Volume 27, Issue 6 pp. 1996-2006
ORIGINAL ARTICLE
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Effect of lipid sources on growth performance, muscle composition, haemolymph biochemical indices and digestive enzyme activities of red swamp crayfish (Procambarus clarkii)

Fan Wu

Fan Wu

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China

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Zhimin Gu

Zhimin Gu

Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Key Laboratory of Freshwater Aquaculture Genetic and Breeding of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou, China

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Xiaoru Chen

Xiaoru Chen

Healthy Aquaculture Key Laboratory of Sichuan Province, Tongwei Co., Ltd., Chengdu, China

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Lijuan Yu

Lijuan Yu

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China

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Xing Lu

Xing Lu

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China

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Lu Zhang

Lu Zhang

Healthy Aquaculture Key Laboratory of Sichuan Province, Tongwei Co., Ltd., Chengdu, China

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Hua Wen

Corresponding Author

Hua Wen

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China

Correspondence

Hua Wen, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, Hubei, China.

Email: [email protected]

Juan Tian, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, Hubei, China.

Email: [email protected]

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Juan Tian

Corresponding Author

Juan Tian

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China

Correspondence

Hua Wen, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, Hubei, China.

Email: [email protected]

Juan Tian, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, Hubei, China.

Email: [email protected]

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First published: 01 September 2021
Citations: 9

Fan Wu and Zhimin Gu equally contributed to this study.

Abstract

This study was conducted to determine the effects of different dietary lipid sources on red swamp crayfish (Procambarus clarkii). Six diets were formulated with the same lipid level (85 g/kg) to contain fish oil (FO), soybean oil (SO), corn oil (CO), palm oil (PO), lecithin oil (LO) and pork lard (PL) respectively. Each diet was fed to triplicate of 18 crayfish with an initial weight of (13.05 ± 0.60) g for 8 weeks. The results showed that crayfish fed diet SO had a significantly higher weight gain rate and specific growth rate than those fed diet CO, PO and PL, but there was no significant difference among crayfish fed diet FO, SO and LO. Crayfish fed diet PL had the highest feed conversion ratio and the lowest survival rate. Significantly increased muscle lipid contents were observed in groups PO and PL compared with other groups. Group FO had the highest values of EPA, DHA and n-3/n-6 ratio in muscle, followed by groups LO and SO. The haemolymph triacylglycerol content was the highest, whereas alkaline phosphatase activity was the lowest in crayfish fed diet PL. Moreover, a significant increase of total protein content was observed in the haemolymph of crayfish fed diet FO, SO and LO. In conclusion, crayfish fed diet FO, SO and LO showed better growth than those fed diet PL and PO without any signs of essential fatty acid deficiency.

1 INTRODUCTION

Red swamp crayfish (Procambarus clarkii), which is native to North America (Huner, 1988) and introduced to China in the 1930s from Japan (Yue et al., 2010), has become one of the most important economic aquaculture shrimp species in China due to its high fecundity, strong adaptability, delicious flavour and rich nutrition (Gherardi, 2006; Shen et al., 2014; Tan et al., 2018; Yta et al., 2012). In recent years, booming consumer demand for the red swamp crayfish has promoted the development of intensive culture. However, the research on aquaculture technique of red swamp crayfish starts relatively late, especially little attention has been placed on nutrition and feed. Thus, it becomes a rising need for research on the nutrition and practical feed formulations for red swamp crayfish in aquaculture.

Aquatic animals, including crustaceans, require certain essential lipids to satisfy their growth and support metabolic functions. Lipids play important roles and offer important nutritional functions such as the source of energy, essential fatty acids (EFA), phospholipids and sterols, carriers for fat-soluble vitamins, components in hormones and precursors for active substances etc. (Deering et al., 1997; Mcdonald & Eskin, 2007; Sargent et al., 2002; Vikas et al., 2018). In addition, lipids also play an important role in maintaining the integrity and fluidity of cell biomembrane, determining the texture and palatability of diet (NRC, 2011; Sargent et al., 2002). Jover et al. (1999) and Xu et al. (2013) reported that the optimal dietary lipid level of red swamp crayfish was 6% and 4–7% respectively. Peng et al.(2019) suggested that appropriate lipid level for red swamp crayfish broodstock was 7.60–7.89% based on growth performance, muscle composition, reproductive performance, digestive enzyme activities and biochemical indexes.

