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

Yuyu Wang1,2, Mingzhu Li 3,4, Gang Lin4, Xiaohua Guo5, Aihua Sun5, Hao Dong5, Qinghui Ai1,* and Kangsen Mai1,*

1The Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, P.R. China

2College of Marine Engineering, Rizhao Polytechnic, Rizhao 276826, China

3College of agriculture, Ludong University, Yantai Shandong 264025, P.R. China

4Alltech, 3031 Catnip Hill Pike Road, Nicholasville, Kentucky 40356, USA

5Shandong Meijia Group Co. Ltd., Rizhao 276800, China

ABSTRACT

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.

Article Information

Received 22 September 2021

Revised 18 October 2021

Accepted 06 November 2021

Available online 11 August 2022

(early access)

Published 04 September 2023

Authors’ Contribution

YYW and MZL performed the experiments, analyzed the data, wrote the manuscript. XHG, AHS and HD analyzed the samples and data. GL helped in preparation of the first draft of the manuscript. QHA and KSM conceived and designed the project and revised the manuscript.

Key words

Schizochytrium meal, Growth, Fatty acid, Scophthalmus maximus L.

DOI: https://dx.doi.org/10.17582/journal.pjz/20220401080451

* Corresponding author: qhai@ouc.edu.cn, kmai@ouc.edu.cn

0030-9923/2023/0005-2417 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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 marine fish larvae is lack of essential fatty acids. Moreover, the larvae had incomplete digestive tract and lack of digestive enzymes (Kolkovski et al., 1997). It is known that nutritional imbalances play a key role in morphogenesis and skeletogenesis at early stages and several dietary components have been identified that affect larval development (Boglino et al., 2012). Therefore, it would be ideal to produce nutritionally balanced microparticulate diets to replace zooplankton. However, ingestion rates of microparticulate diet are often lower than those of live prey during the first feeding, which may limit the availability of nutrients for proper growth and development (Lauff and Hoffer, 1984; Kolkovski et al., 1993).

Microalgae are prokaryotic (eg., Cyanobacteria) or eukaryotic (eg., green algae and diatoms) photosynthetic 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., 2009, 2012; Patterson 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). Thraustochytrid-derived 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.

MATERIALS AND METHODS

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.

 

Table I. Ingredients and nutrients of the experimental diets.

Ingredient	Diet no. (Schizochytrium meal level, g kg-1 dry matter)
	S0	S5	S10	S15
Fish meal1	550	550	550	550
Shrimp meal1	100	100	100	100
Squid meal1	50	50	50	50
Bear Yeast meal1	30	30	30	30
Mussel meal1	50	35	25	15
Schizochytrium meal2	0	50	100	150
Fish oil1	64	44	24	5
Wheat flour1	63	48	28	7
Sodium alginate	10	10	10	10
Vitamin premix3	10	10	10	10
Mineral premix3	15	15	15	15
Choline chloride	2	2	2	2
Antioxidant	0.5	0.5	0.5	0.5
Attractant	15	15	15	15
Lecithin	40	40	40	40
Sodium benzoate	0.5	0.5	0.5	0.5
Proximate analysis (% dry matter basis)
Crude protein	534.2	527.2	530.9	536.5
Crude lipid	111.1	108.2	118.9	117.1

 

1Those ingredients were supplied by Qingdao Great Seven Bio-Tech, Co., Ltd. (Qingdao China). 2Schizochytrium meal was supplied by Alltech Inc., Kentucky, USA. 3Vitamin premix and Mineral premix were supplied by Qing Dao Master Bio-Tech, Co., Ltd. (Qingdao China).

 

Fish rearing

Fish larvae were obtained from a commercial hatchery (Shandong, China) and reared at Haiyang Yellow Sea Aquatic Product Co., Ltd (Yantai, Shandong, China). Larvae were fed with rotifers Brachionus plicatilis (5–10 ind./ml) from mouth opening (3 DAH) to 20 DAH, Artemia nauplii (0.1–0.2 ind./ml to 1–2 ind./ml) from 6 to 22 DAH, microparticulate diets (10.0–20.0 mg/fish/d) from 15 DAH to the end. Both the rotifers and Artemia nauplii had been enriched with Chlorella, yeast and refined fish oil to increase EPA and DHA contents. The Chlorella (8–10×104 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.

 

Table II. Fatty acid composition of dried Schizochytrium meal and experimental diets.

