Dietary Krill Meal Inclusion Contributes to Better Growth Performance of Gilthead Seabream Juveniles

Received: 26 January 2018 | Revised: 28 June 2018 | Accepted: 7 July 2018 DOI: 10.1111/are.13792

ORIGINAL ARTICLE

Dietary krill meal inclusion contributes to better growth performance of gilthead seabream juveniles

1Aquaculture Research Group (GIA), Institute of Sustainable Aquaculture and Abstract Marine Ecosystems (ECOAQUA), There is a need to find sustainable alternatives to fishmeal (FM) and fish oil (FO) in Universidad de Las Palmas de Gran Canaria, Telde, Spain feed formulations to support the continued growth of aquaculture. FM is mostly 2Oceanography Department, Faculty of produced from mass‐caught pelagic species, but the production has been relatively Science, Alexandria University, Alexandria, constant for several decades. The aim of this study was to investigate the potential Egypt 3Aker BioMarine Antarctic AS, Lysaker, of dietary krill meal (KM) inclusion as a sustainable alternative to FM. In view of Norway that, a feeding trial with gilthead seabream juveniles was conducted to evaluate

Correspondence whether dietary KM at 3%, 6% and 9% inclusion improves growth performance in Lena Burri, Aker BioMarine Antarctic AS, comparison with a control diet. At the end of the study, fish in the 9% KM group Oksenøyveien 10, NO‐13327 Lysaker, Norway. showed significantly higher body weight (32.76 g) compared with fish fed the con- Email: [email protected] trol diet (30.30 g). Moreover, FM replacement by 9% KM indicated a reduction in the accumulation of lipid droplets in the hepatocytes and around the pancreatic islets. In summary, this study suggests that FM can be reduced in diets for seabream without negatively affecting growth performance, when KM is added. On the contrary, KM enhances gilthead seabream growth and reduces lipid accumulation and damage of hepatocytes, which will open an interesting innovation line to completely replace FM by alternative terrestrial protein sources and the partial inclusion of KM.

KEYWORDS fishmeal replacement, growth performance, krill meal, seabream juvenile

1 | INTRODUCTION are required for the further expansion of aquaculture. FM can be partially replaced in the diets of many fish species, but in most cases, Fishmeal (FM) and fish oil (FO) used to be the principal ingredients high or complete replacements have detrimental effects mainly due in formulated marine species and salmon feeds, but high prices and to imbalanced amino acids and the presence of toxins or antinutri- sustainability concerns have stimulated the search for novel feed tional factors. Another key factor is lower feed intake (FI) of fish, ingredients. In 2008, the aquaculture industry used 68% and 88% of because of decreased diet palatability or acceptability as the level of the world’s production of FM and FO respectively (Tacon & Metian, alternative protein sources increases, especially plant proteins 2008). The availabilities of FM and FO for feed inclusion are (Chatzifotis, Polemitou, Divanach, & Antonopoulou, 2008; Kissil, decreasing or at best stagnating (Food of Agriculture Organization of Lupatsch, Higgs, & Hardy, 2000; Kubitza, Lovshin, & Lovell, 1997). United Nations [FAO], 2016). However, an increase in the size of Considerable progress has been made in replacing FM with a variety the global aquaculture industry and the fact that FM production is of plant protein ingredients such as soybeans, lupins, peas and limited have resulted in raising prices, as high as $2,000 per ton. canola (Gatlin et al., 2007; Torrecillas et al., 2015, 2017 ). Almost Therefore, alternative sources of protein and lipids derived from fish

