J Appl Phycol DOI 10.1007/s10811-015-0590-9
Carotenoid deposition, flesh quality and immunological response of Nile tilapia fed increasing levels of IMTA-cultivated Ulva spp. Luísa M. P. Valente 1,2 & Mariana Araújo 1,2 & Sónia Batista 1,2 & Maria J. Peixoto 1 & Isabel Sousa-Pinto 1,3 & Vanda Brotas 4 & Luís M. Cunha 3,5 & Paulo Rema 6
Received: 3 October 2014 / Revised and accepted: 6 April 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Increasing levels of a mixture of Ulva spp. (Ulva rigida and Ulva lactuca) produced in an integrated multitrophic aquaculture (IMTA) system were evaluated in Nile tilapia. A control diet (CTRL) was compared with two experimental isonitrogenous (36 %) and isoenergetic (20 kJ g−1) diets containing 5 % (U5) and 10 % (U10) of Ulva spp. meal. Duplicate groups of 18 fish (255 g initial body weight) were reared at 25 °C and fed each diet for 68 days to evaluate carotenoid deposition, flesh organoleptic properties and immunological response. By the end of the trial, all groups of fish showed similar final body weight and specific growth rate. Whole body composition was also similar among treatments. The dietary incorporation of Ulva spp. meal increased total carotenoid content in the skin, with fish fed U5 displaying significantly higher levels (6.5 μg g−1) than the CRTL (1.4 μg g−1). No carotenoids could be found in tilapia muscle. Muscle colour determined
* Luísa M. P. Valente
[email protected] 1
CIIMAR/CIMAR—Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas 289, 4050-123 Porto, Portugal
2
ICBAS—Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
3
FCUP—Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
4
Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal
5
REQUIMTE/DGAOT—Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
6
CECAV/UTAD—Universidade de Trás os Montes e Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal
using a spectrocolorimeter indicated that tilapia fillets from fish fed U10 showed the highest lightness (highest L* value) and yellowness (less negative b* value), but the lowest redness (lowest a* value). Furthermore, sensory attributes of flesh showed no significant effects of dietary treatments on visual, olfactory, texture and flavour parameters, with the exception of sour parameter that was lowest in U10-fed tilapia. The dietary inclusion of Ulva spp. meal had no beneficial effect on lysozyme or peroxidase activity, but the complement activity (ACH50), an important component of innate immune system in fish, increased concomitantly with the dietary inclusion level of Ulva spp. meal. The inclusion of Ulva spp. meal in diets for Nile tilapia seems to be possible up to 10 % without major effects on growth performance or flesh organoleptic properties but enhancing the innate immune response of the fish. Keywords IMTA-cultivated Ulva spp. . Muscle and skin pigmentation . Seaweed . Sensory evaluation . Tilapia innate immune response
Introduction Nile tilapia, Oreochromis niloticus, is one of the most commonly farmed species worldwide (Ng and Romano 2013). The European production of tilapia is minimal (0.05 % of global tilapia production), but European Union fillets imports during the first quarter of 2013 were 14 % higher than the same period of 2012 with 4560 tonnes (FAO 2013). Although the organoleptic quality of aquaculture products is generally acceptable, studies on fillet quality issues are required to develop the aquaculture industry and increase consumer acceptance of tilapia in new markets. Nile tilapia is a tropical species with an omnivorous feeding habit. Protein requirement for optimum growth has been
J Appl Phycol
estimated between 20 and 30 % (Hafedh 1999; Sweilum et al. 2005). Although fishmeal is the best protein source for tilapia and other fish due to its complete amino acid profile, alternative protein sources have been widely considered (El-Sayed 1999). Aquatic plants such as seaweeds are regarded as an important resource for aquaculture as they are rich in proteins, lipids, pigments, vitamins and minerals able to modulate flesh quality improving its nutritional value. Seaweeds also provide bioactive compounds with immunity stimulating capacity that may contribute to fish well-being (reviewed by Holdt and Kraan 2011). Moreover, integrated multi-trophic aquaculture (IMTA) systems have been successfully employed to improve the nutritional quality of seaweed biomass (Schuenhoff et al. 2003; Abreu et al. 2011). In tilapia juveniles, Pereira et al. (2012) evaluated the apparent digestibility coefficients (ADC) of several seaweed species and reported the highest protein ADC values with IMTA-produced Ulva spp. Moreover, Ulva