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Effects of dietary phospholipid levels on growth performance, fatty acid composition and antioxidant responses of Dojo loach Misgurnus anguillicaudatus larvae
Aquaculture 426–427 (2014) 304–309
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Effects of dietary phospholipid levels on growth performance, fatty acid composition and antioxidant responses of Dojo loach Misgurnus anguillicaudatus larvae Jian Gao a, Shunsuke Koshio b, Weimin Wang a,c, Yang Li a, Songqian Huang a, Xiaojuan Cao a,c,⁎ a College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China b Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056, Japan c Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, Hubei, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 13 February 2014 Accepted 14 February 2014 Available online 25 February 2014 Keywords: Misgurnus anguillicaudatus Larvae Growth Fatty acid composition Antioxidant enzyme activities
a b s t r a c t A 30-day feeding trial was conducted to determine the effects of different levels of dietary phospholipids (PLs) on growth performance, fatty acid composition and antioxidant enzyme activities of loach Misgurnus anguillicaudatus larvae. After 15 days post-hatching, larvae were fed five isoproteic and isolipidic formulated diets with different levels of PLs: 0% (PLs0.8), 2% (PLs1.6), 4% (PLs1.9), 6% (PLs2.3) and 8% (PLs3.0). Results showed that dietary PLs supplementation significantly improved survival and growth of larvae. Survival rate (SR) and body weight gain (BWG) in larvae fed the PLs2.3 diet were significantly higher than those fed PLs0.8, PLs1.6 and PLs1.9 diets, and no differences on SR and BWG were observed between PLs2.3 and PLs3.0 groups, indicating that a supplement of more than 2.3 g 100 g−1 of PLs in diet was required for loach larvae. Dietary PLs supplementation significantly increased fat content and the ratio of neutral lipid (NL)/polar lipid (PL) in the whole body. Concentrations of 20:5n-3 and total n-3 fatty acids in the whole body significantly increased in NL and significantly decreased in PL with incremental dietary PLs levels. Dietary PLs supplement significantly increased superoxide dismutase activity and reduced activities of catalase and glutathione peroxidase in the whole body. These results indicated that supplementation of more than 2.3 g 100 g−1 of PLs in diet could improve growth and survival of loach larvae. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dojo loach or weather fish Misgurnus anguillicaudatus is one of the most important cultured freshwater fish in several East Asian countries, including China, Korea, and Japan. Recently, market demand for this species has increased as a result of increased human consumption. However, the supply of wild loach has been declining due to water pollution and overfishing (Gao et al., 2012). On the other hand, cultured loach suffers high mortality during the early development stages which has completely inhibited the development of loach culture. Although several works have tried to use microparticle diets for the replacement of live prey feed for loach larvae culture (Wang et al., 2008, 2009), no research on the nutritional requirements for this species has been carried out yet. Several studies have documented that supplementation of phospholipids (PLs) in diet is needed for good survival and growth of fish larvae
⁎ Corresponding author at: No.1 Shizishan Stress, Hongshan District, Wuhan 430070, Hubei Province, China. Tel.: +86 27 87282113. E-mail address: [email protected] (X. Cao).
http://dx.doi.org/10.1016/j.aquaculture.2014.02.022 0044-8486/© 2014 Elsevier B.V. All rights reserved.
(Geurden et al., 1995; Hamza et al., 2008; Kanazawa et al., 1981, 1983; Zhao et al., 2013). PLs play a major role in maintaining the structure and function of cellular membranes (Kanazawa, 1985). Moreover, fatty acids play a crucial role in membrane structure (Tocher, 2003). It has been reported that dietary PLs supplementation influences the fatty acid compositions of fish tissues (Hamza et al., 2008; Zhao et al., 2013). In addition to that, the n-3 and n-6 series of PUFA are important nutrients for the freshwater fish species; and 18:2n-6 and 18:3n-3 can be converted to the long chain n-6 and n-3 highly unsaturated fatty acids (HUFA), respectively. Therefore, knowledge of the effects of dietary PLs supplementation on fatty acid composition of fish during the early life stages is needed. The antioxidant system of aerobic organisms prevents the deleterious effects of reactive oxygen species (ROS), playing an important role in protecting cells against oxidative stress, preventing or repairing oxidative damage. The antioxidant system involves several enzymes such as superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx) that act in detoxifying the ROS generated. The activities of antioxidant enzymes have been used as effective biomarkers to evaluate the effects of dietary lipid on the biochemical pathways and enzymatic function in many fish species (Mourente et al., 2002).
J. Gao et al. / Aquaculture 426–427 (2014) 304–309
PLs is important for fish during the early life stages. No information is available on the physiological effects of PLs and optimal dietary levels of PLs for loach M. anguillicaudatus larvae. Therefore, the present study was conducted to investigate the effects of different levels of dietary PLs on growth performance, fatty acid composition and antioxidant enzyme activities of loach larvae.
Table 2 Lipid composition of test diets.a
Lipid composition NLb (% of total lipid) PLb(% of total lipid) NL/PL a b
2. Materials and methods
305
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
91.8 8.2 11.2
83.8 16.2 5.2
81.5 18.5 4.4
77.1 22.9 3.4
70.3 29.7 2.4
Values are means of triplicate measurements. NL, Neutral lipid; PL, Polar lipid.
2.1. Experimental diets
2.2. Fish and sampling procedures
The formulation of the experimental diets is presented in Table 1. The formulation of the test diet in this study was designed according to un-publication data in our laboratory. A supplement of more than 40% of crude protein and 10% of crude lipid could respectively meet the protein and lipid requirements for loach juvenile. The test diets were prepared in the Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan. Five diets were formulated to be isonitrogenous and isolipidic to contain 40.8% crude protein and 10.5% crude lipid, respectively. Defatted fish meal and casein were the main protein. The lipid sources were pollock liver oil, soybean lecithin and palm oil. The PLs levels in test diets were obtained by adding a PLs source of soybean lecithin at 0%, 2%, 4%, 6% and 8% diets. The analyzed PLs levels were 0.8% (PLs0.8), 1.6% (PLs1.6), 1.9% (PLs1.9), 2.3% (PLs2.3) and 3.0% (PLs3.0) of the diets. Palm oil was used to keep the diets isolipidic. Microbound diets for larvae were prepared according to the methods of our previous study (Gao et al., 2013). The lipid composition and both neutral lipid (NL) and polar lipid (PL) fractions of fatty acid compositions of the test diets are shown in Tables 2 and 3. All test diets were stored at − 20 °C prior to feeding trail.
The wild-adult Dojo loaches were collected from waters near Ezhou City, Hubei province in China. All loaches were subjected to flow cytometry to estimate their ploidy levels. Ovulation and spermiation were induced by the injection of human chorionic gonadotropin (15 IU/g body weight, Ningbo Renjian Pharmaceutical Co. Ltd, Ningbo City, China). After rearing at 28 °C for 12 h, the fish were anesthetized in 0.1% MS222 (Sigma) and the gametes were collected according to the methods described by Morishima et al. (2011). Loach sperms were added to eggs and were mixed well. The fertilized eggs of loach were incubated at temperatures of 25–28 °C. The feeding regimes of the stock larvae were as follows: larvae aged days 1–4 were fed exclusively with rotifer, and at days 4–10 they were fed with artemia. Then commercial microbound diets (crude protein, 52%; crude lipid, 8.9%, Ayusoft A, Nosan Corporation Co., Ltd. Yokohama, Japan) were gradually introduced prior to the start of the feeding trial to ensure that larvae could easily adjust to the microbound test diets. From day 15 after hatching, the feeding trial was initiated. Larvae with an average initial body weight of 10 mg were stocked in a 50 L tank (40 L water volume) with a flow-through system at a stocking density of 100 larvae/tank with triplicate. Feeding was done four times per day at 0800, 1200, 1600, and 2000 (ad libitum). Diet particle sizes were adjusted according to fish mouth sizes, based on visual observation. Uneaten diets were collected and dried to determine feed intake (FI). The supply of fresh water to the tanks was 0.1 L/min and a photoperiod of 12 h light/12 h dark was maintained throughout the feeding trail. The water temperature was 24.7 ± 0.5 °C (mean ± sd) throughout the trial. After 30 days, all larvae were fasted for 24 h prior to the final sampling. Larvae were sampled for weight and length. Body weights were measured by weighing (wet weight, blotted dry) with an electronic microbalance with the same fish measured for total length with a vernier caliper. Ten fish from each replicate tank were randomly collected and stored at −20 °C for final whole body analysis. Ten fish were collected from each replicate tank, pooled and stored at −80 °C until analysis for antioxidant enzyme activities. The rest of the larvae from each tank were immediately freeze dried, pooled and stored at −80 °C fatty acid composition analysis.
Table 1 Formulation (%) and proximate composition (%) of the experimental diets.