| Aquaculture Research. 2018;1–7. wileyonlinelibrary.com/journal/are © 2018 John Wiley & Sons Ltd 1 2 | SALEH ET AL. complete replacement can be achieved by a balanced supplementa- promote the reduced use of FM and FO in commercial feed for the tion of specific micronutrients and feed attractants (Torrecillas et al., further sustainable development of aquaculture. 2017). Such an attractant is Antarctic krill (Euphausia superba), with a high protein content, favourable amino acid and fatty acid profiles, 2 | MATERIALS AND METHODS as well as enhanced palatability properties. It was suggested that the low molecular weight soluble compounds of krill meal (KM), such as 2.1 | Feeding trial nucleotides, amino acids and high levels of trimethylamine N‐oxide, all act together to make KM an effective attractant and flavouring Gilthead seabream juveniles were obtained from the hatchery at the agent (Ogle & Beaugz, 1991; Shimizu, Ibrahim, Tokoro, & Shirakawa, GIA facilities (Grupo de Investigación en Acuicultura, Las Palmas de 1990). This has been confirmed in various species, such as seabream Gran Canaria, Spain), where the experiment was carried out. A feed- (Pagrus major; Shimizu et al., 1990) and largemouth bass (Micropterus ing trial was conducted testing 9% dietary KM versus a control diet salmoides; Kubitza & Lovshin, 1997). In addition, freeze‐dried krill, for fingerlings with an initial body weight of 12.71 ± 0.11 g and ini- including the soluble protein fraction, stimulated feeding activity of tial fork length of 8.7 ± 0.10 cm. Triplicate groups of fingerlings juvenile Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglos- were randomly distributed in six experimental tanks (at a density of sus hippoglossus), which resulted in increased growth performance 55 fish tank−1) and were fed manually one of the experimental diets and nutrient utilization (Tibbetts, Olsen, & Lall, 2010). Olsen et al. until visual apparent satiety, three times a day, for 12 weeks. FI was (2006) concluded that KM could fully replace FM in diets to Atlantic calculated by recording diet uptake every day, as well as uneaten salmon (Salmo salar) without negatively affecting growth, feed utiliza- pellets at each feeding point. tion and fish health. Salmon fed diets, where 20% or 40% of the FM All tanks (500‐L fibreglass cylinder tanks with conical bottom and was replaced with KM, had a better specific growth rate (SGR) com- painted with light grey colour) were installed in an open system and pared with the FM group. Similarly, no growth reduction was appar- were supplied with filtered sea water (34 ppm salinity) at an increas- ent when rainbow trout (Oncorhynchus mykiss) were fed a diet with ing rate of 100% h−1 along the feeding trial to ensure good water total FM replacement and the addition of deshelled KM (Yoshitomi, quality. Water was continuously aerated attaining 6.7 ± 0.54 mg/Lto Aoki, & Oshima, 2007; Yoshitomi, Aoki, Oshima, & Kazuhiko, 2006). assure good water quality during the entire trial. Water quality was Moreover, Suontama et al. (2007) described no negative effects on tested daily, and no deterioration was observed. Average water tem- Atlantic salmon reared in sea water, when fed diets where FM was perature along the trial was 19.6 ± 1.0°C, and a natural photoperiod replaced with up to 60% Northern krill (Thysanoessa inermis), 40% was adopted. Antarctic krill or 40% Arctic amphipod (Themisto libellula) respec- Two isoproteic and isolipidic experimental diets (pellet size of 2 tively. The authors found that the Atlantic salmon fed a diet, where and 3.2 mm) were formulated containing different KM levels: 0% 40% of the FM was replaced with Antarctic KM, obtained better (control group) and 3%, 6% and 9% KM (QrillTM Aqua; Aker BioMar- SGR than fish fed a control diet. In red porgy (Pagrus pagrus), FM ine Antarctic AS, Norway) (Table 1). The proximate analysis and fatty substitution by KM had no negative effect on growth or feed utiliza- acid composition of the experimental diets are listed in Table 2. tion (Izquierdo, Kalinovski, Thongrod, & Robaina, 2005). In addition, At the beginning and end of the trial, all fish were weighed and because of its content in esterified astaxanthin, KM gave a natural the fork length was measured. Before sampling, fish were always reddish skin coloration to red porgy, in contrast to the lower pig- submitted to 24 hr of fasting. Biological parameters of the growth mentation of fish fed purified canthaxanthin. In another study, the performance of fish were calculated, namely SGR, FI and feed con- inclusion of krill oil high in phospholipids (PL) in larval seabream diets version ratio (FCR). In addition, the hepatosomatic index (HSI) was improved fish growth and survival more effectively than soybean determined. Samples were kept at −80°C until the analysis of bio- PL. It enhanced omega‐3 polyunsaturated fatty acids (n‐3 PUFA) and chemical composition. particularly eicosapentaenoic acid (EPA) incorporation into larval tissues and upregulated the gene expression of bone development 2.2 | Biochemical analysis biomarkers as well as bone mineralization (Izquierdo et al., 2016). Moreover, an increase in dietary krill oil up to 120 g/kg improved Prior to biochemical analysis, samples of 5 fish per tank (in total 15 seabream larval digestive functioning, growth and survival (Saleh fish per treatment) were homogenized (T25 Digital Ultra‐Turrax; et al., 2013). Some environmental NGOs have expressed concerns IKA®, Germany) and analysed in triplicate. Moisture (A.O.A.C., 1995), over the use of KM, which is considered to be essential to all crude protein (A.O.A.C., 1995) and crude lipid (Folch, Lees, & Stan- trophic marine food webs. However, recently the International ley, 1957) contents of fish and diets were analysed. Fatty acid Union for Conservation of Nature has recommended the inclusion methyl esters (FAMES) were obtained by transmethylation of crude of certified krill to reduce the use of FM and FO to produce more lipids as described by Christie (1982). FAMES were separated by sustainable aquafeeds, particularly in larval or broodstock condition- gas‐liquid chromatography (GLC) under the conditions described by ing diets. Izquierdo, Watanabe, Takeuchi, Arakawa, and Kitajima (1990), quan- The objective of this study was to determine the potential bene- tified by a flame ionization detector (FID, Finnigan Focus SG; ficial effects of KM inclusion in feed for juvenile seabream and to Thermo Electron Corporation, Milan, Italy) and identified by SALEH ET AL. | 3