spp. meal has been incorporated in Nile tilapia diets from 5 to 30 %, at different life stages, but results largely differ among studies (Güroy et al. 2007; Azaza et al. 2008; Ergün et al. 2009; Silva et al. 2014). Ergün et al. (2009) reported improved growth and feed efficiency in Nile tilapia fed 5 % Ulva rigida, whereas Marinho et al. (2013) observed no impact of the dietary incorporation of IMTA-produced Ulva spp. meal up to 10 %, but a growth reduction at higher inclusion levels. These studies suggest that Ulva spp. meal may be of interest for use in tilapia feed formulations at low inclusion levels. Flesh quality issues have gained importance in the aquaculture industry as they can affect consumer acceptance and human health. Several studies reported the use of microalgae as natural sources of pigments able to alter fish quality traits (Gomes et al. 2002; Lu et al. 2003; Promya and Chitmanat 2011; Teimouri et al. 2013a; 2013b), but the use of seaweeds has been poorly evaluated in fish species (Araújo et al. 2015; Ribeiro et al. 2015). The main pigments present in green seaweeds like Ulva spp. are photosynthetic pigments (chlorophyll a and b) and β-carotene. In addition, other carotenoids like violaxanthin, lutein, neoxanthin and zeaxanthin can be found in different contents depending on environmental conditions (El-Baky et al. 2009; Takaichi 2011). Carotenoids play an important role in biological systems. In fish, muscle and skin pigmentation are important quality parameters for consumers (Sylvia et al. 1996; Hardy and Lee 2010; Sefc et al. 2014). Moreover, some carotenoids function as vitamins and/ or have antioxidant properties able to prevent diseases related to oxidative stress. The inclusion of seaweeds in aquafeeds, likewise microalgal biomass (Gouveia et al. 1998; 2002; Güroy et al. 2012), could be a cost-effective alternative to the synthetic carotenoids inclusion and an effective way of enhancing the fish fillet quality and nutritional value for human consumption. Moreover, previous studies showed that the flesh deposition of low concentrations of bromophenol
resulted in improved flavour properties, by providing a desirable sea-like and fish-like flavour to fish and shrimp (Boyle et al. 1993; Ma et al. 2005). Seaweeds, and in particular, Ulva spp., with their high concentration of bromophenol (428– 1229 ng g−1), mainly in the form of 2,4,6-tribromophenol (Whitfield et al. 1999), could be used as feed ingredients to increase consumer acceptance of tilapia fillets and benefit the aquaculture industry by providing a value-added product. Moreover, enhancing the immunological response by dietary seaweed inclusion seems a promising method of disease prevention in cultured fish by strengthening the defence mechanisms of fish (reviewed by Holdt and Kraan 2011). Algal compounds, mainly polysaccharides like β-glucans, alginates and carrageenan have been used to enhance immunity and disease resistance in a variety of fish species (Fujiki and Yano 1997; Castro et al. 2004; Cheng et al. 2008; Vetvicka et al. 2013) including Nile tilapia (Wang and Wang 1997; ElBaky et al. 2009). In the present study, increasing levels of IMTA-produced Ulva spp. meal (0, 5 and 10 %) were tested in diets for largesized Nile tilapia. Carotenoid deposition in muscle and skin, and flesh organoleptic properties were evaluated after feeding fish for 68 days. Finally, the modulation of some innate immune parameters (lysozyme, peroxidase and ACH50) by dietary seaweeds was also accessed in order to evaluate its beneficial effect in Nile tilapia.
Material and methods All experiments were performed by trained scientists following the European Directive 2010/63/EU of European Parliament and of the Council of European Union on the protection of animals used for scientific purposes. Ingredients and experimental diets The mixture of Ulva spp. (50 % U. rigida and 50 % Ulva lactuca) was raised in an integrated multi-trophic aquaculture (IMTA) system, with aeration and free-floating in fibreglass circular tanks (volume 1200 L), in the facilities of A. Coelho e Castro, Lda. (Póvoa de Varzim, Portugal) following methods proposed by Abreu et al. (2011). The seaweeds were collected and dried for 48 h, in an oven at 50 °C, and ground to 1.5–2mm-sized particles. A control diet (CTRL) was compared with two experimental isonitrogenous (36 %) and isoenergetic (20 kJ g−1) diets containing 5 % (U5) and 10 % (U10) of Ulva spp. meal mainly at the expense of full-fat rice bran and wheat bran (Table 1). All ingredients were milled to 0.05). However, whole body protein deposition tended to be the highest in fish fed seaweed-rich diets. Total carotenoid concentration