Substitution of Chaetoceros muelleri by Spirulina platensis meal in diets for Litopenaeus schmitti larvae

Aquaculture 260 (2006) 215 – 220 www.elsevier.com/locate/aqua-online

Substitution of Chaetoceros muelleri by Spirulina platensis meal in diets for Litopenaeus schmitti larvae Barbarito J. Jaime-Ceballos a,1 , Alfredo Hernández-Llamas b , Tsai Garcia-Galano c , Humberto Villarreal b,⁎ a b

Departamento de Maricultivo, Centro de Investigaciones Pesqueras (CIP), 5ta Avenida y 248, Barlovento, Santa Fe, Ciudad de la Habana, Cuba Programa de Acuacultura, Centro de Investigaciones Biológicas del Noroeste, S. C. (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, México c Centro de Investigaciones Marinas, Universidad de la Habana, Calle 16 entre 1ra y 3ra. Miramar Ciudad de la Habana, Cuba Received 15 November 2005; received in revised form 3 June 2006; accepted 4 June 2006

Abstract The nutritional response of Litopenaeus schmitti larvae to substitution of Chaetoceros muelleri by Spirulina platensis meal (SPM) was evaluated. The substitution levels (S) were 0%, 25%, 50%, 75% and 100%, dry weight basis. Final larval length (FL) ranged from 1.98 to 3.16 mm for the different substitution levels. There was a significant relationship between S and FL, described by the following quadratic equation: FL = 2.853 + 0.01598S − 0.000233S2. The substitution level (S) yielding maximum FL was 34.2%. Development index (DI) values ranged from 2.84 to 3.93 and were dependent on substitution level. The corresponding equation was DI = 3.799 + 0.00945S − 0.000189S2 (P < 0.01). Maximum DI was obtained at 25.0% substitution. Survival was high (82–87%) and no significant differences were found between treatments. Protein digestibility of either microalgae was high, with 92% for SPM and 94% for C. muelleri, with no significant differences between them. The results in this study indicate that an adequate balance of nutrients in relation to the requirements of the species is critical. To simultaneously improve FL and DI, a 30% substitution of C. muelleri by SPM is suggested. This is equivalent to feeding 0.15 mg larvae− 1 day− 1 dry weight basis of a 70% C. muelleri/30% SPM diet, representing 0.078 mg protein larvae− 1 day− 1, 0.026 mg lipids larvae− 1 day− 1 and 2.732 J larvae− 1 day− 1. © 2006 Elsevier B.V. All rights reserved. Keywords: Litopenaeus schmitti; Spirulina meal; Feeding; Shrimp larvae

1. Introduction Feeding of shrimp larvae in aquaculture largely depends on live feeds. However, these are still expensive ⁎ Corresponding author. Tel.: +52 612 123 8484; fax: +52 612 125 3625. E-mail address: [email protected] (H. Villarreal). 1 Tel.: +53 7 209 71 07. 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.06.002

to produce (Jeffrey and Garland, 1987; D'Souza et al., 2000; Fegan, 2004; Robinson et al., 2005). Larvae of Litopenaeus schmitti are normally fed with Chaetoceros sp. (30–50 cells ml− 1) and Tetraselmis sp. (2–5 cells ml− 1). Artificial diets constitute an alternative to reduce high costs of microalgae. Efforts to develop adequate artificial diets for shrimp larvae are being carried out by scientists and the industry (Gallardo et al., 2004).

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Table 1 Proximate composition (dry weight percentage) and energy level (J/ dry weight) in C. muelleri and SPM

Proximate composition (g 100 g− 1 dry matter) Crude protein Crude lipid Carbohydrate Ash Energy (J g− 1): Protein Lipid Carbohydrates Total