Fatty acids	Schizochytrium	Diet no. (Schizochytrium meal level, 10 g kg-1 of total fatty acids)
		S0	S5	S10	S15
14:0	6.43	5.68	5.05	4.91	3.60
16:0	35.43	23.66	26.73	33.18	40.59
18:0	1.43	4.21	3.47	3.10	2.91
20:0	0.19	0.41	0.33	0.28	0.23
∑SFA	43.48	33.97	35.58	41.46	47.33
16:1n-7	0.18	4.64	4.01	3.07	2.13
18:1n-9	—	13.68	10.93	9.00	8.25
∑MUFA	0.18	18.33	14.95	12.07	10.38
18:3n-3	0.66	1.96	1.80	1.48	1.30
20:5n-3 (EPA)	0.38	3.65	2.93	1.97	1.07
22:6n-3 (DHA)	40.64	7.78	10.41	13.57	15.55
∑n-3 PUFA	41.68	13.39	15.15	17.02	17.92
18:2n-6	0.53	13.69	12.32	11.53	10.88
20:4n-6	1.17	0.51	0.58	0.65	0.73
∑n-6 PUFA	1.7	14.19	12.90	12.18	11.60
∑PUFA	43.38	27.58	28.05	29.20	29.52
n-3/n-6	24.52	0.94	1.17	1.41	1.55
DHA/EPA	106.95	2.13	3.56	6.89	14.55
Total fatty acids	87.04	79.87	78.57	82.72	87.23

 

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 CaCl2 solution addition, 1 mL homogenate was diverted for intestinal enzyme assays. After addition of 0.1 M CaCl2, 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 (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.

RESULTS

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 leucine-aminopeptidase (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).

Fatty acid composition

The percentages of all the identified fatty acids in the muscle of fish fed with graded levels of algae meal are shown in Table V. Fish fed the 50 and 100 g kg−1 algae meal diets had significantly higher C18:0, C22:6n-3, 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 (P<0.05). Fish fed the control diet had significant higher C14:0, C16:1n-7, C18:1n-9, MUFA, C18:3n-3, C18:2n-6, n-6 PUFAs content in the muscle than fish fed the 50, 100 and 150 g kg−1 algae meal diets (P<0.05), however, no significant differences were observed in these fatty acids among fish fed 50, 100 and 150 g kg−1 algae meal diets (P>0.05). C16:0, SFA content and DHA/EPA ratio increased, while EPA decreased in muscle as dietary algae meal level increased; fish fed the 150 g kg−1 algae meal diet had significant lower C20:0, C20:4n-6 and PUFA content than fish fed the 0, 50 and 100 g kg−1 algae meal diets (P<0.05), however, no significant difference was observed among fish fed the 0, 50 and 100 g kg−1 algae meal diets (P>0.05).

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).

 

Table III. Effect of dietary Schizochytrium meal levels on growth and survival of turbot (Scophthalmus maximus L.) larvae.

Diet no.	S0	S5	S10	S15
FBW (g)	0.44±0.02ab	0.48±0.02a	0.44±0.01ab	0.42±0.02b
SGR (%·day-1)	7.19±0.18	7.46±0.15	7.22±0.09	7.05±0.13
FBL (mm)	24.73±0.38b	25.90±0.31a	25.44±0.23ab	24.87±0.33b

 

Note: Data represent as means ± S.D; Values in the same row with different superscripts are significantly different (P<0.05). Specific growth rate (SGR, % day-1) = (Ln FBW-Ln IBW) ×100/ experimental duration (d). IBW, Initial body weight; FBW, Final body weight; FBL, Final body length.

 

Table IV. Effects of dietary Schizochytrium meal levels on activities of digestive enzymes of turbot (Scophthalmus maximus L.) larvae.

Digestive enzymes	Diet no. (Schizochytrium meal level, %)
		S0	S5	S10	S15
Trypsin(mU/ mg•protein)	PS	75.74±2.84	87.08±4.83	84.88±5.01	76.40±4.58
	IS	77.78±4.08ab	82.02±3.86a	75.34±0.61ab	70.96±3.89b
Trypsin (I)/trypsin (P)b		1.03±0.01a	0.94±0.07ab	0.89±0.03b	0.93±0.00ab
Amylase (U/ mg•protein)	PS	0.61±0.04	0.57±0.03	0.63±0.03	0.62±0.05
	IS	0.60±0.03	0.52±0.03	0.59±0.01	0.59±0.02
Lipase (mU/ mg•protein)	PS	0.63±0.04	0.66±0.06	0.64±0.03	0.69±0.01
	IS	0.68±0.02	0.69±0.02	0.67±0.05	0.67±0.01
Specific activities of digestive enzymes in purified brush border membrane of intestine (U/ mg•protein)
Leucine-aminopeptidase		227.89±3.53ab	233.54±7.17ab	239.53±9.23a	214.01±7.80b
Alkaline-phosphatase		375.39±17.55ab	415.78±22.97a	386.93±22.00ab	343.12±19.71b
Specific activities of digestive enzymes in intestine (U/ mg•protein)
Leucine-aminopeptidase		84.86±3.87ab	86.83±4.44ab	88.46±2.29a	78.95±1.90b
Alkaline-phosphatase		95.50±3.85ab	101.10±2.37a	100.09±4.26ab	91.12±2.29b