Ingredient Fish meal (defatted)a Casein Zein Starch Dextrin α-Cellulose Fish oilb Palm oil Soybean lecithinc Attractantsd Vitaminse Mineralsf VCg Proximate composition Moisture Total lipid (dry mass) Crude ash (dry mass) Crude protein (dry mass) a
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
30 30 10 5 5 0.5 2 8 0 1 4 4 0.5
30 30 10 5 5 0.5 2 6 2 1 4 4 0.5
30 30 10 5 5 0.5 2 4 4 1 4 4 0.5
30 30 10 5 5 0.5 2 2 6 1 4 4 0.5
30 30 10 5 5 0.5 2 0 8 1 4 4 0.5
8.1 10.5 9.2 39.8
7.1 10.2 10.8 42.3
7.1 10.7 9.5 40.1
7.5 10.8 10.4 40.5
7.2 10.4 10.1 41.1
Nippon Suisan Co. Ltd., Tokyo, Japan. Riken Vitamin, Tokyo, Japan. c Wako Pure Chemical Ind., Osaka, Japan. Phospholipid concentration is 76.6%. d Attractants (g/kg diet): Taurine, 5; Betain, 4; inosine-5-monophoshate, 1. e Vitamin mixture (mg/kg diet): β-Carotene 96.26; Vitamin D3 9.68; Menadione NaHSO 3 ·3H 2 O (K 3 ) 45.83; Thiamine–Nitrate (B 1 ) 57.75; Riboflavin (B 2 ) 192.37; Pyridoxine–HCl (B6) 45.83; Cyanocobalamine (B12) 0.07; d-Biotin 5.78; Inositol 3212.83; Niacine (Nicotic acid) 769.73; Ca Panthothenate 269.49; Folic acid 14.40; Choline choloride 7869.30; ρ-aminobenzoic acid 383.25. f Mineral mixture (mg kg− 1 diet): MgSO4 5070; Na2HPO4 3230; K2HPO4 8870; Fe Citrate 1100; Ca Lactate12090; Al (OH)3 10; ZnSO4 130; CuSO4 4; MnSO4 30; Ca (IO3)2 10; CoSO4 40. g L-Ascorbyl-2-monophosphate (AMP-Na/Ca, DSM Nutrition Japan K.K.). b
2.3. Analysis The proximate composition of the test diets and whole body samples was analyzed by following A.O.A.C. (1990). Moisture for diets was determined by drying the sample at 120 °C to constant weight. The fish samples were freeze dried (Freezone 2.5, Labconco, Co., Ltd. USA) prior to proximate composition analysis. Loss in weight represented whole body moisture content. The Kjeldahl method was used to determine nitrogen levels, and crude protein was calculated by multiplying by 6.25. The ash was determined by combustion at 550 °C in a muffle furnace. Total lipid was measured following the method of Bligh and Dyer (1959) and further separated into NL and PL fractions by column chromatography (Sep-Pak Silica Cartridges; Waters Corp. Milford, MA) according to Juaneda and Rocquelin (1985). The fatty acid composition of both NL and PL fractions of test diets and whole body samples was determined using gas chromatography (Agilent Technologies Inc., Santa Clara, California, USA; column: OmegawaxTM320) according
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J. Gao et al. / Aquaculture 426–427 (2014) 304–309
Table 3 Fatty acid compositions (% total fatty acids) of NL and PL fractions of test diets.a Fatty acid
14:0 16:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σn-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σn-3 fatty acids a b
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
NLb
PLb
NL
PL
NL
PL
NL
PL
NL
PL
4.4 34.1 6.7 45.2 4.6 31.5 3.2 2.1 41.4 5.1 0.2 0.2 5.5 0.4 0.1 0.4 1.11 0.5 1.8 4.31
2.6 35.1 5.4 43.1 4.6 21.54 2.3 1.3 29.74 4.5 0.5 2.1 7.1 0.5 0.2 0.5 8.1 0.5 7.9 17.7
4.2 34.5 6.73 45.43 4.8 32.6 3.2 2.4 43 5.8 0.3 0.2 6.3 0.5 0.2 0.5 1.4 0.6 1.9 5.1
2.4 29.5 5.3 37.2 4.8 22.35 2.4 1.2 30.75 9.6 0.4 2.2 12.2 0.5 0.4 0.4 7.85 0.6 8.13 17.88
4.1 32.1 6.5 42.7 4.5 30 3.4 2.3 40.2 5.9 0.2 0.3 6.4 0.3 0.2 0.5 1.12 0.4 1.7 4.22
2.3 25.1 5.1 32.5 4.5 22.56 2.3 1.5 30.86 13.7 0.6 2 16.3 0.4 0.5 0.5 7.98 0.4 8.14 17.92
3.9 32.8 6.8 43.5 4.7 30.5 3.5 2.2 40.9 6.4 0.2 0.2 6.8 0.3 0.2 0.5 1.31 0.5 1.8 4.61
2.5 22.8 5.2 30.5 4.7 21.98 2.3 1.3 30.28 15.3 0.7 2.1 18.1 0.5 0.5 0.65 8.23 0.5 7.98 18.36
3.5 31.2 6.4 41.1 4.9 34.5 3.4 2.3 45.1 6.8 0.2 0.4 7.4 0.4 0.1 0.5 1.17 0.5 1.8 4.47
2.0 20.2 5 27.2 4.9 23.04 2.2 1.2 31.34 17.5 0.6 2.3 20.4 0.5 0.3 0.5 8.12 0.5 7.91 17.83
Values are means of triplicate measurements. NL, Neutral lipid; PL, Polar lipid.
3. Result
to the method of Teshima et al. (1986a). The activities of antioxidant enzymes (CAT, SOD and GPx) of whole body larvae were all analyzed spectrophotometrically using diagnostic reagent kits (Nanjing Jiancheng Bioengineering Institute, China). Whole body samples were homogenized in ice-cold, 0.65% physiological saline using a tissue homogenizer. The supernatant was used to determine SOD (EC 1.15.1.1), CAT (EC 1.11.1.6) and GPx (EC 1.11.1.9), SOD activity was assayed according to a SOD kit protocol (No. A001) at 550 nm according to Bayer and Fridovich (1987). CAT activity was assayed according to a CAT kit protocol (No. A007) at 405 nm according to Claiborne (1985). GPx activity was assayed according to a GPx kit protocol (No. A005) at 412 nm according to Wheeler et al. (1990).
Growth performance of loach fed test diets for 30 days is shown in Table 4. The final weight (FW), body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) significantly increased and feed conversion ratio (FCR) significantly decreased with dietary PLs increasing from 0.8 to 2.3%. However, there were no significant differences in these parameters (FW, BWG, SGR, FCR and SR) between the PLs2.3 and PLs3.0 groups. Feed intake (FI) was not affected by dietary PLs levels.
2.4. Statistical analysis
3.2. Lipid composition and proximate of whole body
All data were subjected to Levene's test of equality of error variances and one-way ANOVA followed by Tukey's test using SPSS 16.0 (SPSS 16.0, Michigan Avenue, Chicago, IL, USA). Probability values of b0.05 were considered statistically significant.
The whole body NL fraction significantly increased with increasing dietary PLs levels (Table 5). The highest whole body NL fraction was observed in larvae fed the PLs2.3 diet. However, whole body PL fraction decreased with incremental dietary PLs levels. Larvae fed the PLs0.8
3.1. Growth performance
Table 4 Growth performance of loach larva fed test diets for 30 days. PLs0.8 Final weight (mg) BWG1 (%) SGR2 (%) FCR3 SR4 (%) FI5 (g/fish)
84.6 605.3 6.5 4.9 33.7 0.4
± ± ± ± ± ±
PLs1.6 a
6.6 55.3a 0.3a 0.8b 12.1a 0.02
116.8 873.3 7.6 3.7 47.7 0.4
± ± ± ± ± ±
PLs1.9 a
15.2 126.5a 0.4a 1.0ab 5.9a 0.03
112.8 840.3 7.5 3.7 50.7 0.4
± ± ± ± ± ±
PLs2.3 a
10.3 86.2a 0.3a 0.8ab 3.5ab 0.04
152.3 1168.9 8.5 2.3 67.3 0.3
PLs3.0 ± ± ± ± ± ±
b
16.4 137.0b 0.4b 0.3a 3.8b 0.01
164.3 1268.9 8.7 2.4 72.0 0.4
± ± ± ± ± ±
10.7b 89.2b 0.2b 0.3a 5.3b 0.02
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments. 1 BWG, body weight gain (%) = 100 × (final weight − initial weight) / (initial weight). 2 SGR, specific growth rate = 100 × (ln final weight − ln initial weight) / (duration). 3 FCR, feed conversion ratio = dry feed intake (g) / weight gain (g). 4 SR, survival rate (%) = 100 × (initial fish number − dead fish number) / (initial fish number). 5 FI, feed intake (g/fish) = (total feed intake (g) / number of fishes) in 30 days' feeding period.
J. Gao et al. / Aquaculture 426–427 (2014) 304–309
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Table 5 Whole body proximate analysis (%) of loach larva fed with different diets for 30 days. PLs0.8 Total lipid (dry mass) NL1 (% of total lipid) PL1 (% of total lipid) NL/PL Moisture Crude ash (dry mass) Crude protein (dry mass)
12.8 56.7 43.4 1.3 83.7 11.0 69.1
PLs1.6
± ± ± ± ± ± ±
0.3a 1.9a 1.9b 0.1a 2.5 0.7 2.6
13.7 65.5 34.5 1.9 84.3 9.7 71.1
± ± ± ± ± ± ±
PLs1.9 0.3a 1.8b 1.8a 0.2ab 1.8 0.2 1.7
13.0 68.3 31.7 2.2 83.8 9.7 72.3
± ± ± ± ± ± ±
PLs2.3 1.1a 1.5b 1.5a 0.2b 0.7 0.2 1.4
13.8 70.0 30.0 2.4 82.9 9.7 72.9
± ± ± ± ± ± ±
PLs3.0 0.4a 5.5b 5.5a 0.6b 1.1 0.3 0.5
15.5 66.6 33.4 2.0 82.0 9.9 71.5
± ± ± ± ± ± ±
0.3b 1.6b 1.6a 0.1ab 0.5 0.6 1.0
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments. 1 NL, Neutral lipid; PL, Polar lipid.
diet had significantly higher PL fraction than those fed other diets. The ratio of NL/PL fraction of whole fish body significantly increased with the increase of dietary PLs from 0.8 to 2.3%. Whole body proximate composition of larvae is also reported in Table 4. A significant effect of dietary PLs levels was found on whole body total lipid content at the end of the feeding trial, while whole body moisture, protein and ash content were unaffected by dietary PLs levels. Larvae fed the PLs3.0 diet had significantly higher fat content than those fed other diets.