C. muelleri

SPM

43.11 ± 0.54 21.48 ± 0.27 17.07 ± 0.32 18.33 ± 0.43

65.0 ± 4.92 6.0 ± 1.0 10.5 ± 3.5 12.2 ± 3.4

7760 7560 2930 18,200

11,700 2110 1720 15,530

Spirulina is generally regarded as a rich source of protein, vitamins, essential amino acids, minerals, essential fatty acids (gamma-linolenic acid GLA), and antioxidant pigments, such as carotenoids (Belay et al., 1996; De Lara Andrade et al., 2005). In addition to its good nutritional value, it is effective for the modulation of the immune response and for protection against radiation (Takeuchi et al., 2002). Several studies have been conducted using Spirulina dried as a supplement in diets for crustaceans (Person, 1976; Tsai, 1979; Cuzon et al., 1981; Castro, 1993; Narciso, 1995). Spirulina platensis meal (SPM) is presently available at a commercial scale; thus, its use in feeds for aquaculture is possible. There are no antecedents on the use of SPM in diets for larvae of the Caribbean white shrimp L. schmitti. Chaetoceros muelleri is a microalgae rich in essential nutrients for shrimp larvae and is commonly used in shrimp hatcheries (Brown et al., 1997). For conditions prevailing in Cuba, the cost of SPM and C. muelleri was estimated at 0.010 and 0.015 US dollars per billion cells, respectively. In this study, an investigation was conducted to evaluate the response of L. schmitti zoea and mysis to partial and total substitution of C. muelleri by SPM as part of a feeding protocol for these stages. 2. Materials and methods

A commercial brand of SPM (Empresa de Producción y Comercialización de Microalgas y sus Derivados GENIX, Cuba) was used for the trial. S. platensis was cultured using the protocol of Zarrouk (1966), before drying at 70 °C. A meal was produced by grinding to a particle size of <30 μm, packed in plastic bags and refrigerated at 10 °C. Culture samples of C. muelleri were centrifuged and lyophilized to determine proximal composition (AOAC, 1995). Proximal SPM composition was determined accordingly (Table 1). Available energy was calculated using the energy conversion factors 18.0, 35.2 and 17.2 kJ/g dry weight for protein, lipid and carbohydrate, respectively (Baukema and De Buin, 1979). In vitro protein digestibility of feeds was determined following Hsu et al. (1977), using hepatopancreatine as a reactant (Carrillo et al., 1994). Chemical scores were also determined according to Garcia (1993), where amino acid composition of L. schmitti postlarvae served as reference (Gallardo et al., 1989). 2.2. Feeding trial Partial and total substitution of C. muelleri by SPM was 0%, 25%, 50%, 75% and 100% dry weight basis (diets S0, S25, S50, S75 and S100, respectively; Table 2). Diet composition is presented in Table 3. L. schmitti nauplii were stocked at 150 organisms l− 1 in 50 l fiberglass tanks, using three replicates per substitution level. Seawater at 35 ppt was filtered (0.5 μm) and UV sterilized to reduce bacterial contamination. Constant aeration was provided via air stones using a 5 hp turbo blower. Na2 EDTA was added to the water at 10 mg l− 1 (Lawrence et al., 1981). To maintain microalgae concentrations and eliminate faecal residues or food remains, a 20–50% water exchange was done daily. Constant illumination was provided with fluorescent lights; temperature (°C), oxygen (mg l− 1), salinity (psu), pH and ammonium (μg l− 1) were registered daily before the water exchange. Table 2 Feeding protocol used during the experimental trial

2.1. Feeds preparation

Diet

C. muelleri (cells ml− 1)

SPM (μg ml− 1)

Microalgae C. muelleri was produced in 80 l tanks at room temperature (28 ± 1 °C), 35 ± 0.2 ppt salinity and using natural light (14 h L:10 h D). The microalgae was used for feeding after 3–4 days, when population growth was at an exponential stage, following Alfonso et al. (1988).

S0 (100% C. muelleri) S25 (75% C. muelleri and 25% SPM) S50 (50% C. muelleri and 50% SPM) S75 (25% C. muelleri and 75% SPM) S100 (100% SPM)

40,000 30,000 20,000 10,000 0

0 5 10 15 20

Diets were offered three times a day. For conversion purposes, 40,000 cells of C. muelleri are dry weight equivalents to 20 μg ml− 1 of SPM.

B.J. Jaime-Ceballos et al. / Aquaculture 260 (2006) 215–220 Table 3 Available protein, lipid and energy for L. schmitti larvae, based on a mean daily feeding rate of 20 μg ml− 1of C. muelleri and SPM, according to the experimental treatment Diet

Protein (μg)

Lipid (μg)

Energy (J)

S0 S25 S50 S75 S100

8.62 9.71 10.81 11.90 13.00

4.29 3.52 2.75 1.97 1.20

0.364 0.348 0.336 0.322 0.310

The feeding trial covered from protozoea substage I (P1) to mysis substage I (M1). Larvae substages of L. schmitti were identified according to Garcia (1972). A stereoscopic microscope (OLYMPUS VM, 1×– 4×) and a Neaubauer chamber were used to adjust the concentration of C. muelleri in the tanks daily following the formula (Alfonso et al., 1988): Va ¼ Vt ðCd  Cr Þ=Ca  Cr

ð1Þ

where Va = volume to be added, Vt = volume in the tank, Cd = desired concentration, Cr = residual concentration in the tank, and Ca = concentration in Va.