of Ulva spp. was 239.0± 125.8 μg g−1 DM and the main pigments detected were lutein (8.92 μg g−1) followed by neoxanthin (1.04 μg g−1), fucoxanthin (0.11 μg g−1) and violanxanthin (0.05 μg g−1) (Table 1). The inclusion of Ulva spp. in the experimental diets resulted in increasing levels of total carotenoid content from 0 in CTRL to 77.2 and 180.2 μg g−1 DM in U5 and U10 diet, respectively. The effects of dietary treatments in total carotenoid concentration in Nile tilapia skin are shown in Fig. 2. Total carotenoid concentration was the highest in the skin of fish fed U5 (6.5± 3.3 μg g−1) followed by U10 (5.1±3.1 μg g−1). Fish fed the CTRL diet (1.4±0.4 μg g−1) had significantly lower skin total carotenoid than fish fed U5. In muscle samples, no carotenoids could be detected. The different carotenoids extracted from the skin were identified by HPLC (Table 3). The major carotenoid detected in the skin could not be clearly identified, but its maximum absorption wave length of 493 nm suggests the presence of astaxanthin degradation products (against the expected 471 nm of this xanthophyll). Ulva spp. meal increased violaxanthin deposition in the skin but without statistical significance (P>0.05). Colour evaluation of the tristimulus L*, a* and b* in tilapia flesh indicated significant differences between dietary
Table 1 diets
Formulation and proximate composition of the experimental Dietary treatments CTRL
U5
U10
5.0 0.0 7.8 26.0 10.0 3.0 10.0 5.0 9.5 14.5 4.0 1.0 2.5 0.4 0.3 1.0
5.0 5.0 6.6 26.0 10.0 3.0 10.0 5.3 8.5 11.0 4.4 1.0 2.5 0.4 0.3 1.0
5.0 10.0 6.3 26.0 10.0 3.0 10.0 5.0 4.6 12.7 4.2 1.0 2.5 0.4 0.3 1.0
Feed ingredients (%) Fishmeal 65 LTa Ulva spp. mealb Corn gluten Soybean meal 48c Soybean meal 44c Full-fat soybean meal Rapeseed meal Whole wheat (Firmos) Wheat bran Full-fat rice bran Rapeseed oil Vitf and ming premix Dicalcium phosphate L-lysine DL-methionine Binder (guar gum)i Proximate composition Dry matter (DM, %) Ash (% DM) Crude protein (% DM) Crude fat (% DM) Gross energy (kJ g−1 DM) Total carotenoid (μg g−1 DM)
93.9
96.4
94.5
8.9 35.8 8.3 20.0 0.0
9.7 36.3 8.2 19.6 77.2
10.6 36.0 8.3 19.5 180.2
LT low temperature a
Peruvian fishmeal, EXALMAR, Peru
b
Ulva meal (50/50 % mixture of U. rigida and U. lactuca): dry matter 91.7 %; protein 26.7 %; lipid: 2.7 %; total carotenoids 239.02 μg g−1 (fucoxanthin 0.11 μg g−1 DM; neoxanthin 1.04 μg g−1 DM, violaxanthin 0.05 μg g−1 DM, lutein 8.92 μg g−1 DM) c
Solvent extracted dehulled soybean meal, SORGAL SA, Portugal
d
Dehulled grinded pea grits, Sotexpro, France
e
European origin, Cargill France SAS, France
f Vitamins (in mg or IU kg−1 diet): vitamin A (retinyl acetate); 20,000 UI; vitamin D3 (DL-cholecalciferol), 2000 UI; vitamin E (Lutavit E50), 100 mg; vitamin K3 (menadione sodium bisulfitete), 25 mg; vitamin B1(thiamine hydrochloride), 30 mg; vitamin B2 (riboflavin), 30 mg; calcium pantothenate, 100 mg; nicotinic acid, 200 mg; vitamin B6 (pyridoxine hydrochloride), 20 mg; vitamin B9 (folic acid), 15 mg; vitamin B12 (cyanocobalamin), 100 mg; vitamin H (biotin), 3000 mg; vitamin C (Lutavit C35), 1000 mg; inositol, 500 mg; colin chloride, 1000 mg; betaine (Betafin S1), 500 mg
Minerals (% or mg kg−1 diet): Co (cobalt carbonate), 0.65 mg; Cu (cupric sulphate), 9 mg; Fe (iron sulphate), 6 mg; I (potassium iodide), 0.5 mg; Mn (manganese oxyde), 9.6 mg; Se (sodium selenite), 0.01 mg; Zn (zinc sulphate) 7.5 mg; Ca (calcium carbonate), 18.6 %; KCl, 2.41 %; NaCl, 4.0 %
g
h i
Hilyses (TM), ICC (Brasil)
Carboxymetilcellulose
J Appl Phycol Fig. 1 Tilapia stored in ice and filleted for sensory evaluation
treatments (Table 4). Tilapia fillets from fish fed U10 diet showed the highest lightness (higher L* value) and yellowness (less negative b* scale), but the lowest redness (lower a* value). Fish fed U5 had a significantly lower yellowness than fish fed U10. As all sensory evaluation scores were between the scale anchors, values were measured considering the left anchor as zero and the right anchor as 10. For each sample and each panellist, results were presented as average values across sessions. Results showed no significant effects of the dietary treatments (Table 5) on visual, olfactory and texture parameters (P>0.05). Flavour was the only exception, as fish fed the U10 diet resulted in fillets with the lowest sour value (P≤0.05). Innate immunological parameters in plasma are presented in Fig. 3. The peroxidase and lysozyme activities were similar among treatments (P>0.05). However, the alternative
complement pathway (ACH50) increased proportionally to dietary seaweed inclusion, and tilapia fed U10 showed a significantly higher activity than CTRL fed fish (P≤0.05).