Fish oil, rich in n-3 highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is widely utilized in the formulation of commercial aquafeeds (Sargent et al., 2002) and also believed to be the more suitable lipid source for crustacean diets (González-Félix et al., 2009). However, due to the increasing prices and limited supplies of fish oil (Tacon & Metian, 2008), it is imperative for the aquafeed industry to find alternative lipid sources. Moreover, it is necessary to diversify dietary lipid sources of either plant or animal origin from sustainability standpoints (Huang et al., 2008).

Several studies have investigated limitations and benefits of replacing fish oil (FO) in shrimp feed with alternative lipid (González-Félix et al., 2010; Ju et al., 2012; Kim et al., 2013; Li et al., 2011). Partial or total replacement of fish oil with soy oil and poultry fat had no effect on growth performance, but affected cholesterol metabolism, fatty acid profiles of Pacific white shrimp (Litopenaeus vannamei) (Cheng & Hardy, 2004). Thompson et al. (2010) have concluded that diets for juvenile red claw crayfish (Cherax quadricarinatus) can be formulated to replace menhaden oil with linseed oil, canola oil and corn oil; however, crayfish fed the beef tallow diet with higher contents of saturated fatty acid (SFA), performed poor weight gain compared with those fed linseed oil and corn oil diet. However, according to the results of Gao et al. (2020), beef tallow is the more suitable candidate for FO replacement in diets of red swamp crayfish (Procambarus clarkia), whereas palm oil is not recommended. So far, there are few researches on the dietary lipid source in the culture of red swamp crayfish and the reported results were inconsistent.

Therefore, the aim of this study was to assess the effects of different dietary lipid sources on growth performance, muscle proximate and fatty acid composition, and digestive enzymes activities of red swamp crayfish.

2 MATERIALS AND METHODS

2.1 Ethics statement

Red swamp crayfish is widely farmed in China and is not listed as an endangered or protected species. This study was approved by the Institutional Animal Care and Use Committee of Yangtze River Fisheries Research Institute under permit number YFI 2018–40.

2.2 Experimental diet

Six semi-purified diets were formulated to be isonitrogenous (approximately 320 g/kg crude protein) and isolipidic (approximately 85 g/kg crude lipid), containing protein from soybean meal, fish meal, wheat gluten and shrimp meal. Flour and corn starch were used as carbohydrate sources. Fish oil (FO), soybean oil (SO), corn oil (CO), palm oil (PO), lecithin oil (LO) and pork lard (PL) were used as the lipid sources in six experiment diets respectively. The formulation and proximate analysis of the experimental diets are provided in Table 1. The fatty acid composition of the six experimental diets is presented in Table 2. Before oil addition, all dry ingredients were ground, passed through a 180-µm mesh screen and thoroughly mixed in a mixer. Then, approximately 250 ml of distilled water per kg of diet was added into the mixture. A twin-screw extruder (Model F26; South China University of Technology Science and Technology Industrial Plant, Guangzhou, China) was used to extrude the mixture into 2-mm diameter pellets. The pellets were cured in an oven (DHG-9423BS-Ⅲ, Shanghai CIMO Medical Instrument Manufacturing Co., Ltd.) at 95°C for 20 min and then dried at room temperature to the moisture content less than 70 g/kg. Subsequently, pellets were cut into appropriate sizes of 3–5 mm in length and stored at −20°C until use.