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3. Results Water quality parameters were maintained within the range recommended by Vega and de la Cruz (1988) for the species. Dissolved oxygen was 7.25 ± 0.25 mg l− 1, temperature was maintained at 28.5 ± 0.5 °C, and pH was 8.25 ± 0.05. NH4–N had a range between 0.013 + 0.005 mg l− 1. There were no significant differences in final length up to 75% substitution. However, a regression analysis showed that there was a significant relationship between mean FL and percent substitution (Fig. 1, P < 0.01), described by the quadratic equation: FL ¼ 2:853 þ 0:01598S  0:000233S 2 From this equation, the percent substitution yielding maximum final length (Sm) of larvae was Sm ¼ 0:01598ð2ð0:000233ÞÞ1 ¼ 34:2 For DI values, no significant differences were found up to 50% substitution. A significant response to percent

2.3. Data collection and analysis One hundred organisms were sampled per treatment at the end of the experiment to estimate final length (FL), survival, and the development index (DI) as defined by Villegas and Kanazawa (1979). Normality and variance homogeneity of data were evaluated using the Kolmogorov–Smirnov and Bartlett's tests. The dependence of final size and DI on the percent of substitution was determined using a quadratic equation (Shearer, 2000): Y ¼ a0 þ a1 S þ a2 S 2

ð2Þ

where Y represents FL (or DI), a0, a1, and a2 are regression coefficients, and S is the percent of substitution. The percent of substitution yielding the optimum values of FL or DI (Ym) was calculated from the previous equations as Ym ¼ a1 ð2a2 Þ1

ð3Þ

ANOVA was used to test for possible significant differences in mean FL, ID and survival values. A t-test was used to compare protein digestibility between both feeds. Significance level was set at P = 0.05 for all cases. Procedures available in STATISTICA 6.0 were used to conduct analyses.

Fig. 1. Nutritional response of L. schmitti larvae to percentage of substitution of C. muelleri by SPM. (a) Final length. (b) Development index. The dashed lines indicate the percentages of substitution where the maximum response occurs.

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Table 4 Chemical score (%) of C. muelleri and SPM in relation to amino acid composition of L. schmitti EAA a

L. schmitti (g 100 g− 1 dry weight)

C. muelleri

SPM

Arg Met + Cys His Ile Lys Phe + Tyr Thr Trp Val Leu

8.96 2.66 3.36 4.80 8.89 4.72 3.06 1.14 5.0 7.9

71.4 b 86 68 c 114 82.1 220 192.8 – 118 115

106 151 63 c 178 74.5 b 252 221 175 168 153

a b c

EAA: Essential amino acid. Second limiting EAA. First limiting EAA.

substitution was described by the equation (Fig. 1, P < 0.01): DI ¼ 3:799 þ 0:00945S  0:000189S 2 ; the percent substitution (S′m) yielding maximum DI was S Vm ¼ 0:00945ð2ð0:000189ÞÞ1 ¼ 25:0 Overall survival was high (82–87%) and there were no significant differences between treatments. Protein digestibility was 92% for SPM and 94% for C. muelleri (P > 0.05). The chemical score (Table 4) showed that the first and second limiting amino acids were histidine and lysine for SPM and histidine and arginine for C. muelleri. To simultaneously improve FL and DI, a 30% substitution of C. muelleri by SPM is suggested. This is equivalent to feeding 0.15 mg larvae− 1 day− 1 dry weight basis of a 70% C. muelleri/30% SPM diet, representing 0.078 mg protein larvae − 1 day − 1 , 0.026 mg lipids larvae− 1 day− 1 and 2.732 J larvae− 1 day− 1. 4. Discussion High survival in this investigation is similar to that reported (Gelabert et al., 1988; Jaime et al., 1996; Márquez, 1997) for L. schmitti and other penaeid species (Galgani and AQUACOP, 1988; Ottogalli, 1991; Kumlu and Jones, 1995). Several authors, working with larvae of other penaeid species, have found the substitution of live feed is feasible (Jones et al., 1989; Biedenbach et al., 1990; Ottogalli, 1991; Amjad and Jones, 1992; Sunilkumar, 1996; D'Souza et al., 2000; Boeing, 2005). Replace-

