need to be enriched further. Van der Meeren et al. (2008)

R- α-tocopherol (mg kg-1 DW) = -12.4 + 0.078 * Dietary α-tocopherol (mg kg-1 DW)) Correlation: r = .96

80

500 450

70

400

60

R-iodine (mg kg-1 DW)

R- α-tocopherol (mg kg-1 DW)

7,5

4,5

100 -1000

350 300 250 200 150 100

R-iodine (mg kg-1 DW) = 4.6 + 0.88 * Dietary iodine (mg kg-1 DW) Correlation: r = .95514

50 40 30 20 10 0

50 0

Correlation: r = .57

8,5

0

1000

2000

3000

Dietary a-tocopherol (mg

4000

kg-1

5000

6000

DW))

Fig. 5. Relationship between dietary and rotifer α-tocopherol content (mg kg− 1 DW). R-α-tocopherol — rotifer α-tocopherol content.

-10 -10

0

10

20

30

40

50

60

70

80

Dietary iodine (mg kg-1 DW) Fig. 7. Relationship between dietary and rotifer iodine content (mg kg− 1 DW). R-iodine — rotifer iodine content.

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and Lie et al. (1997) reported up to 51.4 mg kg − 1 DW thiamine in rotifers enriched with I. galbana. The present study and other studies on thiamine fortification in rotifers indicate that copepod thiamine level can be achieved easily with either enrichment with pure thiamine or with an algae based diet. In our previous study, the vitamin C content in rotifers fed with a yeast based diet ranged between 100 and 200 mg kg − 1 DW (Hamre et al., 2008a), while rotifers fed with the commercial diet Culture Selco contained 576 mg kg − 1 DW vitamin C. In the present study, in spite of very high analyzed dietary vitamin C content (6215 mg kg − 1 DW; Table 3), the rotifer level reached only up to 262 mg kg − 1 DW, which is below the copepod content (500 mg kg − 1 DW). A possible reason for this observation might be the source of vitamin C. In our study we used Stay C, which contains L-ascorbyl 2-poly phosphate as the vitamin C source. Stay C is a stable ascorbic acid source containing a minimum 35% ascorbic acid activity and is commonly used in artificial diet production. Some Artemia enrichment studies with ascorbyl 2-poly phosphate have been reported earlier (Smith et al., 2004) but studies on enrichment of rotifers with Stay C are scarce. Moreover, how rotifer enzymes act on L-ascorbyl 2-poly phosphate for the removal of the phosphate group is also unknown. Ascorbyl palmitate (AP) is an alternative source of ascorbic acid, which is used along with lipid emulsions. Different studies with AP shows variation in vitamin C concentration in Artemia. Merchie et al. (1995) reported 1200 and 2500 mg kg− 1 DW vitamin C level in Artemia after 24 h enrichment with 10 and 20% AP/Lipid emulsions, respectively. By contrast, Lim et al. (2002) report 10.9 and 19.9 mg kg− 1 DW vitamin C in juvenile Artemia with 10 and 20% AP. In a study reported by Smith et al. (2004), ascorbic acid levels of N12,000 mg kg− 1 DW were obtained in juvenile Artemia after 24 h enrichment. According to Smith et al. (2004), the uptake efficiency (%) at which ascorbyl-2-monophosphate was removed from the media and incorporated into Artemia as an active form of ascorbic acid, varied from a low efficiency of 0.017% at 0.2 g L− 1 to a maximum efficiency of 0.045% at 1.2 g L− 1. Such type of uptake efficiency studies are lacking in rotifers. In the present study, the vitamin C level in rotifers was still below copepod levels (500 mg kg − 1 DW) but was higher than the requirement in cold water fish given by NRC, 1993 (50 mg kg − 1 DW). The vitamin C requirement varies among species and is dependent on nutrient interactions, metabolic functions and various stages of development and stress (Lall and Lewis-McCrea, 2007). The basal ascorbic acid requirement for many commercially important aquaculture species is generally in between 20 and b100 mg kg − 1 DW (Smith et al., 2004). In some species, high doses of vitamin C have reduced the effect of stress (Azad et al., 2007; Lim et al., 2002; Ortuño et al., 2003; Smith et al., 2004; Zhou et al., 2005), improved resistance to bacterial pathogens and improved the immunological response (Azad et al., 2007; Garcia et al., 2007). α-tocopherol is the most active form of vitamin E (Hamre, 2011; Lampi et al., 1999). The present study and our previous study (Hamre et al., 2008a) shows that rotifers fed with a diet based mainly on yeast, have lower α-tocopherol concentration than copepods, and need to be enriched with α-tocopherol supplements. In the present study, rotifers were able to gain 405 mg kg− 1 DW α-tocopherol, which is approximately 4 times higher than the copepod level (110 mg kg− 1 DW; Hamre et al., 2008a). Linear regression revealed a positive relationship between dietary and rotifer α-tocopherol concentration. The dietary concentration of vitamin E was also mirrored in our previous study (Hamre et al., 2008a) and another study by Brown et al. (2005b). Lie et al. (1997) found 692 mg kg− 1 α-tocopherol in rotifers enriched with the algae I. galbana for 72 h, but these rotifers were initially fed with baker's yeast and high DHA Super Selco and were having 641 mg kg− 1 tocopherol before enrichment with the algae. 110–290 mg kg− 1 vitamin E was reported by Yamamoto et al. (2009) after enrichment with algae and DHA emulsions for 17–28 h. In our study, the maximum level of α-tocopherol was 406± 27 mg kg− 1 DW in rotifers fed with diet containing 0.5% α-tocopherol,