3.4. Antioxidant enzyme activities Activities of antioxidant enzymes of larvae are presented in Table 8. SOD activity significantly increased with incremental dietary PLs level. However, the activities of CAT and GPx were significantly decreased by dietary PLs. The highest CAT and GPx activities were observed in larvae fed the PLs0.8 diet, which was significantly higher than the other groups. 4. Discussion
3.3. Fatty acid compositions Fatty acid compositions of NL and PL in whole body larvae were reflected by dietary fatty acid compositions (Tables 6 and 7). In NL fraction, dietary PLs supplementation significantly increased the percentages of 18:1n-9, total monoene fatty acids, 20:5n-3 (Eicosapentaenoic acid, EPA) and total n-3 fatty acids and decreased 16:0, 17:0, 18:0, 16:1n-7 and total saturated fatty acids. In terms of PL fraction, dietary PLs supplementation significantly increased 18:1n-9 and total monoene fatty acids and decreased 16:1n-7, 22:1n-11, 18:3n-3, 18:4n-3, EPA, 22:6n-3 (Docosapentaenoic acid, DHA) and total n-3 fatty acids. In a comparison between the NL and PL fraction fatty acid compositions, the percentage of 18:2n-6, 18:3n-3, 18:4n-3, EPA and DHA was higher in NL than in PL fraction of whole body of larvae.
The importance of supplementation of dietary PLs on optimal growth has been well studied not only in crustaceans such as Vannamei Litopenaeus vannamei (Niu et al., 2011), crayfish Cherax quadricarinatus (Thompson et al., 2003) and Kuruma shrimp Penaeus japonicus (Teshima et al., 1986a, 1986b, 1986c), but also in both marine and freshwater species including large yellow croaker Larmichthys crocea (Zhao et al., 2013), amberjack Seriola dumerili (Uyan et al., 2009), rainbow trout Oncorhynchus mykiss (Poston, 1990, 1991) and pikeperch Sander lucioperca (Hamza et al., 2008), ayu Plecoglossus altivelis (Kanazawa et al., 1985), the common carp Cyprinus carpio (Geurden et al., 1995). In this study, BWG, SGR and SR significantly increased from 605.3% to 1168.9% with increasing dietary PLs levels from 0.8 to 2.3%, when dietary PLs content exceeded 2.3 mg 100 g− 1 diet (PLs2.3), the BWG
Table 6 Fatty acid composition (%) in NL fraction of whole body of loach larvae fed test diets for 30 days.
Table 7 Fatty acid composition (%) in PL fraction of whole body of loach larvae fed test diets for 30 days.
Fatty acid 14:0 16:0 17:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σn-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σn-3 fatty acids
PLs0.8 2.2 29.7 0.5 0.9 33.2 9.9 44.5 0.6 0.9 56.2 3.2 0.3 0.1 3.6 0.1 0.3 0.4 1.9 0.1 0.2 3.0
PLs1.6 2.0 28.0 0.4 0.5 30.9 6.0 50.1 0.6 1.0 58.0 3.4 0.2 0.1 3.7
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.4c 0.0b 0.2ab 0.3cd 0.2ab 2.9b 0.1 0.1 2.7a 0.3 0.1 0.1 0.3
1.9 27.6 0.2 0.4 30.1 7.1 49.7 0.8 0.7 58.6 3.4 0.2 0.1 3.7
0.0 0.1 0.0 0.1a 0.1 0.1 0.3a
0.2 0.4 0.3 2.1 0.1 0.4 3.5
± ± ± ± ± ± ±
0.0 0.0 0.2 0.1a 0.1 0.1 0.2ab
0.2 0.4 0.6 2.1 0.1 0.3 3.6
± 0.0 ± 1.2c ± 0.1b ± 0.1b ± 1.3d ± 0.6c ± 1.7a ± 0.5 ± 0.6 ± 0.9a ± 0.1 ± 0.1 ± 0.0 ± 0.3 ± ± ± ± ± ± ±
PLs1.9
c
b
PLs2.3 b
± 0.1 ± 1.2c ± 0.1a ± 0.1a ± 1.2c ± 0.4b ± 0.6ab ± 0.2 ± 0.2 ± 0.7a ± 0.4 ± 0.1 ± 0.1 ± 0.4 ± ± ± ± ± ± ±
0.1 0.2 0.2 0.1ab 0.1 0.1 0.4ab
1.6 ± 24.8 ± 0.2 ± 0.3 ± 27.0 ± 5.6 ± 55.4 ± 0.8 ± 0.5 ± 62.6 ± 3.8 ± 0.5 ± 0.2 ± 4.4 ± 0.2 0.4 0.5 2.5 0.1 0.3 4.3
± ± ± ± ± ± ±
PLs3.0 a
a
0.0 0.6b 0.0a 0.2a 0.5b 0.6a 1.4bc 0.0 0.0 0.9b 0.2 0.2 0.0 0.3
1.5 ± 21.7 ± 0.1 ± 0.4 ± 23.7 ± 5.7 ± 56.9 ± 0.7 ± 0.5 ± 64.3 ± 3.8 ± 0.7 ± 0.2 ± 4.5 ±
0.1 1.5a 0.0a 0.1a 1.5a 0.5a 2.7c 0.1 0.0 2.2b 0.2 0.3 0.0 0.2
0.1 0.2 0.2 0.1b 0.0 0.1 0.2b
0.1 ± 0.4 ± 0.5 ± 2.4 ± 0.1 ± 0.3 ± 3.8 ±
0.0 0.1 0.1 0.2ab 0.0 0.1 0.4ab
Values are expressed as mean ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
Fatty acid
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
14:0 16:0 17:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σ n-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σ n-3 fatty acids
0.5 ± 0.0 23.0 ± 0.9a 0.6 ± 0.2 0.2 ± 0.0 24.2 ± 0.9a 5.7 ± 0.1d 36.2 ± 0.6a 3.0 ± 0.2a 1.4 ± 0.1d 46.3 ± 0.8a 7.4 ± 0.4 0.5 ± 0.5 0.4 ± 0.2 8.3 ± 0.4
0.4 ± 0.0 23.4 ± 1.3a 0.5 ± 0.0 0.2 ± 0.0 24.5 ± 1.3a 3.7 ± 0.1c 38.6 ± 0.4b 3.4 ± 0.0b 1.0 ± 0.1bc 46.0 ± 0.6a 8.3 ± 0.4 0.7 ± 0.0 0.5 ± 0.1 9.5 ± 0.5
0.6 ± 0.1 25.7 ± 0.6b 0.9 ± 0.2 0.3 ± 0.0 27.9 ± 1.4b 3.6 ± 0.1bc 38.8 ± 0.3b 3.5 ± 0.1b 0.9 ± 0.1b 46.8 ± 0.3a 8.1 ± 0.3 0.5 ± 0.2 0.4 ± 0.1 9.0 ± 0.5
0.5 ± 0.1 26.8 ± 0.5b 1.3 ± 0.2 0.2 ± 0.0 28.8 ± 0.8b 3.1 ± 0.5ab 41.8 ± 1.3c 3.0 ± 0.0a 0.7 ± 0.1ab 48.6 ± 0.9b 8.3 ± 0.3 0.3 ± 0.2 0.2 ± 0.1 8.8 ± 0.4
0.5 ± 0.0 22.0 ± 0.4a 0.8 ± 0.3 0.2 ± 0.0 23.4 ± 0.2a 2.7 ± 0.2a 43.3 ± 0.1c 2.9 ± 0.1a 0.7 ± 0.1a 49.6 ± 0.3b 8.4 ± 0.5 0.8 ± 0.3 0.6 ± 0.6 9.8 ± 1.4
1.8 ± 0.1c 2.9 ± 0.4c 0.5 ± 0.3 11.0 ± 0.4b 0.6 ± 0.2 1.1 ± 0.1b 17.9 ± 0.6c
1.7 ± 0.0c 1.9 ± 0.0b 0.3 ± 0.2 11.4 ± 2.0b 0.6 ± 0.2 0.9 ± 0.1ab 16.8 ± 2.7bc
1.4 ± 0.2b 1.5 ± 0.2ab 0.5 ± 0.2 9.0 ± 0.2ab 0.6 ± 0.2 0.8 ± 0.2ab 13.8 ± 0.5ab
1.1 ± 0.1ab 1.3 ± 0.1a 0.3 ± 0.2 8.5 ± 0.3a 0.8 ± 0.2 0.6 ± 0.3a 12.7 ± 0.3a
1.1 ± 0.1a 1.4 ± 0.1a 0.4 ± 0.3 9.2 ± 0.5ab 0.6 ± 0.2 0.7 ± 0.2a 13.4 ± 0.1a
Values are expressed as mean ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
308
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Table 8 Activities of antioxidant enzymes (U/mg prot) of loach larva fed test diets for 30 days.