TABLE 1. Composition and proximate analysis of the experimental diets (dry matter, g/kg)
Ingredient Dietary lipid sources
FO SO CO PO LO PL
Fish meal 80.0 80.0 80.0 80.0 80.0 80.0
Soybean meal 200.0 200.0 200.0 200.0 200.0 200.0
Wheat gluten 100.0 100.0 100.0 100.0 100.0 100.0
Shrimp meal 60.0 60.0 60.0 60.0 60.0 60.0
Flour 300.0 300.0 300.0 300.0 300.0 300.0
Corn starch 50.0 50.0 50.0 50.0 50.0 50.0
Yeast extract 20.0 20.0 20.0 20.0 20.0 20.0
Micro-cellulose 70.5 70.5 70.5 70.5 70.5 70.5
Fish oil 70.0
Soybean oil 70.0
Corn oil 70.0
Palm oil 70.0
Lecithin oil 70.0
Pork lard 70.0
Sodium alginate 10.0 10.0 10.0 10.0 10.0 10.0
Monocalcium phosphate 20.0 20.0 20.0 20.0 20.0 20.0
Vitamin premix 10.0 10.0 10.0 10.0 10.0 10.0
Mineral premix 5.0 5.0 5.0 5.0 5.0 5.0
Allicin 1.0 1.0 1.0 1.0 1.0 1.0
Astaxanthin (10%) 1.0 1.0 1.0 1.0 1.0 1.0
Choline chloride 2.5 2.5 2.5 2.5 2.5 2.5
Nutrient levels
Dry matter 946.8 947.2 943.2 949.1 941.5 945.9
Crude protein 319.6 322.7 318.5 321.3 323.6 317.3
Crude lipid 85.1 86.7 87.2 85.9 84.8 87.4
Ash 65.3 64.8 65.8 66.1 68.2 64.6
  • a Vitamin premix contained (g/kg premix): vitamin A 4; vitamin D 0.02; vitamin E 20; vitamin K3 10; vitamin B1 10; vitamin B2 20; nicotinic acid 40; calcium pantothenate 25; vitamin B6 20; biotin 0.2; folic acid 1; vitamin B12 0.01, vitamin C 20; inositol 400.
  • b Mineral premix contained (g/kg premix): KH2PO4 320; NaCl 200; MgSO4 200; FeSO4·7H2O 50; ZnSO4·7H2O 60; CuCl2·2H2O 2; MnSO4·H2O 20; CoCl2·6H2O 2; KIO3 0.6; Na2SeO3·5H2O 0.2.
TABLE 2. Fatty acid composition (% total fatty acids) of the experimental diets
Fatty acid Dietary lipid sources
FO SO CO PO LO PL
14:0 5.49 1.24 1.37 2.43 1.19 3.75
16:0 19.09 14.37 14.51 34.74 15.74 27.5
18:0 6.53 4.31 3.92 8.32 3.89 17.74
ΣSFA 32.41 20.83 20.24 48.11 22.24 51.53
16:1 4.38 1.10 1.62 1.54 1.39 1.23
18:1 18.92 21.51 22.78 24.63 18.16 18.25
ΣMUFA 24.09 23.75 25.79 26.86 21.89 20.29
18:2n-6 15.21 44.31 45.02 21.31 44.47 24.62
20:4n-6 2.16 0.35 0.37 0.31 1.18 0.85
18:3n-3 2.49 3.92 3.27 1.88 3.15 0.94
20:5n-3 EPA 7.97 1.69 1.45 0.28 2.07 0.48
22:6n-3 DHA 13.51 3.61 2.58 0.41 3.14 0.21
ΣPUFA 43.50 55.42 53.97 25.03 55.87 28.18
Σn-3 PUFA 24.02 9.46 7.32 2.61 8.48 1.65
Σn-6 PUFA 18.15 44.94 45.52 21.81 46.35 25.85
Σn-3/Σn-6 1.32 0.21 0.16 0.12 0.18 0.06

Notes

  • Some fatty acids, of which the contents are minor, trace amount or not detected(such as 12:0, 13:0, 15:0, 17:0, 20:0, 21:0, 22:0, 23:0, 24:0, 14:1, 17:1, 20:1, 22:1, 24:1, 20:2, 20:3), are not listed in Table 2.
  • Abbreviations: MUFA, monounsaturated fatty acids; n-3 PUFA, omega 3 polyunsaturated fatty acids; n-6 PUFA, omega 6 polyunsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids.

