i l Effects of Dietary Supplement of Schizochytrium Meal on Growth, Fatty Acid Profile and Activities of Digestive Enzymes in Turbot ( Scophthalmus maximus L.) Larvae

A feeding trial was conducted to investigate the effects of supplementation of Schizochytrium meal on growth, digestive enzymes activities and fatty acid composition of turbot ( Scophthalmus maximus L.) larvae. Four isonitrogenous and isolipidic diets were formulated to contain 0 (S0, control diet), 50 (S5), 100 (S10) and 150 (S15) g kg −1 Schizochytrium meal. Fish (initial body weight, 0.06 g) were randomly allotted to 12 square fiberglass tanks. Fish were fed 5 times daily (7:00, 10:00, 14:00, 17:00 and 21:00) for 28 days. No significant differences were observed in survival and intestinal morphology among fish fed various levels of algae meal ( P >0.05). Fish fed diet S5 had significantly higher final body weight than that of fish fed diet S15, and no significant differences were observed among fish fed diets S0, S5 and S10 ( P >0.05). Trypsin in intestinal segments, specific activities of alkaline phosphatase (AKP) in intestine and purified brush border membrane (BBM) of intestine were significantly higher in fish fed diet S5 than that of fish fed diet S15 ( P <0.05). Specific activities of leucine-aminopeptidase (LAP) in intestine and purified BBM of intestine was significantly higher in fish fed diet S10 than that of fish fed diet S15 ( P <0.05). Fish fed diets S5 and S10 had significantly higher docosahexaenoic (DHA, 22:6n-3), n-3 PUFAs content and n-3/n-6 ratio in muscle than fish fed diets S0 and S15 ( P <0.05). Fish fed diets S5, S10 and S15 had significant lower C18:3n-3, eicosapentaenoic (EPA, 20:5n-3), C18:2n-6, n-6 polyunsaturated fatty acids (PUFAs) content in muscle than fish fed the control diets ( P <0.05). No significant differences were observed in intestinal morphology ( P >0.05). In conclusion, 50-100 g kg −1 dry matter Schizochytrium meal in microdiets can improve growth performance and may be a valuable additive in microdiets of turbot larvae.


INTRODUCTION
A current major bottleneck in marine fish hatcheries is the dependence on live prey, such as rotifers and Artemia nauplii, which cannot be manipulated as desired (Lazo et al., 2000). In addition, live prey that used to feed O n l i n e

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microorganisms that can grow rapidly and proliferate in a wide range of environmental conditions due to their unicellular or simple multicellular structure (Mata et al., 2010). Microalgae contain many valuable nutrients for aquafeeds, such as protein, essential amino acids, minerals, water-soluble vitamins, sterols and bioactive compounds (Ju et al., 2012;Atkinson, 2013). Microalgae are also rich in antioxidant pigments such as carotenoids, chlorophylls and phycobiliproteins, and had commonly been added in diets as pigmentation sources for shrimp, salmon and trout (Chien and Shiau, 2005;Güroy et al., 2012). Moreover, most microalgae are rich in n-3 long-chain polyunsaturated fatty acids (LC-PUFAs), particularly eicosapentaenoic (EPA, 20:5n-3), docosahexaenoic (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6). The beneficial effects of microalgae have been shown to be particularly important on survival, growth, feed utilization, immune responses, metamorphosis, appetites and early maturation of larval and juvenile finfish, crustacean and mollusk (Atkinson, 2013;Güroy et al., 2012;Ju et al., 2009Ju et al., , 2012Patterson and Gatlin III, 2013;Shan and Lin, 2014;Vizcaíno et al., 2014) when these essential fatty acids are provided in sufficient amount or in adequate form in feed. Unfortunately, the use of algal meal to replace fish-based ingredients in aquatic feeds is challenged mostly because of the high cost of production and culture inefficiency. Schizochytrium is a unicellular, heterotrophic thraustochytrid, which containing substantial amounts of lipid (~10-50%), and produce a high level of total lipids as DHA (30-70%) (Lewis et al., 1999;Arney et al., 2015). Schizochytrium can be used as a feed to effectively enriching both n-3 and n-6 LC-PUFAs contents of rotifers and Artemia nauplii prior to feeding to fish and shrimp larvae (Lewis et al., 1999;Li et al., 2009). Thraustochytridderived products could be used as promising alternatives, valuable additive or sustainable sources of LC-PUFAs in aquafeeds without detriment to growth of Atlantic salmon (Salmo salar) (Carter et al., 2003;Miller et al., 2007), white shrimp (Litopenaeus vannamei) (Patnaik et al., 2006;Ju et al., 2009) and sea bream (Sparus aurata) (Ganuza et al., 2008). Schizochytrium meal has never been investigated as an additive in turbot (Scophthalmus maximus L.) microdiets. Therefore, the aim of this study was to evaluate the effects of supplementation of Schizochytrium meal on survival, growth, digestive enzymes activities and fatty acid composition of turbot larvae.