Discussion The inclusion of Ulva spp. meal in tilapia diets up to 10 % resulted in similar dietary protein (36 %) and energy levels (20 kJ g−1 DM), but increased levels of total carotenoid. After feeding fish with the experimental diets for 68 days, the growth parameters remained similar among dietary treatments and all fish reached commercial size (≈300 g). These results confirm previous data obtained in Nile tilapia juveniles showing that the dietary incorporation of 10 % Ulva spp. meal, either produced in IMTA systems (Marinho et al. 2013) or wild caught (Güroy et al. 2007; Azaza et al. 2008), does not
Table 2 Growth performance and feed intake of Nile tilapia fed the experimental diets after 68 days
Dietary treatments
Initial body weight (g) Final body weight (g) Specific growth rate (SGR) Voluntary feed intake (g kg−1 ABW day−1) Hepatosomatic index (HSI) Whole body composition (% WW) Moisture Ash Protein Lipids Energy (kJ g−1)
CTRL
U5
U10
255.8±0.1 305.9±10.1 0.3±0.1 6.7±0.3 0.8±0.1
256.3±0.1 294.1±15.3 0.2±0.1 6.5±0.3 0.8±0.1
255.9±0.2 307.9±30.8 0.3±0.2 6.9±0.1 0.7±0.01
76.3±2.6
74.0±1.4
73.2±1.6
4.0±0.4 12.9±0.2 4.7±2.4 4.5±0.8
4.4±0.1 14.0±0.6 5.8±0.5 5.4±0.4
3.7±0.01 14.0±0.5 6.9±0.4 5.8±0.4
Values are means±standard error (n=2). Initial body composition (%): moisture 74.82; protein 13.01; lipid 5.62; ash 3.25; energy 5.08. In each line, absence of superscript letters indicate no significant differences between treatments (P>0.05) ABW average body weight
J Appl Phycol
Total carotenoids (µg.g-1 skin)
compromise dry feed intake, growth performance or protein utilisation. The dietary inclusion of Ulva spp. meal in Nile tilapia diets was effective in producing a protein-rich product as whole body composition was similar among treatments (13– 14 %w/w), confirming previous data on Oreochromis niloticus fed Ulva spp. meal up to 20 % (Azaza et al. 2008). The nutritional modulation of flesh quality traits has been considered in several fish species as an effective way of enhancing the fillet quality and its nutritional value for human consumption (Holdt and Kraan 2011; Ribeiro et al. 2015). Colour evaluation using the tristimulus L*, a* and b* in tilapia flesh indicated significant differences between dietary treatments (Table 4), but the same trend could not be perceived by the sensory panel (Table 5). As no carotenoids could be detected in tilapia flesh, other traits, like fibre density or fillet fat content must be affecting the instrumental colour analyses, but further studies are needed to clarify this result. A previous study with Nile tilapia from Poland demonstrated that the flesh dominant carotenoids were astaxanthin and canthaxanthin (Czeczuga et al. 2005). However, the absence of these two pigments in the Ulva spp. meal included in tilapia diets may explain the lack of detectable carotenoids in muscle, as cichlid like other fish cannot synthesise them (Sefc et al. 2014). Very few studies evaluated the effects of algae on sensory attributes and flesh quality of fish (Nandeesha et al. 1998; Lu et al. 2003; Ma et al. 2005; Senthil et al. 2005), and many were related to the use of extracts or purified compounds (Boyle et al. 1993; El-Baky et al. 2009), rather than its use as feed ingredient (Araújo et al. 2015). The dietary effects of Ulva spp. meal on Nile tilapia organoleptic characteristics has never been assessed before, and the present study revealed no major effects of this seaweed on visual, olfactory and texture parameters, in spite of the lowest sour value observed in the fillets of fish fed the U10 diet. The incorporation of 30 % Sargassum siliquastrum in diets for Silver seabream (Sparus sarba) significantly increased flesh total bromophenol content providing the desirable sea-like flavour (Ma et al. 2005). However, in the present study, no major sensorial differences could be perceived by the sensory panel. Possibly, higher
7
Dietary treatments CTRL
U5
U10
89.5±5.3 3.9±2.0 1.2±1.2 8.8±0.6
84.9±1.4 128.5±76.4 0.0±0.0 1.6±1.6
86.5±2.5 72.8±21.9 7.4±7.4 1.4±1.4
HPLC analysis in skin Astaxanthin† (relative area %) Violaxanthin (μg kg−1) Lutein (μg kg−1) Zeaxanthin (μg kg−1)
† The main peak, with similar spectrum and retention time as astaxanthin standard, was not identified as astaxanthin since its maximum absorption wavelength was 493 nm (expected 472 nm). However, peak area was quantified and expressed as percent of total area from chromatograms of carotenoid extracts
Values are mean±standard error (n=2). In each line, absence of superscript letters indicate no significant differences between treatments (P>0.05)
inclusion levels of Ulva spp. meal would be necessary to produce detectable sensorial differences. Moreover, previous studies showed that the concentration of bromophenol content in U. lactuca has an extreme seasonal variation (Flodin et al. 1999) so optimised IMTA-systems and/or collection periods should be considered in future studies as a primary tool to modulate seaweed chemical composition. Skin pigmentation is perceived as an important quality factor in several fish species dictating market value (Gomes et al. 2002; Hardy and Lee 2010; Sefc et al. 2014). Total skin carotenoid reported for Nile tilapia vary widely among studies (0.2–43 μg g−1) (Matsuno et al. 1986; Czeczuga et al. 2005). Previous reports in Nile tilapia have shown that dietary supplementation of carotenoids may increase skin deposition but its effects depend on the type of carotenoid added and on the fish population (Kaisuyama and Matsuno 1988; Czeczuga et al. 2005; Sefc et al. 2014). In the present study, the dietary incorporation of Ulva spp. meal resulted in a selective deposition of carotenoids in the skin as no pigments could be detected in Nile tilapia flesh. Skin total carotenoid increased Table 4 Colour measurement in the flesh of Nile tilapia fed the experimental diets
a
8
Table 3 Carotenoid composition in the skin of Nile tilapia fed the experimental diets
ab
6
Dietary treatments
5
CTRL
4 3 2
b
1 0 CTRL
U5
U10
Fig. 2 Total carotenoid content (μg g−1 wet weight) in the skin of Nile tilapia fed the experimental diets. Values are mean±standard error (n=6). Different superscript letters indicate significant differences between treatments (P≤0.05)
Colour variable L* 40.94b ±0.40 a* 1.01a ±0.07 b* −2.63ab ±0.11
U5
U10
42.06a,b ±0.46 0.79a,b ±0.08 −2.91b ±0.10
42.11a ±0.25 0.64b ±0.07 −2.35a ±0.11
Values are means±standard error (n=20). In each line, different superscript letters indicate significant differences between treatments (P≤0.05) L* lightness, a* redness, b* yellowness