2.3 Experimental procedure

Red swamp crayfish were obtained from Xianning Yuxiangyuan Agricultural Development Co., Ltd. (Xianning, HubeiProvince, China) and transferred to the farming base. The crayfish were reared in freshwater tanks (120 cm length × 90 cm width × 35 cm height) and fed with an equal mixture of the experimental diets for two weeks to adapt to the experimental diets and experimental conditions. After two-week acclimation, 324 crayfish (the initial weight: 13.05 ± 0.60 g) were randomly assigned to 18 tanks with 18 crayfish per tank. Each tank was equipped with 18 polyvinyl chloride pipes to act as shelter. Each experimental diet was randomly assigned to triplicate tanks. Diet allowance daily was 3–5% of total body weight of each tank. Crayfish were hand-fed with experimental diets twice a day, at 08:00 h (30% of daily feeding amount) and 19:00 h (70% of daily feeding amount) for 8 weeks. Uneaten feed was sucked out with a siphon tube after 2h feeding and then oven-dried 105°C to calculate feed consumption. The daily rations of feed were adjusted daily based on the feeding situation by crayfish on the previous day. Approximately 30% of the water in each tank was replenished daily at 10:00 am. Water quality parameters were monitored every morning. Water temperature was from 22 to 28°C, pH ranged from 7.3 to 7.9, dissolved oxygen was above 5 mg/L, and total ammonia nitrogen was no more than 0.2 mg/L. Mortality was checked daily. The dead crayfish per tank were weighted and recorded on a daily basis.

2.4 Sample collection and analysis

At the end of the feeding trail, all crayfish were deprived of diet for 24 h and anaesthetized with 30 mg/L of eugenol, and then counted and weighed. Six crayfish from each tank were randomly selected and weighted individually. Blood samples were collected from the pericardial cavity of these six crayfish using 1-mL syringe, allowed to clot at room temperature for 2 h and then centrifuged at 12,000 r/min at 4°C for 20 min. The resulting haemolymph was stored at −80°C until used. After haemolymph samples obtained, the sampled crayfish were dissected on ice plate. Hepatopancreas was separated and weighted to calculate the hepatosomatic index (HSI). Intestine was separated and collected to determine the digestive enzymes activities. Then, the tail muscle was removed and weighed to determine the flesh content (FC) and proximate composition and fatty acid composition. All samples were stored at −80°C until used.

For moisture content determination, diet samples were dried at 105°C to constant weight, while muscle samples were freeze-dried for 48 h in a vacuum freeze dryer (Christ Beta 2–4 LD plus LT, Marin Christ Corporation, Osterode, Germany). Crude protein, crude lipid and ash contents of diets and muscle samples were measured by standard methods (AOAC, 2005). Crude protein content was determined by Kjeldahl (N × 6.25; K324 nitrogen analyzer, BUCHI, Flawil, Switzerland) following acid digestion, distillation and titration. Crude lipid content was measured by Soxhlet extraction using petroleum ether (40 to 60°C boiling point). Ash content was determined through incineration at 550°C for 6 h in a muffle furnace.

The haemolymph samples were thawed at 4°C and then measured using an automatic biochemical analyser (Sysmex-800, Sysmex Corporation, Kobe, Japan) with commercial diagnostic reagent kits (Sysmex Wuxi Co., Ltd., Wuxi, China). The contents of total cholesterol (TCHO), triacylglycerol (TG) and glucose (GLU) were measured with the CHOD-PAP (cholesteroloxidase-para-amino-phenazome) method, ADP-HK (adenosine 5′-diphosphate-hexokinase) method and hexokinase method respectively. Total protein (TP) content was determined by the biuret method. The activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in haemolymph were detected with commercial kits based on the AMP (2-amino-2-methyl-1-propanol) buffer method, MDH-UV (malate dehydrogenase-ultraviolet) method and LDH-UV (lactate dehydrogenase-ultraviolet) method respectively.

The frozen hepatopancreas and intestinal samples were thawed in ice, weighted and thoroughly homogenized in 1:10 (w/v) saline solution, then centrifuged at 12,000 r/min at 4°C for 10 min. The supernatants were collected for enzymatic determinations. The protein concentrations and the activities of protease, amylase and lipase of hepatopancreas and intestinal homogenates were determined by commercial kits (Nanjing Jiancheng Bioengineering Institute, China) and conducted according to the manufacture instruction.