Experimental diets
The Schizochytrium meal (crude protein, 120.6 g kg −1 ; crude lipid, 408.0 g kg −1 ) was supplied by Alltech ® Company (Nicholasville, Kentucky, USA). Four isonitrogenous and isolipidic diets were formulated to contain 0 (S0), 50 (S5), 100 (S10) and 150 (S15) g kg −1 dry matter of Schizochytrium meal. Ingredients and proximate composition of the experimental diets are presented in Table I and fatty acid composition of Schizochytrium meal and diets are shown in Table II. Microdiets were manufactured by micro-bonding technology as described by Wang et al. (2017). The dry pellets were ground into 150-250 μm and 250-380 μm particle sizes subsequently stored at −20 °C until used.

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been enriched with Chlorella, yeast and refined fish oil to increase EPA and DHA contents. The Chlorella (8-10×10 4 cells/ml) was supplied in the rearing pool at the first 20 days. Larvae (23 days after hatching, initial body weight 0.06±0.00 g) were randomly distributed into 12 square fiberglass tanks (65×65×90 cm, water volume 200 L) with 800 fish each tank. Each diet was randomly assigned to triplicate tanks. The fish were hand fed five times daily (07:00, 10:00, 14:00, 17:00 and 21:00). During the rearing period, each tank was provided with continuous aeration to maintain the dissolved oxygen level above 6.5 mg L −1 , water temperature ranged from 18 to 20°C, pH from 6.8 to 7.2, ammonia-N was less than 0.1 mg L -1 , photoperiod was set at 12-h light and 12-h dark. Feeding behavior and mortality were monitored every day. The feeding trial lasted for 28 days. SFA, saturated fatty acids; MUFA, mono-unsaturated fatty acids; n-3 PUFA: n-3 polyunsaturated fatty acids; n-6 PUFA: n-6 polyunsaturated fatty acids.

Sample collection
At the end of the feeding trial, the fish were fasted for 24 h before harvest. Total number and weight of fish in each tank were measured. Twenty fish were randomly collected from each tank to monitor final body weight and final body length. Fifty individuals were randomly collected from each tank, and then immediately stored at −80°C for enzyme activity assays. Twenty fish from each tank were collected and stored frozen (−20°C) for determination of fatty acid.

Activities of digestive enzyme analysis
Trypsin activity was assayed according to Holm et al. (1988). The fish were dissected to separate pancreatic and intestinal segments as described in Cahu and Zambonino-Infante (1994). Dissection was conducted on a glass plate maintained at 0°C. The dissected samples were weighed and homogenized in cold ultrapure water (tissue: water, 1:5). The homogenates were centrifuged at 3300 ×g at 4°C for 10 min, and then the supernatant was gently collected and frozen at −80°C for digestive enzyme activity analysis. Purified brush border membranes (BBM) from homogenate of intestinal segment were obtained according to a method described by Crane et al. (1979). Briefly, before CaCl 2 solution addition, 1 mL homogenate was diverted for intestinal enzyme assays. After addition of 0.1 M CaCl 2 , the homogenates were centrifuged at 3300 ×g for 10 min in a centrifuge at 4°C. The supernatants were collected and stored frozen (−80°C) for digestive enzymes activities or protein content analysis. Trypsin activity was assayed according to Holm et al. (1988). Leucine-aminopeptidase (LAP) and alkaline phosphatase (AKP) were assayed both in intestinal segment and BBM according to Bessey et al. (1946) and Maroux et al. (1973), respectively. Protein concentration was determined according to Bradford (1976), and using bovine serum albumin (BSA; Sigma, Saint Louis, MO, USA) as a standard. All the enzyme activity assays were carried out in triplicate.