J Appl Phycol Table 5
Sensory evaluation of Nile tilapia fed the experimental diets Dietary treatments
Visual Light/dark colour Brown colour intensity Grey colour intensity Black threads Olfactory Boiled potatoes Boiled milk Earth Musty Rancid Texture Flakiness Firm/soft Dry/juicy Fibre Mushy Sticky Flavour Arctic char, new trout Sweet Metallic Earthy Musty Sour Pungent Rancid Spoilage
CTRL
U5
U10
3.0±0.5 2.6±0.4 1.5±0.3 2.2±0.4
4.1±0.5 3.6±0.5 1.2±0.2 3.0±0.5
3.5±0.4 3.2±0.5 1.7±0.3 2.5±0.4
4.3±0.5 1.2±0.3 5.6±0.5 3.2±0.5 0.4±0.1
4.3±0.5 1.9±0.4 5.9±0.5 3.4±0.6 0.3±0.1
4.3±0.5 1.3±0.3 5.8±0.5 4.7±1.5 0.3±0.1
6.3±0.5 7.3±0.4
6.2±0.5 7.1±0.3
5.9±0.5 7.0±0.4
6.7±0.4 3.8±0.5 2.8±0.4 2.6±0.4
6.3±0.4 3.4±0.5 2.9±0.4 3.0±0.5
6.3±0.4 3.9±0.5 2.8±0.4 3.0±0.5
3.0±0.4 0.8±0.2 2.9±0.5 6.7±0.5 4.4±0.5 0.4±0.1ª.b
3.2±0.5 1.0±0.2 2.8±0.5 6.7±0.5 4.3±0.6
3.0±0.4 0.9±0.2 3.6±0.6 6.7±0.5 4.2±0.5
0.5±0.1a 0.6±0.2 0.4±0.1 0.5±0.2
0.2±0.1b 0.9±0.3 0.5±0.2 0.4±0.1
0.8±0.3 0.4±0.1 0.5±0.2
Values are means±standard error (n=20). In each line, different superscript letters indicate significant differences between treatments (P≤0.05)
from 1.4 μg g−1 in fish fed the CTRL to 6.4 μg g−1 in fish fed U5. Previous studies in Nile tilapia identified astaxanthin as the major pigment deposited in the skin (Czeczuga et al. 2005), but in the present study, the presence of astaxanthin degradation products is suggested, and could not be related
Lysozyme
EU mL-1
EU mL-1
20 15 10
2000
40
1500
30
Units ml-1
Peroxidase
25
to the dietary inclusion level of Ulva spp.. Violaxanthin was the second most important pigment identified in Nile tilapia skin followed by zeaxanthin. The metabolism of carotenoids has been reviewed in cichlid fishes by Sefc (2014) and to date the types of carotenoids identified in integument include α and β-carotene, tunaxanthin, canthaxanthin, astaxanthin, lutein, zeaxanthin, rhodoxanthin and canary-xanthophyll B. According to Kaisuyama and Matsuno (1988), the carotenoid metabolic pathways involve epimerization, reduction or oxidation of dietary canthaxanthin, astaxanthin, zeaxanthin and lutein. Lutein is the major carotenoid identified in Ulva spp., but in the present study the levels of this pigment in Nile tilapia skin were highly variably among individuals, not being even identified in some fish. Moreover, it does not seem to have been converted into tunaxanthin, which could not be detected at all, contrarily to previous observations reporting high levels of this pigment in Nile tilapia skin (Matsuno et al. 1986; Czeczuga et al. 2005). In the present study, the violaxanthin identified in Ulva spp. seems to have been preferentially deposited in tilapia skin and together with lutein contributed to the increased carotenoid content observed in fish fed seaweed-rich diets. However, no significant differences could be depicted among dietary treatments probably due to the extremely high variation within individuals in each treatment. Marine algae, including seaweeds, contain several bioactive compounds that possess antibacterial, antiviral, immunoenhancing and anti-tumour properties (Okai et al. 1997; Hudson et al. 1998; El-Baky et al. 2009; Holdt and Kraan 2011; Pohlenz and Gatlin Iii 2014). The stimulation of innate humoral and cellular defence mechanisms induce and enhance resistance against infectious diseases, representing the primary tools in modern fish farming (Vetvicka et al. 2013). In the present report, the humoral immune parameters were measured as indicators of innate immune response of Nile tilapia to dietary seaweed inclusion. The dietary inclusion of Ulva spp. meal had no beneficial effect on lysozyme or peroxidase activities. Different results were observed in rainbow trout (Oncorhynchus mykiss) where the dietary inclusion of 5 % Gracilaria vermiculophylla meal enhanced fish innate immune response, inducing the highest peroxidase and lysozyme activities (Araújo et al. 2015). Elevated
1000 500
5
0
0 CTRL
U5
U10
ACH50
b
a
ab
20 10 0
CTRL
U5
U10
CTRL
U5
U10
Fig. 3 Peroxidase, lysozyme and alternative complement pathway (ACH50) in Nile tilapia plasma. Values are means±standard error (n=6). In each line, different superscript letters indicate significant differences between treatments (P≤0.05)
J Appl Phycol
lysozyme activity has also been reported in African sharptooth catfish (Clarias gariepinus) (Promya and Chitmanat 2011) and in tilapia (Ardó et al. 2008; Zahran et al. 2014) after dietary supplementation with microalgae (Arthrospira (Spirulina) platensis and Cladophora) and Chinese herbs (Astragalus membranaceus and Lonicera japonica), respectively. However, a different herb (Scutellaria extract) had inhibitory or no effect on phagocytic cell activities or lysozyme production (Yin et al. 2006) in tilapia. These distinct results seem to be dose and species dependent. In the present study, the complement activity (ACH50), an important component of innate immune system in fish increased concomitantly to the dietary inclusion level of Ulva spp. meal. The maximal activity was registered at the highest seaweed inclusion level (10 %). Similarly, supplementation of 5 % Ulva spp. meal in the diet for red sea bream (Pagrus major) enhanced complement activity and disease resistance without impairment of growth (Satoh et al. 1987). This suggests that dietary inclusion of seaweeds initiates activation of Nile tilapia innate defence mechanisms, which may be due to its high content of carbohydrates. El-Boshy et al. (2010) have previously reported immunostimulant properties of both β-glucan and laminaran in farmed Nile tilapia, suggesting its use under immune depressive stressful condition to increase their resistance to diseases. This preliminary study indicates that Ulva spp. could be used to promote the health status of tilapia in intensive finfish aquaculture, but further studies testing its resistance against bacterial infection and/or following stress conditions are needed. In conclusion, the inclusion of Ulva spp. meal in diets for Nile tilapia is possible up to 10 % without affecting growth performance or flesh organoleptic properties. The violaxanthin identified in Ulva spp. seems to have been preferentially deposited in tilapia skin and together with lutein contributed to increased carotenoid content in fish fed seaweed-rich diets. Moreover, this seaweed enhanced the innate immune response of Nile tilapia by increasing complement activity (ACH50), promoting the health status of the fish.