TABLE 1 Formulation and proximate composition of the TABLE 2 Fatty acids composition and proximate composition of experimental diets (g/100 g diet) control and 9% krill experimental diets for gilthead sea bream (% total fatty acids) 3% krill 6% krill 9% krill Ingredients Control meal meal meal Fatty acids Control 9% krill meal Fishmeala 20.0 18.5 17.0 16.0 14:0 2.18 3.36 Blood meal 5.0 5.0 5.0 5.0 15:0 0.22 0.26 Krill mealb 0.0 3.0 6.0 9.0 16:0 18.56 5.55 Rapeseed powder 12.8 12.8 12.8 12.8 17:0 0.21 0.24 Corn gluten 12.0 12.0 12.0 12.0 18:0 3.66 4.05 Soybean 14.2 14.2 14.2 14.2 SFA 24.83 13.46 concentrate 16:1n‐7 2.33 3.2 Wheat meal 10.0 10.0 10.0 10.0 18:1n‐9 33.06 36.83 Wheat gluten 5.0 3.5 2.0 0.0 MUFA 35.39 40.03 Fish oilc 6.0 6.0 6.0 6.0 18:2n‐6 13.61 14.33 Rapeseed oil 4.0 4.0 4.0 4.0 18:3n‐6 8.41 9.05 Linseed oil 2.0 2.0 2.0 2.0 20:2n‐6 0.17 0.18 Palm oil 4.0 4.0 4.0 4.0 20:3n‐6 0.06 0.06 Vitamin mix 2.0 2.0 2.0 2.0 20:2n‐6 0.17 0.18 Mineral mix 2.5 2.5 2.5 2.5 20:3n‐6 0.06 0.06 CMC 0.5 0.5 0.5 0.5 20:4n‐6 0.39 0.43 Proximate analysis (% dry weight) 22:5n‐6 0.02 0.02 Crude lipid 25.0 24.4 24.6 24.2 PUFA n‐6 22.89 24.31 Crude protein 43.6 43.0 43.3 43.6 18:3n‐3 0.01 0.01 Ash 7.1 7.3 7.4 7.5 18:4n‐3 0.53 0.91 Moisture 9.0 8.6 8.8 8.5 20:3n‐3 0.06 0.07 aSouth American Super prime, Bergen, Norway. 20:5n‐3 3.69 5.55 ™ bQRILL Aqua, Oslo, Norway. 22:5n‐3 0.67 0.72 cSouth American Super prime, Bergen, Norway. 22:6n‐3 5.31 6.36 PUFA n‐3 10.27 13.62 ‐ comparison with previous characterized standards and GLC‐MS. HUFA n 3 9.9 12.0 Retention of the most relevant fatty acids was calculated. Total n‐3 19.3 23.49 Total n‐6 14.4 15.21