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which is higher than the maximum level of 144 mg kg− 1 α-tocopherol, with dietary supplement of 0.5% α-tocopherol reported by Brown et al. (2005b). The same study found maximum 1040 mg kg− 1 DW αtocopherol in rotifers fed with a diet supplemented with 4% αtocopherol. These results show that it is possible to increase the αtocopherol level further with increasing α-tocopherol concentration in diet. High vitamin E level, in the absence of a sufficient amount of vitamin C, has been shown to increase mortality and tissue lipid oxidation in Atlantic salmon and Atlantic halibut juveniles (Hamre et al., 1997; Hamre et al., unpublished data). Hamre et al. (2010a) suggested using the concentration ratio of the two vitamins in copepods (110 mg kg -1 vitamin E and 500 mg kg − 1 Vitamin C) as a guideline for the supplementation of the larval diet. Live feed organisms contain very little vitamin A (Hamre et al., 2010a) but marine fish larvae appear to convert carotenoids to vitamin A (Moren et al., 2004a). Copepods do not contain vitamin A, but the high levels of carotenoids (630–750 mg kg − 1 DW; van der Meeren, 2003) can probably be converted to vitamin A by the larvae at sufficient rates to cover the vitamin A requirement (Moren et al., 2004a). Rotifers, on the contrary, contain low levels of carotenoids (4.3–15 mg kg − 1 DW) and in some cases vitamin A is not detected in rotifers fed with yeast based diets (Hamre et al., 2008a). In the present study vitamin A (4.89 mg kg − 1 DW) was detected in rotifers fed with control diet. This level of retinol was higher than the optimum level of vitamin A (2.4 mg kg − 1 DW; Moren et al., 2004b) in Atlantic halibut juveniles. In the present study rotifers achieved maximum 7 mg kg − 1 DW retinol and there was no correlation in between dietary and rotifer content. Giménez et al. (2007) also reported that total vitamin A did not accumulate in rotifers in a dose dependent manner. The same study reported 6.03 ± 2.78 mg kg − 1 DW retinol in rotifers enriched with emulsions containing 192–327 μg total vitamin A l –1. Monroig et al. (2007) showed some possibility of fortification of vitamin A in Artemia by using liposomes. Fernández et al. (2008) reported graded level of retinol from 7.6 to 68.1 mg kg − 1 DW in rotifers after 2 h enrichment with a retinyl palmitate emulsion containing 6.6–11.4 mg kg − 1 DW retinol. Takeuchi et al. (1998) found that rotifers fortified with a commercial emulsion contained 31 mg kg − 1 DW retinoids, while Giménez et al. (2007) reported 179 mg kg − 1 DW retinoids in rotifers enriched with Easy Selco™. In these studies and in a study by Fernández et al. (2008) the dominating form of vitamin A was retinyl palmitate whereas retinol was present at a minor concentration. In our study we added retinyl palmitate and we estimated free retinol. The analytical method contains a hydrolysis step which transforms retinyl esters to retinol prior to HPLC analyses. Linear regression shows a positive relationship between dietary and rotifer iodine concentration. In the present study, a maximum 63 mg kg− 1 DW iodine in rotifers was achieved. This level of iodine is in the range of copepod levels (50–350 mg kg− 1 DW; Hamre et al., 2008a). Other recent studies on enrichment of rotifers with iodine are Hamre et al. (2008b) and Ribeiro et al. (2011). Ribeiro et al. (2011) reported 48 mg kg− 1 wet weight iodine in the rotifers after 3 h enrichment with NaI (0.1–0.2 g I L − 1 depending on the rotifer density) which was 19 times higher than in the control rotifers. Hamre et al. (2008b) reported 112 mg kg− 1 DW iodine in rotifers enriched with sodium iodide (0.4 g L − 1) along with sodium selenite after 1.5 h enrichment. The biological availability of minerals depends on several factors such as the concentration and form of the nutrient; particle size and digestibility of the food; nutrient interaction and physiological and pathological condition of the organism. In the present study, we added graded levels of a vitamin and mineral mix to the rotifer diet, not single nutrients. This design did not allow us to study interactions between the different nutrients, which would be interesting to do in the future. The micronutrient