Acknowledgments The authors acknowledge M. Pereira and Carolina Beltrán for their valuable help during sampling of fish and laboratorial work. This work was funded by FEDER (European fund for regional development), in the context of the Operational Competitiveness Programme – COMPETE, by FCT—Fundação para a Ciência e a Tecnologia, under the project Project Benefits (PTDC/MAR/105229/2008) n. FCOMP-01-0124FEDER-010622.
References Abreu MH, Pereira R, Yarish C, Buschmann AH, Sousa-Pinto I (2011) IMTA with Gracilaria vermiculophylla: productivity and nutrient removal performance of the seaweed in a land-based pilot scale system. Aquaculture 312:77–87
AOAC (2006) Official methods of analysis of AOAC International, 18th edn. AOAC International, Maryland, USA Araújo M, Rema P, Sousa-Pinto I, Cunha LM, Peixoto MJ, Pires MA, Seixas F, Brotas V, Beltrán C, Valente LMP (2015) Dietary inclusion of IMTA-cultivated Gracilaria vermiculophylla in rainbow trout (Oncorhynchus mykiss) diets: effects on growth, intestinal morphology, tissue pigmentation and immunological response J Appl Phycol Submitted for publication. doi:10.1007/s10811-015-0591-8 Ardó L, Yin G, Xu P, Váradi L, Szigeti G, Jeney Z, Jeney G (2008) Chinese herbs (Astragalus membranaceus and Lonicera japonica) and boron enhance the non-specific immune response of Nile tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila. Aquaculture 275:26–33 Azaza MS, Mensi F, Ksouri J, Dhraief MN, Brini B, Abdelmouleh A, Kraïem MM (2008) Growth of Nile tilapia (Oreochromis niloticus L.) fed with diets containing graded levels of green algae ulva meal (Ulva rigida) reared in geothermal waters of southern Tunisia. J Appl Ichthyol 24:202–207 Boyle JL, Lindsay RC, Stuiber DA (1993) Contributions of bromophenols to marine-associated flavors of fish and seafoods. J Aquat Food Prod T 1(3–4):43–63 Brotas V, Plante-Cuny M-R (2003) The use of HPLC pigment analysis to study microphytobenthos communities. Acta Oecol 24(Supplement 1):S109–S115 Castro R, Zarra I, Lamas J (2004) Water-soluble seaweed extracts modulate the respiratory burst activity of turbot phagocytes. Aquaculture 229:67–78 Cheng A-C, Chen Y-Y, Chen J-C (2008) Dietary administration of sodium alginate and κ-carrageenan enhances the innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and its resistance against Vibrio alginolyticus. Vet Immunol Immunopathol 121:206–215 Choubert G, Storebakken T (1989) Dose response to astaxanthin and canthaxanthin pigmentation of rainbow trout fed various dietary carotenoid concentrations. Aquaculture 81:69–77 Costas B, Conceição LEC, Dias J, Novoa B, Figueras A, Afonso A (2011) Dietary arginine and repeated handling increase disease resistance and modulate innate immune mechanisms of Senegalese sole (Solea senegalensis Kaup, 1858). Fish Shellfish Immunol 31: 838–847 Czeczuga B, Czeczuga-Semeniuk E, Káyszejko B, Szumiec J (2005) Carotenoid content in the Nile tilapia (Oreochromis niloticus L.) cultured in Poland. Acta Sci Pol Piscaria 4:25–32 El-Baky HHA, El-Baz FK, El-Baroty GS (2009) Natural preservative ingredient from marine alga Ulva lactuca L. Int J Food Sci Technol 44: 1688–1695 El-Boshy ME, El-Ashram AM, AbdelHamid FM, Gadalla HA (2010) Immunomodulatory effect of dietary Saccharomyces cerevisiae, βglucan and laminaran in mercuric chloride treated Nile tilapia (Oreochromis niloticus) and experimentally infected with Aeromonas hydrophila. Fish Shellfish Immunol 28:802–808 El-Sayed A-FM (1999) Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture 179 :149–168 Ergün S, Soyutürk M, Güroy B, Güroy D, Merrifield D (2009) Influence of Ulva meal on growth, feed utilization, and body composition of juvenile Nile tilapia (Oreochromis niloticus) at two levels of dietary lipid. Aquacult Int 17:355–361 FAO (2013) Aquaculture production: quantities 1950–2011. In: FISHSTAT Plus—universal software for fishery statistical time series [online]. Food and Agriculture Organization of the United Nations (2010), Fisheries and Aquaculture Information and Statistics Service, Rome, Italy Flodin C, Helidoniotis F, Whitfield FB (1999) Seasonal variation in bromophenol content andbromoperoxidase activity in Ulva lactuca. Phytochemistry 51:135–138