 

Note: Data represent as means ± S.D; Values in the same row with different superscripts are significantly different (P<0.05). aPS, pancreatic segments; IS, intestinal segments; bTrypsin (I), trypsin of intestinal segment; trypsin (P): trypsin of pancreatic segment.

 

 

DISCUSSION

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., 2013, 2015; Kousoulaki 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 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.

 

Table V. Effects of dietary Schizochytrium meal levels on fillet fatty acid composition of turbot (Scophthalmus maximus L.) larvae.

Fatty acids	Diet no. (Schizochytrium meal level, 10 g kg-1 of total fatty acids)
	S0	S5	S10	S15
14:0	3.24±0.01a	2.48±0.13b	2.43±0.31b	2.82±0.05ab
16:0	22.24±0.12c	22.56±0.89c	25.32±0.22b	27.96±0.89a
18:0	7.60±0.00bc	8.33±0.17a	8.30±0.29ab	7.10±0.12c
20:0	0.31±0.00a	0.30±0.01ab	0.31±0.02a	0.26±0.01b
∑SFA	33.39±0.17b	33.66±0.83b	36.36±1.20a	38.14±1.19a
16:1n-7	4.15±0.02a	2.82±0.04b	2.46±0.24b	2.61±0.26b
18:1n-9	15.09±0.03a	11.47±0.25b	10.57±1.12b	10.12±1.16b
∑MUFA	19.24±0.05a	14.29±0.29b	13.03±0.88b	12.73±1.42b
18:3n-3	1.03±0.02a	0.73±0.07b	0.71±0.02b	0.67±0.04b
20:5n-3 (EPA)	2.86±0.12a	2.26±0.09ab	1.73±0.13bc	1.26±0.23c
22:6n-3 (DHA)	9.64±0.56c	13.15±0.26b	15.31±0.33a	10.98±0.34c
∑n-3 PUFA	13.52±1.00b	16.14±0.60a	17.75±0.68a	12.91±0.75b
18:2n-6	11.72±0.10a	9.69±0.19b	9.87±0.37b	9.72±0.41b
20:4n-6	1.18±0.14a	1.11±0.02a	1.14±0.11a	0.70±0.04b
∑n-6 PUFA	12.90±0.35a	10.79±0.30b	11.01±0.36b	10.42±0.64b
∑PUFA	26.42±1.35a	26.93±0.30a	28.76±1.03a	23.33±0.12b
n-3/n-6	1.05±0.04c	1.50±0.07ab	1.61±0.01a	1.24±0.10bc
DHA/EPA	3.37±0.05b	5.82±0.12b	8.88±0.46a	8.97±1.39a
Total fatty acids	79.05	74.88	78.15	74.20

 

Note: Data represent as means ± S.D; Values in the same row with different superscripts are significantly different (P<0.05). For abbreviation see Table II.

 

Table VI. Effect of dietary Schizochytrium meal levels on micromorphology of the intestine of turbot (Scophthalmus maximus) larvae.

Diet groups	S0	S5	S10	S15
HF (μm)	65.59±1.59	69.28±1.62	67.20±1.73	63.84±1.75
HE (μm)	19.19±0.83	21.26±0.76	19.16±0.77	18.89±0.93
HMV (μm)	1.96±0.05	2.11±0.07	2.07±0.07	1.93±0.05

 

Note: Values in the same row with different superscripts are significantly different (P<0.05). HF, fold height; HE, enterocyte height; HMV, microvillus height. Fold height was analyzed in a lower magnification of objective lens of microscope (magnification ×100); enterocytes height and microvilli height were analyzed in a higher magnification of objective lens of microscope (magnification ×200).

 

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 leucine- aminopeptidase (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 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.

ACKNOWLEDGMENTS

This study was financially supported by a grant from Alltech–Ocean University of China Research Alliance, China Agriculture Research System (CARS-47) and the applied research project for Qingdao postdoctoral researchers (Grant No.: 861605040091). The authors thank Renlei Ji, Wei Ren and Jingqi Li for their help in feeding and sampling.

Statement of conflict of interest

The authors have declared no conflict of interest.

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