ment of C. muelleri by SPM to feed L. schmitti larvae results in an optimum response at 25% substitution for DI and 34% in terms of final length (FL). The benefits of using more than one feed for shrimp larvae have been reported. However, it should be noted that optimal values are seldom indicated. On the other hand, the advantages of using regression analysis over ANOVA to determine such an optimum have been addressed by Shearer (2000). From the equations in our study, it can be concluded that the optimum substitution level, to simultaneously improve larvae size and DI, is approximately 30%. Feed use by crustaceans is dependent, among other things, on feed size, digestibility, nutrient biodisponibility and essential nutrients (Kurmaly et al., 1989; Webster et al., 1994). During the protozoa stage, high enzymatic activity maximizes the assimilation of feed (Jones, 1998). However, some authors indicate that microalgae digestibility is low because of the presence of a cellular wall (Rodriguez et al., 1994; Le Vay et al., 2001). In vivo digestibility studies (e.g., Kawamura et al., 1995) provide reliable information on nutrient assimilation. In the present study, estimated nutrient assimilation of C. muelleri and SPM by in vitro digestibility was high. Hence, we consider the differences in larvae response to be a consequence of the nutritional composition of the diet. Feed mixtures are generally recommended because they produce better results than when a particular feed is used alone, as mixtures contain a complementary blend of nutrients, such as amino acids, that meet or exceed nutritional requirements (Lovell, 1998). For our diets, increased substitution of C. muelleri by SPM resulted in higher protein being offered to larvae, while lipids and energy diminished (Table 3). The chemical score shows that histidine and lysine may become limiting at substitution levels over 50%. Shuli and Baoqing (1992) used C. muelleri and Spirulina sp. to feed larvae of Penaeus orientalis and found that, regardless of the protein content, feeds produced poorer results when used alone, rather than in combination. According to the amino acid requirements of the species, the authors consider that this was a consequence of lysine, arginine and methionine being limiting in both feeds. Thus, optimal FL and DI responses can be explained in terms of the nutrient balance achieved with a particular diet. Future research should focus on determining food consumed and nutrient and energy requirements for L. schmitti. The benefit of using dietary mixtures that exceed nutritional requirements should be understood as

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nutrients in excess have also a high energetic cost (Tacon, 1990). Several studies have shown the nutritional importance of lipids for larval development of penaeid species (Ward et al., 1979; Teshima and Kanazawa, 1982; Kanazawa et al., 1985; Teshima et al., 1986; Cahu et al., 1988; Sorgeloos and Léger, 1992). The lack of fatty acids can result in impaired growth and development of shrimp larvae (Kurmaly et al., 1989; Cuzon, 1996; Shuli and Baoqing, 1992). Theoretical composition of diatoms and SPM suggest that an essential fatty acid limitation could impair larval development of L. schmitti. Further research is needed to confirm that the growth reduction observed at substitution levels over 50% is related to this. A possible toxic effect of the marine blue-green algae, Spirulina subsalsa, on the blue shrimp (Litopenaeus stylirostris) was reported by Lightner (1978). However, organisms fed SPM in our investigation did not show evidence of toxicity. There are no reports describing toxic effects of the freshwater microalgae on penaeid shrimp. The results of this investigation contribute to the development of diets that will optimize hatchery production of L. schmitti. It was evidenced that an adequate balance of nutrients in relation to the requirements of the species is critical for optimal larval development. Acknowledgements The authors are grateful to the staff at Centro de Investigaciones Pesqueras, YAGUACAM hatchery and Centro de Investigaciones Marinas (Cuba). Barbarito Jaime is a doctoral fellow of CONACYT (No. 182852) at the Centro de Investigaciones Biológicas del Noroeste, S. C. (México). References Alfonso, E., Martínez, L., Gelabert, R., Leal, S., 1988. Alimentación de larvas de Penaeus schmitti con diatomeas y flagelados. Rev. Invest. Mar. 9, 47–58. Amjad, S., Jones, D., 1992. An evaluation of artificial larval diets used in the culture of penaeid shrimp larvae Penaeus monodon (Fabricius). Pak. J. Zool. 24, 135–142. AOAC, 1995. 16th edn. Official Methods of Analysis of the Association of Official Analytical Chemist, Vol. I, Washington, DC, USA, 1234 pp. Baukema, J.J., De Buin, W., 1979. Calorific values of the soft parts of the tellinid bivalve Macoma baltica (L.) as determined by two methods. J. Exp. Mar. Biol. Ecol. 37, 19–30. Belay, A., Kato, T., Ota, Y., 1996. Spirulina (Arthrospira): potential application as an animal feed supplement. J. Appl. Phycol. 8, 303–311.

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