Catalase (CAT) Superoxide dismutase (SOD) Glutathione peroxidase (GPx)
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
16.1 ± 1.5b 5.6 ± 0.1a 206.4 ± 6.4c
9.1 ± 0.7a 6.8 ± 0.2b 169.8 ± 6.6b
7.8 ± 0.4a 6.9 ± 0.1b 165.9 ± 6.6b
6.4 ± 0.4a 7.0 ± 0.9b 182.0 ± 11.1bc
7.3 ± 0.5a 6.9 ± 0.1b 134.7 ± 13.7a
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
reached a plateau, suggesting that 2.3% of dietary PLs supplementation could maintain the normal physiological function and survival for loach larvae. Our results showed that SGR of larvae fed with PLs2.3 and PLs3.0 reached more than 8.5 from day 15 to day 45 after hatching. This value was similar with Wang et al.'s (2009), who reported that SGR of loach larvae fed microparticle diets was about 9.0 from day 20 to day 60 after hatching. However, the SGR of loach fed microparticle diets was lower than those fed live gray like cladocerans (SGR, 11.9). For successful aquaculture, it is important to know the nutritional requirements for loach larvae. In this study, SR significantly increased from 33.7% to 72.0% with incremental dietary PLs level from 0.8% to 2.3%. A significant effect of dietary PLs supplementation on SR in fish larvae has also been demonstrated in a number of studies (Azarm et al., 2013; Hamza et al., 2008; Zhao et al., 2013). In contrast to that, Uyan et al. (2009) demonstrated that SR of juvenile amberjack was not influenced with increasing levels of dietary PLs. This suggested that PLs were essential to maintain the normal physiological function and survival for only the early development stage of fish. No effects of dietary PLs supplementation on growth at the early juvenile stage have been reported by Coutteau et al. (1997) and Dapra et al. (2011). Dietary PLs are considered as structural lipids that are constituents of cell membranes (Kanazawa, 1985). Thus, dietary PLs are believed to be extremely crucial for larval development in fish. The stimulatory effect of PLs on growth can be explained in different ways (Azarm et al., 2013). Geurden et al. (1995) reported that the stimulating effects of PLs on larval fish growth were due to the fish larvae having a limited ability to biosynthesize phospholipids de novo. In a review, Tocher et al. (2008) demonstrated that diets rich in triacylglycerol and a lack of sufficient dietary PLs could limit lipoprotein synthesis in enterocytes, leading to impaired transport of lipid nutrients to tissues. The stimulating effect of PLs on growth is due to improved transport, assimilation and utilization of dietary lipid. In the present experiment, whole body total lipid content was significantly increased with increasing dietary PLs supplementation. This was in agreement with some previous studies. Uyan et al. (2009) reported that whole body fat content of juvenile amberjack significantly increased with increasing dietary PLs supplementation. Moreover, Zhao et al. (2013) found that whole body fat content of large yellow croaker larvae significantly increased with incremental dietary PLs level from 26.0 g/kg to 85.1 g/kg. The inclusion of dietary PLs influenced lipid deposition of the whole body, resulting in increased lipid retention in the animal (Teshima et al., 1986c). Based on results from the present study, dietary PLs significantly increased the percentage of NL fraction and significantly decreased the percentage of PL fraction. The increase in the ratio of NL/PL might be due to an increase in NL fraction in whole body total lipid content. The likely reasons are related to the following two aspects. The first one is that PL is more well utilized than NL for fish larvae. It is generally believed that PL as main lipid component in PLs plays a crucial role in membrane structure during early life stages. The other is likely that supplementation of dietary PLs promoted incorporation of NL in the tissues (Kanazawa et al., 1985). In contrast, Zhao et al. (2013) found that the ratio of NL/PL of large yellow croaker larvae whole body fat content decreased with incremental dietary PLs level. In the present study, we found that the percentages of DHA in both NL (0.2–0.4%) and PL (0.6–1.1%) of larvae whole body were significantly lower compared with the results from our previous study (Gao et al.,
2012), showing that the percentages of DHA in wild and cultured loach whole body were 6.7% and 10.9%, respectively. Poor DHA concentration may be explained by a lack of essential fatty acid (EFA) concentration in diet. Since n-3 fatty acids can be considered to be the most limiting EFA (Sargent et al., 1999), adequate amounts of fish oil must be incorporated in the diets to cover the EFA requirements, as fish oils are the only dietary source of n-3 HUFA (Sargent et al., 2002). It has been reported that the fatty acid composition of fish tissues was influenced by fatty acid composition of diet. Relatively low DHA concentration of fish tissues indicated in some extent EFA deficiency, which has been reported in several studies (Izquierdo et al., 2005; Ng et al., 2013; Xue et al., 2006). Although there is no information about the EFA requirement of this species, our results indicated that 2% of fish oil in diet did not meet the EFA requirements of loach larvae. Several studies have reported that freshwater fish has capability to elongate and desaturate n-3 and n-6 PUFA (C18) chains to the n-3 and n-6 HUFA (C20 and C22) chains (Bell et al., 1986; Sargent et al., 1999). The main effect of dietary PLs supplementation on larval fatty acid composition was an increase in the percentage of n-6 fatty acid like 18:2n-6 and total n-6 fatty acids in both NL and PL fractions (Hamza et al., 2008). In this study, there were no significant differences in n-6 fatty acids like 18:2n-6 and total n-6 fatty acids in both NL and PL fractions among different treatment groups. However, EPA and total n-3 fatty acids in NL fraction of larvae significantly increased with incremental dietary PLs level. Since all diets were added the same level of fish oil, the increase n-3 HUFA could indicate that loach can convert 18: 2n-6 to the long chain n-6 fatty acids. This was in agreement with the result of Hamza et al. (2008) who demonstrated that a relatively high level of n-3 HUFA in pikeperch Sander lucioperca larvae fed dietary 9% of PLs supplementation contrasting with their low level in the diet. Moreover, Azarm et al. (2013) observed that the percentages of HUFA especially EPA + DHA were higher in rainbow trout O. mykiss fed soybean lecithin diets than those fed soybean lecithin un-supplementation group. On the other hand, results from the present study showed that n-3 fatty acids in PL fraction such as EPA, DHA and total n-3 HUFA decreased with incremental dietary PLs levels, suggesting that dietary PLs supplementation may promote utilization of EPA and DHA efficiently in PL fraction for loach larvae. HUFAs are vital constituents for cell membrane structure and function in fish larvae (Tocher, 2003). It has been reported that fish larvae can more efficiently use n-3 HUFA from the PL fraction than those from the NL fraction (Cahu et al., 2003; Gisbert et al., 2005; Hamza et al., 2008). The present results also confirmed that a high content of n-3 fatty acids was in the PL fraction rather than in the NL fraction in larvae. Dietary PLs supplementation could enhance stress resistance and induce anti-oxidant responses to protect an organ against oxidative damage (Hamza et al., 2008; Kanazawa, 1997; Zhao et al., 2013). The antioxidant defense system of organisms provides a means of dealing with oxidative stress, refers to the disturbance of the equilibrium between antioxidants and pro-oxidants towards oxidants and includes several enzymes and vitamins. In this study, dietary PLs supplementation induced SOD actively in larvae. It is known that SOD value can be believed to be a more reliable and expressive set of oxidative stress indicator. High SOD activity in the whole body might be a consequence of free radicals derived from oxidation of lipids (Zhang et al., 2009). On the contrary, the activities of CAT and GPx showed an opposite tendency with
J. Gao et al. / Aquaculture 426–427 (2014) 304–309
SOD activity in this study, indicating that PLs supplementation might inhibit activities of CAT and GPx in loach larvae. Although the present results indicated that dietary PLs influenced larvae antioxidant enzyme activities, it was difficult to explain the reasons for the changes in activities of CAT, SOD and GPx in whole body of larvae since the activities of antioxidant enzymes in fish always showed different patterns among different organs (Zhang et al., 2009) and growth time (Mourente et al., 2002). The activities of antioxidant enzymes in different organs of fish need to be studied in the future. 5. Conclusions In summary, this is the first study that evaluated a nutritional requirement of loach at their larval stage. Results showed that a supplementation more than 6% of soybean lecithin in diet (2.3 g 100 g−1 of PLs in diet) could improve growth performance and survival of loach larvae. Dietary PLs supplementation increased fat content and the ratio of NL/PL in whole body of larvae. An increase in the percentage of total n-3 fatty acids in NL fraction was found along with increasing dietary PLs levels, suggesting that loach larvae had the capability to elongate n-6 PUFA chains to n-6 HUFA. The dietary requirement of HUFA for loach larvae needs to be researched in future. Acknowledgments The author would like to thank the Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan, for providing the test diets in this study. This study was supported by the National Natural Science Foundation of China (Grant Nos.: 31372180, 34113053 and 31201719), and partly by the Fundamental Research Funds for the Central Universities of China (2013PY074). We are very grateful to three anonymous reviewers for their constructive suggestions and comments. References A.O.A.C, 1990. Official Methods of Analysis of the Association of Official Analytical Chemists, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Azarm, H.M., Kenari, A.A., Hedayati, M., 2013. Effect of dietary phospholipid sources and levels on growth performance, enzymes activity, cholecystokinin and lipoprotein fractions of rainbow trout (Oncorhynchus mykiss) fry. Aquac. Res. 44, 634–644. Bayer, W.F., Fridovich, J.L., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Chem. 161, 559–566. Bell, M.V., Henderson, R.J., Sargent, J.R., 1986. The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol. 83B, 711–719. Bligh, E.G., Dyer, W.J., 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cahu, C.L., Jambonino Infante, J.L., Barbosa, V., 2003. Effect of dietary phospholipid level and phospholipid: neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet. Br. J. Nutr. 90, 21–28. Claiborne, A., 1985. Catalase activity, In: Greenwald, R.A. (Ed.), CRC Handbook of Methods in Oxygen Radical Research, 2nd edn. CRC Press, Boca Raton, FL, pp. 283–284. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P., 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155, 149–164. Dapra, F., Geurden, I., Corraze, G., Bazin, D., Zambonino-Infante, J.L., 2011. Physiological and molecular responses to dietary phospholipids vary between fry and early juvenile stages of rainbow trout (Oncorhynchus mykiss). Aquaculture 319, 377–384. Gao, J., Koshio, S., Nguyen, B.T., Wang, W.M., Cao, X.J., 2012. Comparative studies on lipid profiles and amino acid composition of wild and cultured dojo loach Misgurnus anguillicaudatus obtained from Southern Japan. Fish. Sci. 78, 1331–1336. Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Daisuke, N., Ren, T.J., 2013. Interactive effects of vitamin C and E supplementation on growth, fatty acid composition, and lipid peroxidation of sea cucumber, Apostichopus japonicus, fed with dietary oxidized fish oil. J. World Aquac. Soc. 44, 536–546. Geurden, I., Radunz-Neto, L., Bergot, P., 1995. Essentiality of dietary phospholipids for carp (Cyprinus carpio) larvae. Aquaculture 131, 303–314. Gisbert, E., Villeneuve, L., Zambonino-Infante, J.L., Quazuguel, P., Cahub, C.L., 2005. Dietary phospholipids are more efficient than neutral lipids for long-chain polyunsaturated
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Effects of dietary phospholipid levels on growth performance, fatty acid composition and antioxidant responses of Dojo loach Misgurnus anguillicaudatus larvae Jian Gao a, Shunsuke Koshio b, Weimin Wang a,c, Yang Li a, Songqian Huang a, Xiaojuan Cao a,c,⁎ a College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China b Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056, Japan c Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, Hubei, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 13 February 2014 Accepted 14 February 2014 Available online 25 February 2014 Keywords: Misgurnus anguillicaudatus Larvae Growth Fatty acid composition Antioxidant enzyme activities
a b s t r a c t A 30-day feeding trial was conducted to determine the effects of different levels of dietary phospholipids (PLs) on growth performance, fatty acid composition and antioxidant enzyme activities of loach Misgurnus anguillicaudatus larvae. After 15 days post-hatching, larvae were fed five isoproteic and isolipidic formulated diets with different levels of PLs: 0% (PLs0.8), 2% (PLs1.6), 4% (PLs1.9), 6% (PLs2.3) and 8% (PLs3.0). Results showed that dietary PLs supplementation significantly improved survival and growth of larvae. Survival rate (SR) and body weight gain (BWG) in larvae fed the PLs2.3 diet were significantly higher than those fed PLs0.8, PLs1.6 and PLs1.9 diets, and no differences on SR and BWG were observed between PLs2.3 and PLs3.0 groups, indicating that a supplement of more than 2.3 g 100 g−1 of PLs in diet was required for loach larvae. Dietary PLs supplementation significantly increased fat content and the ratio of neutral lipid (NL)/polar lipid (PL) in the whole body. Concentrations of 20:5n-3 and total n-3 fatty acids in the whole body significantly increased in NL and significantly decreased in PL with incremental dietary PLs levels. Dietary PLs supplement significantly increased superoxide dismutase activity and reduced activities of catalase and glutathione peroxidase in the whole body. These results indicated that supplementation of more than 2.3 g 100 g−1 of PLs in diet could improve growth and survival of loach larvae. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dojo loach or weather fish Misgurnus anguillicaudatus is one of the most important cultured freshwater fish in several East Asian countries, including China, Korea, and Japan. Recently, market demand for this species has increased as a result of increased human consumption. However, the supply of wild loach has been declining due to water pollution and overfishing (Gao et al., 2012). On the other hand, cultured loach suffers high mortality during the early development stages which has completely inhibited the development of loach culture. Although several works have tried to use microparticle diets for the replacement of live prey feed for loach larvae culture (Wang et al., 2008, 2009), no research on the nutritional requirements for this species has been carried out yet. Several studies have documented that supplementation of phospholipids (PLs) in diet is needed for good survival and growth of fish larvae
⁎ Corresponding author at: No.1 Shizishan Stress, Hongshan District, Wuhan 430070, Hubei Province, China. Tel.: +86 27 87282113. E-mail address: [email protected] (X. Cao).