Fatty acid analysis
The fatty acid profiles were determined as described by Zuo et al. (2012) by using HP6890 gas chromatograph (Agilents Technologies Inc., Santa Clara, California, USA) with a fused silica capillary column (007-CW, Hewlett Packard, Palo Alto, CA, USA) and a flame ionization detector.

Histopathological observation
Distal intestine tissue of three fish per tank were cut and immersed in Bouin's fixative solution. After fixation for 24 h, the fixed intestine tissue samples were dehydrated in a graded series of ethyl alcohol, equilibrated in xylene and embedded in paraffin. Tissue sections (5 μm thickness) were cut from each sample and then stained with hematoxylin and eosin (H and E). The fold height

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(HF), enterocyte height (HE) and microvillus height (HMV) were measured using a microscope equipped with a camera (E600, Nikon, Tokyo, Japan) and an image acquiring software (CellSens Standard, Olympus, Tokyo, Japan).

Statistical analysis
The data are presented as means ± S.D (n = 3). All data were analyzed using one-way analysis of variance (ANOVA). Duncan's multiple range test was applied as a multiple sample comparison when significant differences was detected (P<0.05). All statistical analyses were carried out by using SAS 9.12 (Statistical Analysis System Institute, Cary, NC, USA) for Windows.

Growth performance
After the 28-day feeding trial, no significant differences were observed in survival and specific growth rate among fish fed diets with Schizochytrium meal (P>0.05). Fish fed the diet with 50 g kg −1 algae meal had significantly higher final body weight than that of fish fed diet with 150 g kg −1 algae meal, and no significant differences were observed among fish fed diets with 0, 50 and 100 g kg −1 algae meal (P>0.05). Final body length of fish fed diet with 50 g kg −1 algae meal was significantly higher than that of fish fed diets with 0 and 150 g kg −1 algae meal (P<0.05), but no significant differences were observed between fish fed diets with 50 and 100 g kg −1 algae meal (P>0.05) (Table  III).

Specific activities of digestive enzyme
In this study, activity of trypsin in pancreatic segments, and activity of amylase and lipase in pancreatic and intestinal segments were not significantly affected by dietary Schizochytrium meal levels (P>0.05). Activity of trypsin in intestinal segments of fish fed diet with 50 g kg −1 algae meal was significantly higher than that of fish fed diet with 150 g kg −1 algae meal, and no significant difference was observed among fish fed diets with 0, 50 and 100 g kg −1 algae meal. Specific activities of alkaline phosphatase (AKP) in intestine and purified brush border membrane of intestine was significantly higher in fish fed diet with 50 g kg −1 algae meal than that of fish fed diet with 150 g kg −1 algae meal (P<0.05). Specific activities of leucineaminopeptidase (LAP) in intestine and purified brush border membrane of intestine was significantly higher in the diet with 100 g kg −1 algae meal than that of fish fed diet with 150 g kg −1 algae meal (P<0.05). No significant differences were observed in AKP and LAP among fish fed diets with 0, 50 and 100 g kg −1 algae meal (P>0.05) ( Table IV).

Intestinal morphology
As shown in Table VI, there was an increase trend in HF, HE and HMV in fish fed diets with 5% and 10% Schizochytrium meal than 0 and 15% groups, but no significant differences were observed in HF, HE and HMV among fish fed different diets (P>0.05) (Fig. 1, Table VI).