2.3 | Morphological studies

Liver and intestine of six fish per tank (in total 18 fish per treatment) were fixed in 10% buffered formalin. Paraffin‐embedded tis- supranuclear accumulation of lipid droplets in the enterocytes, and sue sections were cut at 4 μm on a microtome (Leica, RM2135; low incidence denotes a normal shape of enterocytes without lipid Leica Instruments, Nussloch, Germany) and stained with haematoxylin droplet retention. and eosin for histopathological evaluation (Martoja & Martoja‐ Pierson, 1970). The mounted sections were examined under light 2.4 | Statistical analysis microscopy using an Olympus CX41 binocular microscope (Olympus, Hamburg, Germany) connected to an Olympus XC30 camera (Olym- All data were tested for normality and homogeneity of variances. pus), which was linked to a computer using image capturing software Means and standard deviations were calculated for each parameter (CellB®; Olympus). Tissue morphology of intestinal and hepatic measured. If necessary, arcsine square root transformation of the steatosis was examined by two independent pathologists and the data was performed, particularly when data were expressed as % evaluation was transformed to relative percentage using levels from (Fowler, Cohen, & Jarvis, 1998). Differences among dietary treat- 1 to 4, where 1 refers to very low incidence and 4 to high incidence. ments were established by one‐way ANOVA. When p‐values were High incidence of hepatic steatosis means an accumulation of large significant (p < 0.05), individual means were compared using Tukey’s vacuoles of lipids in the hepatocytes, and low incidence implies a and Duncan’s multiple range test (Tukey, 1949). Analyses were per- normal shape of hepatocytes without retention of lipids in the formed using the SPSS Statistical Software System v20.0 (SPSS for hepatocytes. High incidence of intestinal steatosis means high Mac; SPSS Inc, Chicago, IL USA). 4 | SALEH ET AL.

3 | RESULTS and 12.1 cm for fish fed 3%, 6% and 9% KM), although no significant differences were obtained. The inclusion of KM led to a pro- 3.1 | Feeding trial gressive increase in growth in terms of final body weight, which was significantly higher in fish fed 9% KM (32.76 g) compared with the During the 12‐week feeding trial, no external signs of abnormality control group (30.30 g; Figure 2a). In agreement, the SGR signifi- were detected. Survival was high in all groups (around 97%) and was cantly increased to 1.31 in the KM group in comparison with the not negatively affected by the FM replacement by dietary KM. There control group, which had an SGR of 1.20 (Figure 2b). Besides, the were no significant differences in FI between control, 3%, 6% and FCR was significantly reduced by the KM inclusion (1.16) compared 9% KM (Figure 1). FM replacement by KM tended to increase fish with the control group (1.31; Figure 2c). The increase or decrease in total length (11.90 cm for fish fed the control diet and 12.01, 11.96 growth parameters was dose‐dependent, but only the 9% KM group reached significance. But no significant difference was observed for the HSI in the 9% KM group, when compared with the control group 0.4 (1.17 and 1.13 respectively). –1 0.35