J Appl Phycol Fujiki K, Yano T (1997) Effects of sodium alginate on the non-specific defence system of the common carp (Cyprinus carpio L.). Fish Shellfish Immunol 7:417–427 Gomes E, Dias J, Silva P, Valente L, Empis J, Gouveia L, Bowen J, Young A (2002) Utilization of natural and synthetic sources of carotenoids in the skin pigmentation of gilthead seabream (Sparus aurata). Eur Food Res Technol 214:287–293 Gouveia L, Gomes E, Empis J (1997) Use of chlorella vulgaris in rainbow trout, oncorhynchus mykiss, diets to enhance muscle pigmentation. J Appl Aquac 7:61–70 Gouveia L, Choubert G, Gomes E, Rema P, Empis J (1998) Use of Chlorella vulgaris as a carotenoid source for rainbow trout: effect of dietary lipid content on pigmentation, digestibility and retention in the muscle tissue. Aquacult Int 6:269–279 Gouveia L, Choubert G, Pereira N, Santinha J, Empis J, Gomes E (2002) Pigmentation of gilthead seabream, Sparus aurata (L. 1875), using Chlorella vulgaris (Chlorophyta, Volvocales) microalga. Aquac Res 33:987–993 Green-Petersen DMB, Hyldig G (2010) Variation in sensory profile of individual rainbow trout (Oncorhynchus mykiss) from the same production batch. J Food Sci 75:S499–S505 Güroy B, Cirik Ş, Güroy D, Sanver F, Tekinay A (2007) Effects of Ulva rigida or Cystoseira barbata meals as a feed additive on growth performance, feed utilization, and body composition in Nile tilapia, Oreochromis niloticus. Turk J Vet Anim Sci 31:91–97 Güroy B, Şahin İ, Mantoğlu S, Kayalı S (2012) Spirulina as a natural carotenoid source on growth, pigmentation and reproductive performance of yellow tail cichlid Pseudotropheus acei. Aquacult Int 20: 869–878 Hafedh YSA (1999) Effects of dietary protein on growth and body composition of Nile tilapia, Oreochromis niloticus L. Aquac Res 30: 385–393 Hardy R, Lee C (2010) Aquaculture feed and seafood quality. Bull Fish Res Agency 31:43–50 Holdt S, Kraan S (2011) Bioactive compounds in seaweed: functional food applications and legislation. J Appl Phycol 23: 543–597 Hudson JB, Kim JH, Lee MK, DeWreede RE, Hong YK (1998) Antiviral compounds in extracts of Korean seaweeds: evidence for multiple activities. J Appl Phycol 10:427–434 Hyldig G, Nielsen D (2001) A review of sensory and instrumental methods used to evaluate the texture of fish muscle. J Texture Stud 32:219–242 Kaisuyama M, Matsuno T (1988) Carotenoid and vitamin A, and metabolism of carotenoids, β-carotene, canthaxanthin, astaxanthin, zeaxanthin, lutein and tunaxanthin in tilapia Tilapia nilotica. Comp Biochem Physiol B: Comp Biochem 90:131–139 Kraay GW, Zapata M, Veldhuis MJW (1992) Separation of chlorophylls c1, c2 and c3 of marine phytoplankton by reversedphase C18 high-performance liquid chromatography. J Phycol 28:708–712 Lie O, Syed M, Solbu H (1986) Improved agar plate assays of bovine lysozyme and haemolytic complement activity. Acta Vet Scand 27: 23–32 Lu J, Takeuchi T, Ogawa H (2003) Flesh quality of tilapia Oreochromis niloticus fed solely on raw Spirulina. Fish Sci 69(3):529–534 Ma WCJ, Chung HY, Ang PO, Kim J-S (2005) Enhancement of bromophenol levels in aquacultured silver seabream (Sparus sarba). J Agric Food Chem 53:2133–2139 Macfie HJ, Bratchell N, Greenhoff K, Vallis LV (1989) Designs to balance the effect of order of presentation and first-order carry-over effects in hall tests. J Sens Stud 4:129–148 Marinho G, Nunes C, Sousa-Pinto I, Pereira R, Rema P, Valente LP (2013) The IMTA-cultivated Chlorophyta Ulva spp. as a sustainable ingredient in Nile tilapia (Oreochromis niloticus) diets. J Appl Phycol 25:1359–1367
Matsuno T, Katsuyama M, Hirono T, Maoka T, Komori T (1986) The carotenoids of tilapia Tilapia nilotica. Bull Jpn Soc Sci Fish 52:115–119 Meilgaard MC, Carr BT, Civille GV (1999) Sensory evaluation techniques, 3rd edn. Taylor & Francis, Boca Raton Nandeesha MC, Gangadhar B, Varghese TJ, Keshavanath P (1998) Effect of feeding Spirulina platensis on the growth, proximate composition and organoleptic quality of common carp, Cyprinus carpio L. Aquac Res 29:305–312 Ng W-K, Romano N (2013) A review of the nutrition and feeding management of farmed tilapia throughout the culture cycle. Rev Aquac 5:220–254 Okai Y, Higashi‐Okai K, Ishizaka S, Yamashita U (1997) Enhancing effect of polysaccharides from an edible brown alga, hijikia