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Schizochytrium Meal in Microdiets of Turbot

Growth performance
This study was conducted to determine the feasibility of Schizochytrium meal use in microdiets for turbot larvae.
The results indicate that 0-100 g kg −1 algae meal could be used as a promising additive in microdiets of turbot larvae. Many studies also indicated that the addition of dried algae meal to aquacfeed has a positive effect on growth and gut health than those fed diets without algae meal (Li et al., 2009;Ju et al., 2009;Güroy et al., 2012;Eryalçın et al., 2013Eryalçın et al., , 2015Kousoulaki et al., 2015).
In this study, growth of fish fed diet with 150 g kg −1 algae meal was significantly lower than that of fish fed diet with 50 g kg −1 algae meal. Similarly, the negative effects on growth and feed intake caused by high inclusion level or long-term utilization of microalgae have been reported for goldfish (Carassius auratus) (Coutinho et al., 2006), Atlantic cod (Gadus morhua) (Walker and Berlinsky 2011) and red drum (Sciaenops ocellatus) (Patterson and Gatlin III, 2013) and Atlantic salmon (Kousoulaki et al., 2015). The reduced growth of fish/shrimp maybe mainly attributed to the depressed palatability (Coutinho et al., 2006;Walker and Berlinsky, 2011;Ju et al., 2012). However, it is difficult to test if the high levels of algae meal affected the palatability and digestibility of fish larvae in this study. In addition, the lack of fatty acids and the lower digestibility may impair growth rate and development of fish larvae (Coutinho et al., 2006;Jaime-Ceballos et al., 2006;Kousoulaki et al., 2015).
The essential fatty acids, particularly DHA, are necessary for the normal growth, survival development of nervous system and sensory organs, behaviour of aquatic animals, particularly critical for marine fish larvae (Navarro et al., 1995;Sargent and Tacon, 1999;Carboni et al., 2012). Inadequate contents of essential fatty acids in diets may resulted in poor feeding, low survival and poor O n l i n e

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W.W. Wang et al. growth, impaired predator behavior, skeletal deformities, abnormal pigmentation and immune-deficiency of marine fish larvae (Glencross and Smith, 2001;Benítez-Santana et al., 2007;Ganuza et al., 2008;Carboni et al., 2012). In present study, fatty acids analysis revealed that dietary DHA content and DHA/EPA ratio increased from 7.78% to 15.55% and 2.13 to 14.55, respectively, with increasing algae meal inclusion level from 0 to 15%. The imbalance n-3 LC-PUFAs may destroy the balance and structure of the cell membrane of fish larvae, subsequently affects the growth, behavior, quality and pigmentation of marine fish larvae (Reitan et al., 1993;Rainuzzo et al., 1994). Therefore, the imbalance DHA/EPA ratio may be one of the reasons responsible for decreased growth of fish or shrimp fed diets that contain high level of microalgae meal.

Specific activities of digestive
Marine fish larvae undergo major changes in morphology and functionality of their digestive tract during the first five weeks of life (Péres et al., 1997), and changes in enzymatic activities had been used as indicators for studying the effects of the dietary additives that might modulate the maturation process of the digestive tract (Gisbert et al., 2009). In this study, activity of trypsin in intestinal segments of fish fed diet with 50 g kg −1 algae meal was significantly higher than that of fish fed diet with 150 g kg −1 algae meal, similarly, the increased trypsin activity may improve growth or survival of sea bass larvae (Cahu and Zambonino-Infante, 1995); but no significant differences in activity of trypsin in the pancreatic segments, and activity of amylase and lipase in pancreatic and intestinal segments were observed among all treatments. Many compounds present in microalgae could potentially influence digestive enzyme activity in fish larvae. Fioramonti et al. (1994) pointed out that algae growth regulators, such as polyamides, can stimulate cholecystokinin release in rats, which mediates the release of pancreatic enzymes.
Brush border membrane (BBM) enzymes assays have been successfully used to determine the degree of the maturation process of the digestive function in intestine in fish larvae (Cahu and Zambonino Infante, 1995;Ma et al., 2005). Alkaline phosphatase (AKP) and leucineaminopeptidase (LAP) are regarded as indicators for a well-differentiated intestinal BBM and have been found to exhibit high activities in fish larvae fed the optimal diets (Cahu et al., 1999;Zambonino-Infante and Cahu, 2001;Ma et al., 2005). In this study, no significant differences were observed in specific activities of AKP and LAP in intestine and purified BBM of intestine among fish fed diets with 0, 50 and 100 g kg −1 algae meal, but higher than that of fish fed diet with 150 g kg −1 algae meal. These results shown that algae meal did not cause negative effects on O n l i n e