http://dx.doi.org/10.1016/j.aquaculture.2014.02.022 0044-8486/© 2014 Elsevier B.V. All rights reserved.
(Geurden et al., 1995; Hamza et al., 2008; Kanazawa et al., 1981, 1983; Zhao et al., 2013). PLs play a major role in maintaining the structure and function of cellular membranes (Kanazawa, 1985). Moreover, fatty acids play a crucial role in membrane structure (Tocher, 2003). It has been reported that dietary PLs supplementation influences the fatty acid compositions of fish tissues (Hamza et al., 2008; Zhao et al., 2013). In addition to that, the n-3 and n-6 series of PUFA are important nutrients for the freshwater fish species; and 18:2n-6 and 18:3n-3 can be converted to the long chain n-6 and n-3 highly unsaturated fatty acids (HUFA), respectively. Therefore, knowledge of the effects of dietary PLs supplementation on fatty acid composition of fish during the early life stages is needed. The antioxidant system of aerobic organisms prevents the deleterious effects of reactive oxygen species (ROS), playing an important role in protecting cells against oxidative stress, preventing or repairing oxidative damage. The antioxidant system involves several enzymes such as superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx) that act in detoxifying the ROS generated. The activities of antioxidant enzymes have been used as effective biomarkers to evaluate the effects of dietary lipid on the biochemical pathways and enzymatic function in many fish species (Mourente et al., 2002).
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PLs is important for fish during the early life stages. No information is available on the physiological effects of PLs and optimal dietary levels of PLs for loach M. anguillicaudatus larvae. Therefore, the present study was conducted to investigate the effects of different levels of dietary PLs on growth performance, fatty acid composition and antioxidant enzyme activities of loach larvae.
Table 2 Lipid composition of test diets.a
Lipid composition NLb (% of total lipid) PLb(% of total lipid) NL/PL a b
2. Materials and methods
305
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
91.8 8.2 11.2
83.8 16.2 5.2
81.5 18.5 4.4
77.1 22.9 3.4
70.3 29.7 2.4
Values are means of triplicate measurements. NL, Neutral lipid; PL, Polar lipid.
2.1. Experimental diets
2.2. Fish and sampling procedures
The formulation of the experimental diets is presented in Table 1. The formulation of the test diet in this study was designed according to un-publication data in our laboratory. A supplement of more than 40% of crude protein and 10% of crude lipid could respectively meet the protein and lipid requirements for loach juvenile. The test diets were prepared in the Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan. Five diets were formulated to be isonitrogenous and isolipidic to contain 40.8% crude protein and 10.5% crude lipid, respectively. Defatted fish meal and casein were the main protein. The lipid sources were pollock liver oil, soybean lecithin and palm oil. The PLs levels in test diets were obtained by adding a PLs source of soybean lecithin at 0%, 2%, 4%, 6% and 8% diets. The analyzed PLs levels were 0.8% (PLs0.8), 1.6% (PLs1.6), 1.9% (PLs1.9), 2.3% (PLs2.3) and 3.0% (PLs3.0) of the diets. Palm oil was used to keep the diets isolipidic. Microbound diets for larvae were prepared according to the methods of our previous study (Gao et al., 2013). The lipid composition and both neutral lipid (NL) and polar lipid (PL) fractions of fatty acid compositions of the test diets are shown in Tables 2 and 3. All test diets were stored at − 20 °C prior to feeding trail.
The wild-adult Dojo loaches were collected from waters near Ezhou City, Hubei province in China. All loaches were subjected to flow cytometry to estimate their ploidy levels. Ovulation and spermiation were induced by the injection of human chorionic gonadotropin (15 IU/g body weight, Ningbo Renjian Pharmaceutical Co. Ltd, Ningbo City, China). After rearing at 28 °C for 12 h, the fish were anesthetized in 0.1% MS222 (Sigma) and the gametes were collected according to the methods described by Morishima et al. (2011). Loach sperms were added to eggs and were mixed well. The fertilized eggs of loach were incubated at temperatures of 25–28 °C. The feeding regimes of the stock larvae were as follows: larvae aged days 1–4 were fed exclusively with rotifer, and at days 4–10 they were fed with artemia. Then commercial microbound diets (crude protein, 52%; crude lipid, 8.9%, Ayusoft A, Nosan Corporation Co., Ltd. Yokohama, Japan) were gradually introduced prior to the start of the feeding trial to ensure that larvae could easily adjust to the microbound test diets. From day 15 after hatching, the feeding trial was initiated. Larvae with an average initial body weight of 10 mg were stocked in a 50 L tank (40 L water volume) with a flow-through system at a stocking density of 100 larvae/tank with triplicate. Feeding was done four times per day at 0800, 1200, 1600, and 2000 (ad libitum). Diet particle sizes were adjusted according to fish mouth sizes, based on visual observation. Uneaten diets were collected and dried to determine feed intake (FI). The supply of fresh water to the tanks was 0.1 L/min and a photoperiod of 12 h light/12 h dark was maintained throughout the feeding trail. The water temperature was 24.7 ± 0.5 °C (mean ± sd) throughout the trial. After 30 days, all larvae were fasted for 24 h prior to the final sampling. Larvae were sampled for weight and length. Body weights were measured by weighing (wet weight, blotted dry) with an electronic microbalance with the same fish measured for total length with a vernier caliper. Ten fish from each replicate tank were randomly collected and stored at −20 °C for final whole body analysis. Ten fish were collected from each replicate tank, pooled and stored at −80 °C until analysis for antioxidant enzyme activities. The rest of the larvae from each tank were immediately freeze dried, pooled and stored at −80 °C fatty acid composition analysis.
Table 1 Formulation (%) and proximate composition (%) of the experimental diets.