For fatty acid analysis, diet and muscle samples were freeze-dried for 48 h in a vacuum freeze dryer (Christ Beta 2–4 LD plus LT, Marin Christ Corporation, Osterode, Germany). Then, the fatty acid profiles were analysed using the procedures described by Peng et al. (2014). Fatty acid methyl esters were separated and quantified by a gas chromatography–mass spectrometer (Thermo Trace1300 - ISQ7000, Thermo Fisher Scientific, USA) with a DB-5 capillary column (60 cm × 0.25 mm × 0.25 µm). The column temperature was initially 140°C for 5 min, raised to 180°C at the rate of 10°C/min and then raised to 210°C at 2°C/min, then programmed to increase at 10°C/min up to 260°C where it was maintained for 10 min. Carrier gas was helium (1.5 ml/min), and the split ratio was 20:1. Injector and interface temperatures were 260°C respectively. Ion trap temperature was 230°C.

2.5 Calculations and statistical analysis

The following variables were calculated:
  • Weight gain rate (WGR, %) = 100 × (final body weight − initial body weight)/initial body weight.
  • Specific growth rate (SGR, %/d) = 100 × ln (final weight/initial weight)/days.
  • Feed conversion ratio (FCR) = dry feed consumed/wet weight gain.
  • Hepatosomatic index (HSI, %) = 100 × (hepatosomatic weight/whole body weight).
  • Flesh content (FC, %) = 100 × abdomen muscle weight/whole body weight.
  • Survival rate (SR, %) = 100 × final number/initial number.

All data were shown as means ± SD of three replications and analysed using one-way analysis of variance (ANOVA) and Duncan's multiple-range test. The level of significance was set at p < .05. Analyses were performed using SPSS 18.0 (SPSS Inc, Chicago, Illinois, USA).

3 RESULTS

3.1 Growth performance, feed utilization and survival

The effects of different dietary lipid sources on growth performance, feed utilization and survival rate of red swamp crayfish are presented in Table 3. Crayfish fed the SO diet had a significantly higher WGR and SGR than those fed diet CO, PO and PL (p < .05). The lowest WGR and SGR were observed at crayfish fed the diet PL. Crayfish fed the PL diet had a significantly higher FCR and lower SR than those fed the other diets. In addition, HSI and FC were not influenced by the different lipid source diets (p > .05).

TABLE 3. Growth performance and feed utilization of red swamp crayfish fed diets with different lipid source for 8 weeks
Item Dietary lipid sources
FO SO CO PO LO PL
IMW (g) 13.03 ± 0.30 13.21 ± 0.82 13.32 ± 0.65 13.16 ± 0.81 12.85 ± 0.78 12.74 ± 0.53
FMW (g) 24.09 ± 1.33b 24.78 ± 2.02b 23.42 ± 1.46b 22.22 ± 1.01ab 23.86 ± 1.83b 20.31 ± 1.14a
WGR (%) 84.80 ± 6.27cd 87.51 ± 4.96d 75.75 ± 5.22bc 68.99 ± 6.80b 85.51 ± 3.96cd 59.41 ± 4.52a
SGR (%/d) 1.09 ± 0.06cd 1.2 ± 0.05d 1.01 ± 0.05bc 0.93 ± 0.04b 1.10 ± 0.04cd 0.83 ± 0.05a
FCR 1.36 ± 0.12a 1.44 ± 1.14a 1.39 ± 0.11a 1.45 ± 0.09a 1.41 ± 0.04a 1.66 ± 0.08b
HSI (%) 5.46 ± 0.29 5.32 ± 0.24 5.52 ± 0.37 5.61 ± 0.45 5.35 ± 0.33 5.74 ± 0.15
FC (%) 11.26 ± 0.32 11.34 ± 0.54 11.09 ± 0.94 11.29 ± 0.84 11.61 ± 0.83 11.60 ± 0.62
SR (%) 83.33 ± 5.56bc 85.19 ± 3.21c 75.93 ± 6.41bc 74.07 ± 3.21b 83.33 ± 5.56bc 62.96 ± 8.47a

Notes

  • Values (Means ± SD, n = 3) in the same row with different superscript letters are significantly different (< .05).
  • Abbreviations: FC, flesh content; FCR, feed conversion ratio; FMW, final mean weight; HSI, hepatosomatic index; IMW, initial mean weight; SGR, specific growth rate; SR, survival rate; WGR, weight gain rate.