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requirements of cod larvae are not well characterized. Therefore, we have used copepod levels of nutrients as a reference, assuming that these levels are at least high enough for cod and also for other fish larvae. However, copepods are very nutritious prey and nutrient levels are most often far above the minimum requirements given for fish by NRC (1993) (Hamre et al., 2008a). It is necessary to run studies with graded levels of micronutrients to find the true requirements. We have done similar studies with enrichment of rotifers with minerals other than iodine (Penglase et al., 2011; Nordgreen et al., unpublished) and requirement studies with cod larvae have been performed with selenium (Penglase et al., 2010) and iodine (Penglase et al., unpublished). Furthermore, we have studied uptake and retention of iodine in rotifers enriched with different iodine sources (Srivastava et al., unpublished). The present experiment thus represents one of several steps which will make it possible to determine the nutrient requirements in fish larvae. 5. Conclusion The present study shows that rotifers can be fortified up to copepod level with thiamine, vitamins C and E, and iodine. Vitamin A enrichment seems difficult in rotifers. More research is needed in this area. Acknowledgements This study was financed by the Norwegian Research Council (Project: 14768/120). Ashutosh Srivastava was supported by a grant given by the Norwegian Research Council in the context of the cultural agreement between Norway and India. References Åkerman, G., Tjärnlund, U., Noaksson, E., Balk, L., 1998. Studies with oxythiamine to mimic reproduction disorders among fish early life stages. Mar. Environ. Res. 46, 493–497. Azad, I.S., Dayal, J.S., Poornima, M., Ali, S.A., 2007. Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos to formalin-killed Vibrio vulnificus. Fish Shellfish Immunol. 23, 154–163. Azzi, A., Stocker, A., 2000. Vitamin E: non-antioxidant roles. Prog. Lipid Res. 39, 231–255. Baer, A., Langdon, C., Mills, S., Schulz, C., Hamre, K., 2008. Particle size preference, feeding and gut emptying rates of the rotifer Brachionus “Cayman” using polystyrene latex beads. Aquaculture 282, 75–82. Blanner, W.S., Olson, J.A., 1994. Retinol and retinoic acid metabolism. In: Sporn, M.B., Roberts, A.B., Goodman, D.S. (Eds.), The Retinoids. Raven Press, New York, pp. 229–255. Brown, S.B., Brown, L.R., Brown, M., Moore, K., Villella, M., Fitzsimons, J.D., Williston, B., Honeyfield, D.C., Hinterkopf, J.P., Tillitt, D.E., Zajicek, J.L., Wolgamood, M., 2005a. Effectiveness of egg immersion in aqueous solutions of thiamine and thiamine analogs for reducing early mortality syndrome. J. Aquat. Anim. Heal. 17, 106–112. Brown, M.R., Dunstan, G.A., Nichols, P.D., Battaglene, S.C., Morehead, D.T., Overweter, A.L., 2005b. Effects of α-tocopherol supplementation of rotifers on the growth of striped trumpeter Latris lineata larvae. Aquaculture 246, 367–378. Busch, K.E.T., Falk-Petersen, I.-B., Peruzzi, S., Rist, N.A., Hamre, K., 2010. Natural zooplankton as larval feed in intensive rearing systems for juvenile production of Atlantic cod (Gadus morhua L.). Aquac. Res. 41 (12), 1727–1740. Butterworth, R.F., 1989. Effects of thiamine deficiency on brain metabolism: implications for the pathogenesis of the Wernicke–Korsakoff syndrome. Alcohol Alcohol. 24 (4), 271–279. CEN (Comite' Europe'en de Normalisation), 2002. TC 275 WI 002750053 N134. 2002. Foodstuffs—determination of vitamin B1 by HPLC. EN14122. Conceição, L.E.C., Yúfera, M., Makridis, P., Morais, S., Dinis, M.T., 2010. Live feeds for early stages of fish rearing. Aquac. Res. 41, 613–640. Dabrowski, K., 1992. Ascorbate concentration in fish ontogeny. J. Fish Biol. 40, 273–279. Dedi, J., Takeuchi, T., Seikai, T., Watanabe, T., Hosoya, K., 1997. Hypervitaminosis A during vertebral morphogenesis in larval Japanese flounder. Fish. Sci. 63, 466–473. Drillet, G., Jorgensen, N.O.G., Sørensen, T.F., Ramløv, H., Hansen, B.W., 2006. Biochemical and technical observations supporting the use of copepods as live feed organisms in marine larviculture. Aquac. Res. 37, 756–772. Everitt, D.W. 2006. Natural reproduction and spawning site characteristics of Chinook salmon (Oncorhynchus tshawytscha) in the Salmon River, New York. Master's thesis. State University of New York at Syracuse, Syracuse. Evjemo, J.O., Reitan, K.I., Olsen, Y., 2003. Copepods as live food organisms in the larval rearing of halibut larvae (Hippoglossus hippoglossus) with special emphasis on the nutritional value. Aquaculture 227, 191–210. Fernández, I., Hontoria, F., Ortiz-Delgado, J.B., Kotzamanis, Y., Estévez, A., ZamboninoInfante, J.L., Gisbert, E., 2008. Larval performance and skeletal deformities in farmed

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