Ingredient Fish meal (defatted)a Casein Zein Starch Dextrin α-Cellulose Fish oilb Palm oil Soybean lecithinc Attractantsd Vitaminse Mineralsf VCg Proximate composition Moisture Total lipid (dry mass) Crude ash (dry mass) Crude protein (dry mass) a
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
30 30 10 5 5 0.5 2 8 0 1 4 4 0.5
30 30 10 5 5 0.5 2 6 2 1 4 4 0.5
30 30 10 5 5 0.5 2 4 4 1 4 4 0.5
30 30 10 5 5 0.5 2 2 6 1 4 4 0.5
30 30 10 5 5 0.5 2 0 8 1 4 4 0.5
8.1 10.5 9.2 39.8
7.1 10.2 10.8 42.3
7.1 10.7 9.5 40.1
7.5 10.8 10.4 40.5
7.2 10.4 10.1 41.1
Nippon Suisan Co. Ltd., Tokyo, Japan. Riken Vitamin, Tokyo, Japan. c Wako Pure Chemical Ind., Osaka, Japan. Phospholipid concentration is 76.6%. d Attractants (g/kg diet): Taurine, 5; Betain, 4; inosine-5-monophoshate, 1. e Vitamin mixture (mg/kg diet): β-Carotene 96.26; Vitamin D3 9.68; Menadione NaHSO 3 ·3H 2 O (K 3 ) 45.83; Thiamine–Nitrate (B 1 ) 57.75; Riboflavin (B 2 ) 192.37; Pyridoxine–HCl (B6) 45.83; Cyanocobalamine (B12) 0.07; d-Biotin 5.78; Inositol 3212.83; Niacine (Nicotic acid) 769.73; Ca Panthothenate 269.49; Folic acid 14.40; Choline choloride 7869.30; ρ-aminobenzoic acid 383.25. f Mineral mixture (mg kg− 1 diet): MgSO4 5070; Na2HPO4 3230; K2HPO4 8870; Fe Citrate 1100; Ca Lactate12090; Al (OH)3 10; ZnSO4 130; CuSO4 4; MnSO4 30; Ca (IO3)2 10; CoSO4 40. g L-Ascorbyl-2-monophosphate (AMP-Na/Ca, DSM Nutrition Japan K.K.). b
2.3. Analysis The proximate composition of the test diets and whole body samples was analyzed by following A.O.A.C. (1990). Moisture for diets was determined by drying the sample at 120 °C to constant weight. The fish samples were freeze dried (Freezone 2.5, Labconco, Co., Ltd. USA) prior to proximate composition analysis. Loss in weight represented whole body moisture content. The Kjeldahl method was used to determine nitrogen levels, and crude protein was calculated by multiplying by 6.25. The ash was determined by combustion at 550 °C in a muffle furnace. Total lipid was measured following the method of Bligh and Dyer (1959) and further separated into NL and PL fractions by column chromatography (Sep-Pak Silica Cartridges; Waters Corp. Milford, MA) according to Juaneda and Rocquelin (1985). The fatty acid composition of both NL and PL fractions of test diets and whole body samples was determined using gas chromatography (Agilent Technologies Inc., Santa Clara, California, USA; column: OmegawaxTM320) according
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Table 3 Fatty acid compositions (% total fatty acids) of NL and PL fractions of test diets.a Fatty acid
14:0 16:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σn-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σn-3 fatty acids a b
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
NLb
PLb
NL
PL
NL
PL
NL
PL
NL
PL
4.4 34.1 6.7 45.2 4.6 31.5 3.2 2.1 41.4 5.1 0.2 0.2 5.5 0.4 0.1 0.4 1.11 0.5 1.8 4.31
2.6 35.1 5.4 43.1 4.6 21.54 2.3 1.3 29.74 4.5 0.5 2.1 7.1 0.5 0.2 0.5 8.1 0.5 7.9 17.7
4.2 34.5 6.73 45.43 4.8 32.6 3.2 2.4 43 5.8 0.3 0.2 6.3 0.5 0.2 0.5 1.4 0.6 1.9 5.1
2.4 29.5 5.3 37.2 4.8 22.35 2.4 1.2 30.75 9.6 0.4 2.2 12.2 0.5 0.4 0.4 7.85 0.6 8.13 17.88
4.1 32.1 6.5 42.7 4.5 30 3.4 2.3 40.2 5.9 0.2 0.3 6.4 0.3 0.2 0.5 1.12 0.4 1.7 4.22
2.3 25.1 5.1 32.5 4.5 22.56 2.3 1.5 30.86 13.7 0.6 2 16.3 0.4 0.5 0.5 7.98 0.4 8.14 17.92
3.9 32.8 6.8 43.5 4.7 30.5 3.5 2.2 40.9 6.4 0.2 0.2 6.8 0.3 0.2 0.5 1.31 0.5 1.8 4.61
2.5 22.8 5.2 30.5 4.7 21.98 2.3 1.3 30.28 15.3 0.7 2.1 18.1 0.5 0.5 0.65 8.23 0.5 7.98 18.36
3.5 31.2 6.4 41.1 4.9 34.5 3.4 2.3 45.1 6.8 0.2 0.4 7.4 0.4 0.1 0.5 1.17 0.5 1.8 4.47
2.0 20.2 5 27.2 4.9 23.04 2.2 1.2 31.34 17.5 0.6 2.3 20.4 0.5 0.3 0.5 8.12 0.5 7.91 17.83
Values are means of triplicate measurements. NL, Neutral lipid; PL, Polar lipid.
3. Result
to the method of Teshima et al. (1986a). The activities of antioxidant enzymes (CAT, SOD and GPx) of whole body larvae were all analyzed spectrophotometrically using diagnostic reagent kits (Nanjing Jiancheng Bioengineering Institute, China). Whole body samples were homogenized in ice-cold, 0.65% physiological saline using a tissue homogenizer. The supernatant was used to determine SOD (EC 1.15.1.1), CAT (EC 1.11.1.6) and GPx (EC 1.11.1.9), SOD activity was assayed according to a SOD kit protocol (No. A001) at 550 nm according to Bayer and Fridovich (1987). CAT activity was assayed according to a CAT kit protocol (No. A007) at 405 nm according to Claiborne (1985). GPx activity was assayed according to a GPx kit protocol (No. A005) at 412 nm according to Wheeler et al. (1990).
Growth performance of loach fed test diets for 30 days is shown in Table 4. The final weight (FW), body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) significantly increased and feed conversion ratio (FCR) significantly decreased with dietary PLs increasing from 0.8 to 2.3%. However, there were no significant differences in these parameters (FW, BWG, SGR, FCR and SR) between the PLs2.3 and PLs3.0 groups. Feed intake (FI) was not affected by dietary PLs levels.
2.4. Statistical analysis
3.2. Lipid composition and proximate of whole body
All data were subjected to Levene's test of equality of error variances and one-way ANOVA followed by Tukey's test using SPSS 16.0 (SPSS 16.0, Michigan Avenue, Chicago, IL, USA). Probability values of b0.05 were considered statistically significant.
The whole body NL fraction significantly increased with increasing dietary PLs levels (Table 5). The highest whole body NL fraction was observed in larvae fed the PLs2.3 diet. However, whole body PL fraction decreased with incremental dietary PLs levels. Larvae fed the PLs0.8
3.1. Growth performance
Table 4 Growth performance of loach larva fed test diets for 30 days. PLs0.8 Final weight (mg) BWG1 (%) SGR2 (%) FCR3 SR4 (%) FI5 (g/fish)
84.6 605.3 6.5 4.9 33.7 0.4
± ± ± ± ± ±
PLs1.6 a
6.6 55.3a 0.3a 0.8b 12.1a 0.02
116.8 873.3 7.6 3.7 47.7 0.4
± ± ± ± ± ±
PLs1.9 a
15.2 126.5a 0.4a 1.0ab 5.9a 0.03
112.8 840.3 7.5 3.7 50.7 0.4
± ± ± ± ± ±
PLs2.3 a
10.3 86.2a 0.3a 0.8ab 3.5ab 0.04
152.3 1168.9 8.5 2.3 67.3 0.3
PLs3.0 ± ± ± ± ± ±
b
16.4 137.0b 0.4b 0.3a 3.8b 0.01
164.3 1268.9 8.7 2.4 72.0 0.4
± ± ± ± ± ±
10.7b 89.2b 0.2b 0.3a 5.3b 0.02
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments. 1 BWG, body weight gain (%) = 100 × (final weight − initial weight) / (initial weight). 2 SGR, specific growth rate = 100 × (ln final weight − ln initial weight) / (duration). 3 FCR, feed conversion ratio = dry feed intake (g) / weight gain (g). 4 SR, survival rate (%) = 100 × (initial fish number − dead fish number) / (initial fish number). 5 FI, feed intake (g/fish) = (total feed intake (g) / number of fishes) in 30 days' feeding period.
J. Gao et al. / Aquaculture 426–427 (2014) 304–309
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Table 5 Whole body proximate analysis (%) of loach larva fed with different diets for 30 days. PLs0.8 Total lipid (dry mass) NL1 (% of total lipid) PL1 (% of total lipid) NL/PL Moisture Crude ash (dry mass) Crude protein (dry mass)
12.8 56.7 43.4 1.3 83.7 11.0 69.1
PLs1.6
± ± ± ± ± ± ±
0.3a 1.9a 1.9b 0.1a 2.5 0.7 2.6
13.7 65.5 34.5 1.9 84.3 9.7 71.1
± ± ± ± ± ± ±
PLs1.9 0.3a 1.8b 1.8a 0.2ab 1.8 0.2 1.7
13.0 68.3 31.7 2.2 83.8 9.7 72.3
± ± ± ± ± ± ±
PLs2.3 1.1a 1.5b 1.5a 0.2b 0.7 0.2 1.4
13.8 70.0 30.0 2.4 82.9 9.7 72.9
± ± ± ± ± ± ±
PLs3.0 0.4a 5.5b 5.5a 0.6b 1.1 0.3 0.5
15.5 66.6 33.4 2.0 82.0 9.9 71.5
± ± ± ± ± ± ±
0.3b 1.6b 1.6a 0.1ab 0.5 0.6 1.0
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments. 1 NL, Neutral lipid; PL, Polar lipid.
diet had significantly higher PL fraction than those fed other diets. The ratio of NL/PL fraction of whole fish body significantly increased with the increase of dietary PLs from 0.8 to 2.3%. Whole body proximate composition of larvae is also reported in Table 4. A significant effect of dietary PLs levels was found on whole body total lipid content at the end of the feeding trial, while whole body moisture, protein and ash content were unaffected by dietary PLs levels. Larvae fed the PLs3.0 diet had significantly higher fat content than those fed other diets.