3.2 Proximate composition and fatty acid composition in muscle

The effects of different lipid sources on proximate composition in the muscle are presented in Table 4. As shown, the dietary lipid sources had no significant effects on the levels of moisture, crude protein and ash in muscle. Crude lipid contents in muscle of crayfish fed diet PO and PL were significantly higher than those of crayfish fed diet FO, SO, CO and LO (p < .05).

TABLE 4. Muscle composition of red swamp crayfish fed diets with different lipid source for 8 weeks
Items (g/kg) Dietary lipid sources
FO SO CO PO LO PL
Moisture 794.3 ± 10.8 791.5 ± 8.9 789.8 ± 15.4 781.5 ± 4.0 793.2 ± 12.9 793.9 ± 18.5
Crude protein 191.9 ± 4.8 190.1 ± 0.65 191.3 ± 7.1 188.5 ± 6.5 186.5 ± 7.0 187.4 ± 11.7
Crude lipid 23.7 ± 2.6a 22.9 ± 2.0a 24.2 ± 2.4a 27.3 ± 1.2b 21.5 ± 1.1a 28.1 ± 1.6b
Ash 1.33 ± 0.12 1.35 ± 0.04 1.28 ± 0.06 1.35 ± 0.06 1.32 ± 0.11 1.25 ± 0.03

Notes

  • Values (Means ± SD, n = 3) in the same row with different superscript letters are significantly different (< .05).

The fatty acid composition in muscle of red swamp crayfish is presented in Table 5. The percentages of SFA in the crayfish fed diet PO were significantly higher than the other treatments, followed by diet PL. Crayfish fed diet LO had significantly higher proportion of 20:4n-6 in muscle than that fed the other diets. Significantly higher EPA contents in muscle were observed in crayfish fed diet FO and LO than in those fed the other diets (p < .05). The content of muscle DHA is closely related to the dietary lipid source of crayfish, and the order of its content was FO > LO > SO > CO > PO, PL. The highest Σn-3 PUFA and Σn-6 PUFA values were found in crayfish fed diet FO and LO respectively. Group FO had higher ratio of Σn-3/Σn-6 in muscle compared with other groups (p < .05), while this ratio in groups SO and LO was significantly higher than that in groups CO, PO and PL (p < .05).

TABLE 5. Fatty acid composition (% total fatty acids) in muscle of red swamp crayfish
Fatty acid Dietary lipid sources
FO SO CO PO LO PL
14:0 2.83 ± 0.11e 0.81 ± 0.05a 1.07 ± 0.07b 1.78 ± 0.11c 0.93 ± 0.05ab 2.56 ± 0.16d
16:0 16.50 ± 0.59b 17.79 ± 0.67c 17.92 ± 0.69c 26.03 ± 1.01e 15.18 ± 0.32a 22.43 ± 0.57d
18:0 10.13 ± 0.42a 10.68 ± 0.32ab 11.21 ± 0.56b 14.59 ± 0.43c 9.96 ± 0.24a 14.51 ± 0.60c
ΣSFA 32.23 ± 0.25b 31.67 ± 0.45b 32.09 ± 1.44b 45.48 ± 0.42d 27.96 ± 0.74a 41.94 ± 1.26c
16:1 2.58 ± 0.18d 1.89 ± 0.06c 1.13 ± 0.04a 2.76 ± 0.15d 1.64 ± 0.04b 1.96 ± 0.06c
18:1 15.06 ± 0.79a 19.03 ± 0.42c 20.38 ± 0.61d 17.93 ± 0.43b 18.79 ± 0.24bc 18.42 ± 0.32bc
ΣMUFA 19.47 ± 0.61a 22.16 ± 0.27bc 23.06 ± 0.83c 23.04 ± 0.36c 21.78 ± 0.41b 21.35 ± 0.46b
18:2n-6 17.25 ± 0.44b 17.50 ± 0.62b 19.49 ± 0.57d 11.19 ± 0.61a 18.74 ± 0.63cd 17.86 ± 0.95bc
20:4n-6 5.14 ± 0.18b 6.09 ± 0.20d 5.63 ± 0.37c 4.94 ± 0.25b 6.86 ± 0.28e 3.85 ± 0.19a
18:3n-3 1.09 ± 0.06c 1.39 ± 0.07d 0.89 ± 0.04b 0.86 ± 0.04b 1.74 ± 0.06e 0.19 ± 0.02a
20:5n-3 EPA 14.60 ± 0.55d 13.15 ± 0.21c 10.96 ± 0.29b 8.66 ± 0.25a 14.13 ± 0.35d 8.53 ± 0.41a
22:6n-3 DHA 7.42 ± 0.36e 5.70 ± 0.26c 4.41 ± 0.18b 3.21 ± 0.12a 6.17 ± 0.26d 3.43 ± 0.25a
ΣPUFA 48.30 ± 0.36de 46.17 ± 0.72cd 44.85 ± 2.26c 31.47 ± 0.99a 50.25 ± 1.69e 36.71 ± 1.71b
Σn-3 PUFA 23.15 ± 0.97e 20.27 ± 0.40c 16.29 ± 0.14b 12.77 ± 0.42a 22.09 ± 0.55d 12.21 ± 0.20a
Σn-6 PUFA 22.86 ± 0.29b 23.98 ± 0.41c 25.88 ± 0.98d 16.84 ± 0.33a 26.12 ± 0.92d 22.53 ± 0.74b
Σn-3/Σn-6 1.01 ± 0.05e 0.84 ± 0.03d 0.63 ± 0.03b 0.75 ± 0.04c 0.85 ± 0.01d 0.54 ± 0.01a