ﬁsh 0.3 –1 0.25 3.2 | Biochemical analysis

0.2 The biochemical and morphological studies were performed on con- 0.15 trol and 9% KM samples only, where significance in growth perfor- 0.1 mance parameters was observed. Seabream fed 9% KM tended to Feed intake g day 0.05 have slightly lower hepatic and higher whole body lipid contents, 0 otherwise no significant differences were noticeable (Table 3). Pro- Control 3% krill meal 6% krill meal 9% krill meal tein, ash and moisture contents in whole body and liver were also

FIGURE 1 Feed intake of gilthead seabream fed either control, similar among fish fed the different diets. In addition, the fatty acid 3%, 6% or 9% krill meal diets for 12 weeks showed no significant content of both, whole fish and liver, did not show a significant dif- differences ference between the two treatments (Table 3).

(a) (b) ab a ab a b ab ab b

(c)

a ab ab b

FIGURE 2 Growth performance of gilthead seabream fed either control, 3%, 6% or 9% krill meal diets for 12 weeks. Significantly changed parameters were body weight (a), specific growth rate (b) and food conversion ratio (c) of the 9% krill meal group, when compared with the control group. Values are presented as mean ± SD (n = 55). Treatments bearing identical lettering above bars were not significantly different (p > 0.05) SALEH ET AL. | 5

TABLE 3 Proximate analysis (% Whole body Liver dry weight) and main fatty acids (% total fatty acids) in total lipids of Proximate analysis Control 9% krill meal Control 9% krill meal whole body and liver of gilthead Crude lipid 35.2 ± 0.9a 37.0 ± 0.9a 38.3 ± 0.3a 34.9 ± 3.8a sea bream (n = 5) fed control and Crude protein 56.5 ± 1.6a 54.9 ± 0.7a 44.48 ± 0.3a 47.58 ± 5.3a 9% krill meal diets for 12 weeks Ash 7.6 ± 0.2a 7.6 ± 0.7a 2.7 ± 0.2a 2.4 ± 0.8a Moisture 35.2 ± 0.9a 37.0 ± 0. 9a 38.3 ± 0.3a 34.9 ± 3.8a Fatty acids Saturated 26.1.±3.0a 25.9±0.2.9a 22.6 ± 2.4a 24.2 ± 2.8a Monounsaturated 46.1 ± 5.1a 46.1 ± 4.7a 45.0 ± 4.1a 44.5 ± 5.3a n‐3 11.3 ± 1.2a 10.5 ± 0.8a 11.7 ± 1.0a 11.8 ± 0.7a n‐6 13.2 ± 4.0a 13.7 ± 0.3a 15.4 ± 0.5a 14.3 ± 0.2a n‐3 HUFA 6.2 ± 3.5a 5.5 ± 0.5a 7.1 ± 0.1a 7.5 ± 0.8a ARA 0.3 ± 0.1a 0.3 ± 0.0a 0.7 ± 0.1a 0.7 ± 0.1a EPA 1.9 ± 0.3a 1.6 ± 0.1a 1.5 ± 0.2a 1.9 ± 0.1a DHA 3.1 ± 0.5a 3.0.0 ± 0.3a 4.5 ± 0.8a 4.7 ± 0.7a

Notes. Values (mean ± standard deviation) with the same letters are not significantly different (p > 0.05). ARA: arachidonic acid; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; n‐3 HUFA: omega‐3 highly unsaturated fatty acids; n‐3: omega‐3 fatty acids; n‐6: omega‐6 fatty acids.