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Schizochytrium Meal in Microdiets of Turbot 7 both enzymatic activities at lower inclusion levels tested. Similarly, Vizcaíno et al. (2014) found that both AKP and LAP activities of gilthead sea bream tended to increased with increasing dietary Scenedesmus almeriensis level. An increase in specific activity of aminopeptidase has been related to maturation of the intestinal membrane and enhanced survival in fish (Cahu and Zambonino-Infante, 1995).

Fatty acid composition
The fatty acid profile of fish closely reflected the composition of diet (Boglino et al., 2012). In this study, fish fed diets with 50 and 100 g kg −1 algae meal had significantly higher DHA in muscle than fish fed diets with 0 and 150 g kg −1 algae meal, denoting the high nutritional value of the algae meal as an alternative source of DHA. Similar results have been reported for channel catfish (Ictalurus punctatus) (Li et al., 2009), olive flounder (Paralichthys olivaceus) (Qiao et al., 2014) and Atlantic salmon (Kousoulaki et al., 2015). The algae meal contains low level of EPA, the EPA levels in muscle decreased as dietary algae meal levels increased. The negative effects of fish fed algae meal based diets on EPA content in flesh has also been reported in Atlantic salmon (Carter et al., 2003;Miller et al., 2007) and seabream (Ganuza et al., 2008).
Fish fed diets with 50 and 100 g kg −1 algae meal had significantly higher n-3 PUFAs content and n-3/n-6 ratio in muscle than fish fed diets with 0 and 150 g kg −1 algae meal. Similarly, many researches had shown that feeding the algae meal increases n-3 PUFAs in fillet of Atlantic salmon (Miller et al., 2007;Kousoulaki et al., 2015) and channel catfish (Li et al., 2009), without adverse effects on flavor quality of fish product, which would benefit for humans. In contrast, no significant differences in muscle total n-3 PUFAs and n-3/n-6 ratio were observed in Atlantic cod (Walker and Berlinsky, 2011) and olive flounder (Qiao et al., 2014) when fish fed diets with various levels of algae meal. The disparate responses may be related to fish species, physiological state, algal products types and processing method.

Intestinal morphology
The intestinal morphology parameters can serve as an index to evaluate the functional structure of the intestine and its ability to maintain optimum nutrient absorption and digestive health (Buddle and Bolton, 1992). In the present study, although an increase trend in HF, HE and HMV were observed in fish fed diets with 5% and 10% Schizochytrium meal than 0 and 15% groups, no significant differences were observed in different groups. The HF, HE and HMV are important for intestinal function. The increased HF, HE and HMV, caused by Schizochytrium meal supplementation, may indicate an increase in the intestinal surface area and consequently increased nutrient absorption. This improvement in intestinal structure may be related to the active substances in Schizochytrium meal, such as spermine. Previous studies have established the positive effect of spermine on growth, pancreatic enzyme secretion, intestinal maturation and health of animals (Osborne and Seidel, 1989;Wild et al., 1993;Peres et al., 1997;Peulen et al., 2000). Futher study is warranted to investigate whether high levels of Schizochytrium meal would have a negative effect on intestinal maturation.

CONCLUSIONS
In conclusion, the results from the present has study shown that 50-100 g kg −1 Schizochytrium meal in microdiets can support better growth performance of turbot larvae. The use of this algae meal in turbot microdiets increased the n-3 PUFAs levels and n-3/n-6 ratios in the muscle, and therefore Schizochytrium meal could be used as a valuable additive in microdiets of turbot.