fusiforme (hijiki), on release of tumor necrosis factor-α from macrophages of endotoxin-nonresponder C3H/HeJ mice. Nutr Cancer 27:74–79 Pereira R, Valente LMP, Sousa-Pinto I, Rema P (2012) Apparent nutrient digestibility of seaweeds by rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis niloticus). Algal Res 1: 77–82 Pocock T, Król M, Huner NA (2004) The determination and quantification of photosynthetic pigments by reverse phase highperformance liquid chromatography, thin-layer chromatography, and spectrophotometry. In: Carpentier R (ed) Photosynthesis Research Protocols, vol 274. Methods In Molecular Biology. Humana Press, pp 137–148 Pohlenz C, Gatlin Iii DM (2014) Interrelationships between fish nutrition and health. Aquaculture 431:111–117 Promya J, Chitmanat C (2011) The effects of Spirulina platensis and Cladophora algae on the growth performance, meat quality and immunity stimulating capacity of the African Sharptooth Catfish (Clarias gariepinus). Int J Agric Biol 13:77–82 Quade MJ, Roth JA (1997) A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Vet Immunol Immunopathol 58:239–248 Ribeiro AR, Gonçalves A, Colen R, Nunes ML, Dinis MT, Dias J (2015) Dietary macroalgae is a natural and effective tool to fortify gilthead seabream fillets with iodine: effects on growth, sensory quality and nutritional value. Aquaculture 437:51–59 Satoh K-i, Nakagawa H, Kasahara S (1987) Effect of Ulva meal supplementation on disease resistance of red sea bream. Nippon Suisan Gakkaishi 53(7):115–1120 Schuenhoff A, Shpigel M, Lupatsch I, Ashkenazi A, Msuya FE, Neori A (2003) A semi-recirculating, integrated system for the culture of fish and seaweed. Aquaculture 221:167–181 Sefc KM, Brown AC, Clotfelter ED (2014) Carotenoid-based coloration in cichlid fishes. Comp Biochem Physiol A-Mol Integr Physiol 173: 42–51 Senthil MA, Mamatha BS, Mahadevaswamy M (2005) Effect of using seaweed (Eucheuma) powder on the quality of fish cutlet. Int J Food Sci Nutr 56:327–335 Silva D, Valente L, Sousa-Pinto I, Pereira R, Pires M, Seixas F, Rema P (2014) Evaluation of IMTA-produced seaweeds (Gracilaria, Porphyra and Ulva) as dietary ingredients in Nile tilapia, Oreochromis niloticus L., juveniles. Effects on growth performance and gut histology. J Appl Phycol. doi:10. 1007/s10811-014-0453-9 Sunyer JO, Tort L (1995) Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are affected by the alternative complement pathway. Vet Immunol Immunopathol 45:333–345 Sweilum MA, Abdella MM, Salah El-Din SA (2005) Effect of dietary protein-energy levels and fish initial sizes on growth rate, development and production of Nile tilapia, Oreochromis niloticus L. Aquac Res 36:1414–1421
J Appl Phycol Sylvia G, Morrissey MT, Graham T, Garcia S (1996) Changing trends in seafood markets. J Food Prod Market 3:49–63 Takaichi S (2011) Carotenoids in algae: distributions, biosyntheses and functions. Marine Drugs 9(6):1101–1118 Teimouri M, Amirkolaie AK, Yeganeh S (2013a) The effects of dietary supplement of Spirulina platensis on blood carotenoid concentration and fillet color stability in rainbow trout (Oncorhynchus mykiss). Aquaculture 414–415:224–228 Teimouri M, Amirkolaie AK, Yeganeh S (2013b) The effects of Spirulina platensis meal as a feed supplement on growth performance and pigmentation of rainbow trout (Oncorhynchus mykiss). Aquaculture 396–399:14–19 Vetvicka V, Vannucci L, Sima P (2013) The effects of β - glucan on fish immunity. N Am J Med Sci 5:580–588 Wang W-S, Wang D-H (1997) Enhancement of the resistance of tilapia and grass carp to experimental Aeromonas hydrophila and
Edwardsiella tarda infections by several polysaccharides. Comp Immunol Microbiol Infect Dis 20:261–270 Whitfield FB, Helidoniotis F, Shaw KJ, Svoronos D (1999) Distribution of bromophenols in species of marine algae from Eastern Australia. J Agric Food Chem 47:2367–2373 Yin G, Jeney G, Racz T, Xu P, Jun X, Jeney Z (2006) Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus. Aquaculture 253(1–4):39–47 Zahran E, Risha E, AbdelHamid F, Mahgoub HA, Ibrahim T (2014) Effects of dietary Astragalus polysaccharides (APS) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol 38(1):149–157 Zar JH (1999) Biostatistical analysis, 4th edn. Prentice Hall, London