TABLE 4 Evaluation of morphological characteristics (relative %) TABLE 5 Evaluation of fat deposition (relative %) in anterior and of liver of gilthead sea bream (n = 6) fed control and 9% krill meal posterior intestine of gilthead sea bream (n = 6) fed control and 9% diets for 12 weeks krill meal diets for 12 weeks

Morphological features (relative %) Control 9% krill meal Morphological 9% krill Nucleus pyknosis 21.7 ± 7.8a 6.67 ± 5.75a feature Control meal a a Fatty hepatocytes 30.0 ± 18.0a 13.3 ± 3.0 Anterior Epithelium 20.0 ± 7.8 52.5 ± 15.0 intestine a a Empty/Broken hepatocytes 26.7 ± 10.5a 16.7 ± 3.0a L. propria 20.0 ± 18.0 12.5 ± 5.0 a a Peripancreatic adipose 10.0 ± 7.8a 8.3 ± 5.8a Posterior Epithelium 35.0 ± 10.5 40.0 ± 5.0 intestine a a Extracellular lipid droplets 11.7 ± 8.8a 20.0 ± 5.5a L. propria 7.5 ± 7.8 7.5 ± 7.5 Bile ducts 11.7 ± 8.8a 35.0 ± 23.0a Note. The evaluation used levels from 1 to 4, where 1 refers to very low incidence and 4 to high incidence, which was transformed to relative Note. The evaluation used levels from 1 to 4, where 1 refers to very low percentage. Values (mean ± standard deviation) with the same letters are incidence and 4 to high incidence, which was transformed to relative not significantly different (p > 0.05). percentage. Values (mean ± standard deviation) with the same letters are not significantly different (p > 0.05). FM and FO in feed for fish (Nicol & Endo, 1999). In the present study, FM substitution by 9% KM increased fish growth with significantly better final body weight and SGR. The 3% and 6% KM inclu- 3.3 | Morphological studies sions showed intermediary values that did not reach significance, but After 12 weeks of feeding, a trend to a reduction in the accumula- showed the same trend. tion of lipid droplets in the hepatocytes and around the pancreatic The results are in agreement with the improved growth obtained islets and reduced hepatic tissue damage was observed in the 9% in other sparids (Chebbaki et al., 2002), as well as in other fish spe- KM group, when compared with the control group (Table 4). cies fed with diets including KM or astaxanthin (Christiansen & Tor- However, the quantitative and statistical analysis did not show rissen, 1996; Hatlen et al., 2016; Izquierdo et al., 2005; Storebakken significant differences. Similarly, fat deposition in anterior and poste- & Choubert, 1991; Torrissen, 1986; Zhang et al., 2012). rior intestine did not demonstrate significant differences between Some authors have related such improved growth to an fish fed the control and 9% KM diets (Table 5). improved appetite based on the high content of feed attractants (Julshamn, Malde, Bjorvatn, & Krogedal, 2004; Oikawa & March, 1997). However, in the present study, there were no significant dif- 4 | DISCUSSION ferences in FI between the two treatments, which could have caused Krill constitutes an interesting marine resource with a large biomass the reduction in FCR and led to better utilization of dietary nutri- that can be promising to contribute to the complete replacement of ents. In addition to its attractants properties, it has been suggested 6 | SALEH ET AL. that KM may contain growth promoting factors or increased miner- ORCID als such as calcium, iron, copper or magnesium (Barrows & Lellis, Lena Burri http://orcid.org/0000-0002-0099-504X 1999). In the present study, the diets were well balanced in mineral and fat‐soluble nutrients, and, therefore, these nutrients do not seem to be related to the growth improvement. Among other nutrients, REFERENCES KM also contains important amounts of astaxanthin and PL (Saito et al., 2002), which are known to be stimulators of growth in larvae A.O.A.C. (1995). Official method of analysis (p. 1018). Washington, DC: and very young fish (Betancor et al., 2011; Saleh et al., 2013, 2015). Association official analytical chemistry. Barrows, F. T., & Lellis, W. A. (1999). 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