3.4. Antioxidant enzyme activities Activities of antioxidant enzymes of larvae are presented in Table 8. SOD activity significantly increased with incremental dietary PLs level. However, the activities of CAT and GPx were significantly decreased by dietary PLs. The highest CAT and GPx activities were observed in larvae fed the PLs0.8 diet, which was significantly higher than the other groups. 4. Discussion
3.3. Fatty acid compositions Fatty acid compositions of NL and PL in whole body larvae were reflected by dietary fatty acid compositions (Tables 6 and 7). In NL fraction, dietary PLs supplementation significantly increased the percentages of 18:1n-9, total monoene fatty acids, 20:5n-3 (Eicosapentaenoic acid, EPA) and total n-3 fatty acids and decreased 16:0, 17:0, 18:0, 16:1n-7 and total saturated fatty acids. In terms of PL fraction, dietary PLs supplementation significantly increased 18:1n-9 and total monoene fatty acids and decreased 16:1n-7, 22:1n-11, 18:3n-3, 18:4n-3, EPA, 22:6n-3 (Docosapentaenoic acid, DHA) and total n-3 fatty acids. In a comparison between the NL and PL fraction fatty acid compositions, the percentage of 18:2n-6, 18:3n-3, 18:4n-3, EPA and DHA was higher in NL than in PL fraction of whole body of larvae.
The importance of supplementation of dietary PLs on optimal growth has been well studied not only in crustaceans such as Vannamei Litopenaeus vannamei (Niu et al., 2011), crayfish Cherax quadricarinatus (Thompson et al., 2003) and Kuruma shrimp Penaeus japonicus (Teshima et al., 1986a, 1986b, 1986c), but also in both marine and freshwater species including large yellow croaker Larmichthys crocea (Zhao et al., 2013), amberjack Seriola dumerili (Uyan et al., 2009), rainbow trout Oncorhynchus mykiss (Poston, 1990, 1991) and pikeperch Sander lucioperca (Hamza et al., 2008), ayu Plecoglossus altivelis (Kanazawa et al., 1985), the common carp Cyprinus carpio (Geurden et al., 1995). In this study, BWG, SGR and SR significantly increased from 605.3% to 1168.9% with increasing dietary PLs levels from 0.8 to 2.3%, when dietary PLs content exceeded 2.3 mg 100 g− 1 diet (PLs2.3), the BWG
Table 6 Fatty acid composition (%) in NL fraction of whole body of loach larvae fed test diets for 30 days.
Table 7 Fatty acid composition (%) in PL fraction of whole body of loach larvae fed test diets for 30 days.
Fatty acid 14:0 16:0 17:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σn-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σn-3 fatty acids
PLs0.8 2.2 29.7 0.5 0.9 33.2 9.9 44.5 0.6 0.9 56.2 3.2 0.3 0.1 3.6 0.1 0.3 0.4 1.9 0.1 0.2 3.0
PLs1.6 2.0 28.0 0.4 0.5 30.9 6.0 50.1 0.6 1.0 58.0 3.4 0.2 0.1 3.7
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.4c 0.0b 0.2ab 0.3cd 0.2ab 2.9b 0.1 0.1 2.7a 0.3 0.1 0.1 0.3
1.9 27.6 0.2 0.4 30.1 7.1 49.7 0.8 0.7 58.6 3.4 0.2 0.1 3.7
0.0 0.1 0.0 0.1a 0.1 0.1 0.3a
0.2 0.4 0.3 2.1 0.1 0.4 3.5
± ± ± ± ± ± ±
0.0 0.0 0.2 0.1a 0.1 0.1 0.2ab
0.2 0.4 0.6 2.1 0.1 0.3 3.6
± 0.0 ± 1.2c ± 0.1b ± 0.1b ± 1.3d ± 0.6c ± 1.7a ± 0.5 ± 0.6 ± 0.9a ± 0.1 ± 0.1 ± 0.0 ± 0.3 ± ± ± ± ± ± ±
PLs1.9
c
b
PLs2.3 b
± 0.1 ± 1.2c ± 0.1a ± 0.1a ± 1.2c ± 0.4b ± 0.6ab ± 0.2 ± 0.2 ± 0.7a ± 0.4 ± 0.1 ± 0.1 ± 0.4 ± ± ± ± ± ± ±
0.1 0.2 0.2 0.1ab 0.1 0.1 0.4ab
1.6 ± 24.8 ± 0.2 ± 0.3 ± 27.0 ± 5.6 ± 55.4 ± 0.8 ± 0.5 ± 62.6 ± 3.8 ± 0.5 ± 0.2 ± 4.4 ± 0.2 0.4 0.5 2.5 0.1 0.3 4.3
± ± ± ± ± ± ±
PLs3.0 a
a
0.0 0.6b 0.0a 0.2a 0.5b 0.6a 1.4bc 0.0 0.0 0.9b 0.2 0.2 0.0 0.3
1.5 ± 21.7 ± 0.1 ± 0.4 ± 23.7 ± 5.7 ± 56.9 ± 0.7 ± 0.5 ± 64.3 ± 3.8 ± 0.7 ± 0.2 ± 4.5 ±
0.1 1.5a 0.0a 0.1a 1.5a 0.5a 2.7c 0.1 0.0 2.2b 0.2 0.3 0.0 0.2
0.1 0.2 0.2 0.1b 0.0 0.1 0.2b
0.1 ± 0.4 ± 0.5 ± 2.4 ± 0.1 ± 0.3 ± 3.8 ±
0.0 0.1 0.1 0.2ab 0.0 0.1 0.4ab
Values are expressed as mean ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
Fatty acid
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
14:0 16:0 17:0 18:0 Σsaturated 16:1n-7 18:1n-9 20:1n-9 22:1n-11 Σmonoenes 18:2n-6 20:2n-6 20:4n-6 Σ n-6 fatty acids 18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Σ n-3 fatty acids
0.5 ± 0.0 23.0 ± 0.9a 0.6 ± 0.2 0.2 ± 0.0 24.2 ± 0.9a 5.7 ± 0.1d 36.2 ± 0.6a 3.0 ± 0.2a 1.4 ± 0.1d 46.3 ± 0.8a 7.4 ± 0.4 0.5 ± 0.5 0.4 ± 0.2 8.3 ± 0.4
0.4 ± 0.0 23.4 ± 1.3a 0.5 ± 0.0 0.2 ± 0.0 24.5 ± 1.3a 3.7 ± 0.1c 38.6 ± 0.4b 3.4 ± 0.0b 1.0 ± 0.1bc 46.0 ± 0.6a 8.3 ± 0.4 0.7 ± 0.0 0.5 ± 0.1 9.5 ± 0.5
0.6 ± 0.1 25.7 ± 0.6b 0.9 ± 0.2 0.3 ± 0.0 27.9 ± 1.4b 3.6 ± 0.1bc 38.8 ± 0.3b 3.5 ± 0.1b 0.9 ± 0.1b 46.8 ± 0.3a 8.1 ± 0.3 0.5 ± 0.2 0.4 ± 0.1 9.0 ± 0.5
0.5 ± 0.1 26.8 ± 0.5b 1.3 ± 0.2 0.2 ± 0.0 28.8 ± 0.8b 3.1 ± 0.5ab 41.8 ± 1.3c 3.0 ± 0.0a 0.7 ± 0.1ab 48.6 ± 0.9b 8.3 ± 0.3 0.3 ± 0.2 0.2 ± 0.1 8.8 ± 0.4
0.5 ± 0.0 22.0 ± 0.4a 0.8 ± 0.3 0.2 ± 0.0 23.4 ± 0.2a 2.7 ± 0.2a 43.3 ± 0.1c 2.9 ± 0.1a 0.7 ± 0.1a 49.6 ± 0.3b 8.4 ± 0.5 0.8 ± 0.3 0.6 ± 0.6 9.8 ± 1.4
1.8 ± 0.1c 2.9 ± 0.4c 0.5 ± 0.3 11.0 ± 0.4b 0.6 ± 0.2 1.1 ± 0.1b 17.9 ± 0.6c
1.7 ± 0.0c 1.9 ± 0.0b 0.3 ± 0.2 11.4 ± 2.0b 0.6 ± 0.2 0.9 ± 0.1ab 16.8 ± 2.7bc
1.4 ± 0.2b 1.5 ± 0.2ab 0.5 ± 0.2 9.0 ± 0.2ab 0.6 ± 0.2 0.8 ± 0.2ab 13.8 ± 0.5ab
1.1 ± 0.1ab 1.3 ± 0.1a 0.3 ± 0.2 8.5 ± 0.3a 0.8 ± 0.2 0.6 ± 0.3a 12.7 ± 0.3a
1.1 ± 0.1a 1.4 ± 0.1a 0.4 ± 0.3 9.2 ± 0.5ab 0.6 ± 0.2 0.7 ± 0.2a 13.4 ± 0.1a
Values are expressed as mean ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
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J. Gao et al. / Aquaculture 426–427 (2014) 304–309
Table 8 Activities of antioxidant enzymes (U/mg prot) of loach larva fed test diets for 30 days.
Catalase (CAT) Superoxide dismutase (SOD) Glutathione peroxidase (GPx)
PLs0.8
PLs1.6
PLs1.9
PLs2.3
PLs3.0
16.1 ± 1.5b 5.6 ± 0.1a 206.4 ± 6.4c
9.1 ± 0.7a 6.8 ± 0.2b 169.8 ± 6.6b
7.8 ± 0.4a 6.9 ± 0.1b 165.9 ± 6.6b
6.4 ± 0.4a 7.0 ± 0.9b 182.0 ± 11.1bc
7.3 ± 0.5a 6.9 ± 0.1b 134.7 ± 13.7a
Values are means ± S.E.M. from triplicate groups. Means in each row with different letters are significantly different (P b 0.05). Absence of letters indicates no significant difference between treatments.
reached a plateau, suggesting that 2.3% of dietary PLs supplementation could maintain the normal physiological function and survival for loach larvae. Our results showed that SGR of larvae fed with PLs2.3 and PLs3.0 reached more than 8.5 from day 15 to day 45 after hatching. This value was similar with Wang et al.'s (2009), who reported that SGR of loach larvae fed microparticle diets was about 9.0 from day 20 to day 60 after hatching. However, the SGR of loach fed microparticle diets was lower than those fed live gray like cladocerans (SGR, 11.9). For successful aquaculture, it is important to know the nutritional requirements for loach larvae. In this study, SR significantly increased from 33.7% to 72.0% with incremental dietary PLs level from 0.8% to 2.3%. A significant effect of dietary PLs supplementation on SR in fish larvae has also been demonstrated in a number of studies (Azarm et al., 2013; Hamza et al., 2008; Zhao et al., 2013). In contrast to that, Uyan et al. (2009) demonstrated that SR of juvenile amberjack was not influenced with increasing levels of dietary PLs. This suggested that PLs were essential to maintain the normal physiological function and survival for only the early development stage of fish. No effects of dietary PLs supplementation on growth at the early juvenile stage have been reported by Coutteau et al. (1997) and Dapra et al. (2011). Dietary PLs are considered as structural lipids that are constituents of cell membranes (Kanazawa, 1985). Thus, dietary PLs are believed to be extremely crucial for larval development in fish. The stimulatory effect of PLs on growth can be explained in different ways (Azarm et al., 2013). Geurden et al. (1995) reported that the stimulating effects of PLs on larval fish growth were due to the fish larvae having a limited ability to biosynthesize phospholipids de novo. In a review, Tocher et al. (2008) demonstrated that diets rich in triacylglycerol and a lack of sufficient dietary PLs could limit lipoprotein synthesis in enterocytes, leading to impaired transport of lipid nutrients to tissues. The stimulating effect of PLs on growth is due to improved transport, assimilation and utilization of dietary lipid. In the present experiment, whole body total lipid content was significantly increased with increasing dietary PLs supplementation. This was in agreement with some previous studies. Uyan et al. (2009) reported that whole body fat content of juvenile amberjack significantly increased with increasing dietary PLs supplementation. Moreover, Zhao et al. (2013) found that whole body fat content of large yellow croaker larvae significantly increased with incremental dietary PLs level from 26.0 g/kg to 85.1 g/kg. The inclusion of dietary PLs influenced lipid deposition of the whole body, resulting in increased lipid retention in the animal (Teshima et al., 1986c). Based on results from the present study, dietary PLs significantly increased the percentage of NL fraction and significantly decreased the percentage of PL fraction. The increase in the ratio of NL/PL might be due to an increase in NL fraction in whole body total lipid content. The likely reasons are related to the following two aspects. The first one is that PL is more well utilized than NL for fish larvae. It is generally believed that PL as main lipid component in PLs plays a crucial role in membrane structure during early life stages. The other is likely that supplementation of dietary PLs promoted incorporation of NL in the tissues (Kanazawa et al., 1985). In contrast, Zhao et al. (2013) found that the ratio of NL/PL of large yellow croaker larvae whole body fat content decreased with incremental dietary PLs level. In the present study, we found that the percentages of DHA in both NL (0.2–0.4%) and PL (0.6–1.1%) of larvae whole body were significantly lower compared with the results from our previous study (Gao et al.,
2012), showing that the percentages of DHA in wild and cultured loach whole body were 6.7% and 10.9%, respectively. Poor DHA concentration may be explained by a lack of essential fatty acid (EFA) concentration in diet. Since n-3 fatty acids can be considered to be the most limiting EFA (Sargent et al., 1999), adequate amounts of fish oil must be incorporated in the diets to cover the EFA requirements, as fish oils are the only dietary source of n-3 HUFA (Sargent et al., 2002). It has been reported that the fatty acid composition of fish tissues was influenced by fatty acid composition of diet. Relatively low DHA concentration of fish tissues indicated in some extent EFA deficiency, which has been reported in several studies (Izquierdo et al., 2005; Ng et al., 2013; Xue et al., 2006). Although there is no information about the EFA requirement of this species, our results indicated that 2% of fish oil in diet did not meet the EFA requirements of loach larvae. Several studies have reported that freshwater fish has capability to elongate and desaturate n-3 and n-6 PUFA (C18) chains to the n-3 and n-6 HUFA (C20 and C22) chains (Bell et al., 1986; Sargent et al., 1999). The main effect of dietary PLs supplementation on larval fatty acid composition was an increase in the percentage of n-6 fatty acid like 18:2n-6 and total n-6 fatty acids in both NL and PL fractions (Hamza et al., 2008). In this study, there were no significant differences in n-6 fatty acids like 18:2n-6 and total n-6 fatty acids in both NL and PL fractions among different treatment groups. However, EPA and total n-3 fatty acids in NL fraction of larvae significantly increased with incremental dietary PLs level. Since all diets were added the same level of fish oil, the increase n-3 HUFA could indicate that loach can convert 18: 2n-6 to the long chain n-6 fatty acids. This was in agreement with the result of Hamza et al. (2008) who demonstrated that a relatively high level of n-3 HUFA in pikeperch Sander lucioperca larvae fed dietary 9% of PLs supplementation contrasting with their low level in the diet. Moreover, Azarm et al. (2013) observed that the percentages of HUFA especially EPA + DHA were higher in rainbow trout O. mykiss fed soybean lecithin diets than those fed soybean lecithin un-supplementation group. On the other hand, results from the present study showed that n-3 fatty acids in PL fraction such as EPA, DHA and total n-3 HUFA decreased with incremental dietary PLs levels, suggesting that dietary PLs supplementation may promote utilization of EPA and DHA efficiently in PL fraction for loach larvae. HUFAs are vital constituents for cell membrane structure and function in fish larvae (Tocher, 2003). It has been reported that fish larvae can more efficiently use n-3 HUFA from the PL fraction than those from the NL fraction (Cahu et al., 2003; Gisbert et al., 2005; Hamza et al., 2008). The present results also confirmed that a high content of n-3 fatty acids was in the PL fraction rather than in the NL fraction in larvae. Dietary PLs supplementation could enhance stress resistance and induce anti-oxidant responses to protect an organ against oxidative damage (Hamza et al., 2008; Kanazawa, 1997; Zhao et al., 2013). The antioxidant defense system of organisms provides a means of dealing with oxidative stress, refers to the disturbance of the equilibrium between antioxidants and pro-oxidants towards oxidants and includes several enzymes and vitamins. In this study, dietary PLs supplementation induced SOD actively in larvae. It is known that SOD value can be believed to be a more reliable and expressive set of oxidative stress indicator. High SOD activity in the whole body might be a consequence of free radicals derived from oxidation of lipids (Zhang et al., 2009). On the contrary, the activities of CAT and GPx showed an opposite tendency with
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SOD activity in this study, indicating that PLs supplementation might inhibit activities of CAT and GPx in loach larvae. Although the present results indicated that dietary PLs influenced larvae antioxidant enzyme activities, it was difficult to explain the reasons for the changes in activities of CAT, SOD and GPx in whole body of larvae since the activities of antioxidant enzymes in fish always showed different patterns among different organs (Zhang et al., 2009) and growth time (Mourente et al., 2002). The activities of antioxidant enzymes in different organs of fish need to be studied in the future. 5. Conclusions In summary, this is the first study that evaluated a nutritional requirement of loach at their larval stage. Results showed that a supplementation more than 6% of soybean lecithin in diet (2.3 g 100 g−1 of PLs in diet) could improve growth performance and survival of loach larvae. Dietary PLs supplementation increased fat content and the ratio of NL/PL in whole body of larvae. An increase in the percentage of total n-3 fatty acids in NL fraction was found along with increasing dietary PLs levels, suggesting that loach larvae had the capability to elongate n-6 PUFA chains to n-6 HUFA. The dietary requirement of HUFA for loach larvae needs to be researched in future. Acknowledgments The author would like to thank the Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan, for providing the test diets in this study. This study was supported by the National Natural Science Foundation of China (Grant Nos.: 31372180, 34113053 and 31201719), and partly by the Fundamental Research Funds for the Central Universities of China (2013PY074). We are very grateful to three anonymous reviewers for their constructive suggestions and comments. References A.O.A.C, 1990. Official Methods of Analysis of the Association of Official Analytical Chemists, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Azarm, H.M., Kenari, A.A., Hedayati, M., 2013. Effect of dietary phospholipid sources and levels on growth performance, enzymes activity, cholecystokinin and lipoprotein fractions of rainbow trout (Oncorhynchus mykiss) fry. Aquac. Res. 44, 634–644. Bayer, W.F., Fridovich, J.L., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Chem. 161, 559–566. Bell, M.V., Henderson, R.J., Sargent, J.R., 1986. The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol. 83B, 711–719. Bligh, E.G., Dyer, W.J., 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cahu, C.L., Jambonino Infante, J.L., Barbosa, V., 2003. Effect of dietary phospholipid level and phospholipid: neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet. Br. J. Nutr. 90, 21–28. Claiborne, A., 1985. Catalase activity, In: Greenwald, R.A. (Ed.), CRC Handbook of Methods in Oxygen Radical Research, 2nd edn. CRC Press, Boca Raton, FL, pp. 283–284. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P., 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155, 149–164. Dapra, F., Geurden, I., Corraze, G., Bazin, D., Zambonino-Infante, J.L., 2011. Physiological and molecular responses to dietary phospholipids vary between fry and early juvenile stages of rainbow trout (Oncorhynchus mykiss). Aquaculture 319, 377–384. Gao, J., Koshio, S., Nguyen, B.T., Wang, W.M., Cao, X.J., 2012. Comparative studies on lipid profiles and amino acid composition of wild and cultured dojo loach Misgurnus anguillicaudatus obtained from Southern Japan. Fish. Sci. 78, 1331–1336. Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Daisuke, N., Ren, T.J., 2013. Interactive effects of vitamin C and E supplementation on growth, fatty acid composition, and lipid peroxidation of sea cucumber, Apostichopus japonicus, fed with dietary oxidized fish oil. J. World Aquac. Soc. 44, 536–546. Geurden, I., Radunz-Neto, L., Bergot, P., 1995. Essentiality of dietary phospholipids for carp (Cyprinus carpio) larvae. Aquaculture 131, 303–314. Gisbert, E., Villeneuve, L., Zambonino-Infante, J.L., Quazuguel, P., Cahub, C.L., 2005. Dietary phospholipids are more efficient than neutral lipids for long-chain polyunsaturated
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