Notes

  • Values (Means ± SD, n = 3) in the same row with different superscript letters are significantly different (p < .05). Some fatty acids, of which the contents are minor, trace amount or not detected(such as 12:0, 13:0, 15:0, 17:0, 20:0, 21:0, 22:0, 23:0, 24:0, 14:1, 17:1, 20:1, 22:1, 24:1, 20:2, and 20:3), are not listed in Table 2.
  • Abbreviations: MUFA, monounsaturated fatty acids; n-3 PUFA, omega 3 polyunsaturated fatty acids; n-6 PUFA, omega 6 polyunsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids.

3.3 Haemolymph biochemical parameters

Haemolymph biochemical parameters were listed in Table 6. The haemolymph TCHO level showed no difference among the six treatments (p > .05). Higher content of TG and activity of ALT were observed in group PL than those in other five groups (p < .05), whereas no significant difference was observed among these five groups (p > .05). Inversely, crayfish fed diet PL had a significant lower TP content (p < .05), whereas no significant difference was observed among crayfish fed diet FO, SO and LO (p > .05). The crayfish fed diet FO, SO and LO had a significantly higher TP content than that fed diet CO, LO and PL (p < .05). Higher GLU content was detected in crayfish from groups CO, PO and PL than that in groups FO, SO and LO (p < .05). The lowest AST activity and the highest AST activity were observed in groups SO and CO respectively. Moreover, AST activity was significantly lower in group SO than in other groups except for group FO (p < .05).

TABLE 6. Haemolymph biochemical parameters of red swamp crayfish fed diets with different lipid source for 8 weeks
Items Dietary lipid sources
FO SO CO PO LO PL
TCHO (mmol/L) 2.55 ± 0.13 2.64 ± 0.09 2.44 ± 0.12 2.48 ± 0.17 2.62 ± 0.15 2.57 ± 0.13
TG (mmol/L) 0.86 ± 0.07a 0.97 ± 0.05a 0.90 ± 0.08a 0.85 ± 0.12a 0.88 ± 0.09a 1.45 ± 0.13b
GLU (mmol/L) 3.10 ± 0.23b 2.64 ± 0.14a 4.78 ± 0.18c 4.68 ± 0.32c 2.95 ± 0.20ab 4.72 ± 0.15c
TP (g/L) 74.00 ± 3.61c 82.33 ± 4.51c 53.67 ± 5.51b 54.33 ± 4.73b 72.00 ± 4.58c 40.33 ± 3.06a
ALP (U/L) 28.00 ± 2.65b 26.33 ± 2.08b 27.67 ± 1.53b 26.00 ± 2.00b 26.33 ± 1.53b 21.67 ± 2.52a
AST (U/L) 49.33 ± 4.04ab 42.67 ± 3.51a 81.00 ± 4.00d 58.67 ± 3.51c 53.00 ± 2.65bc 79.67 ± 5.03d
ALT (U/L) 39.67 ± 3.51a 37.00 ± 2.65a 40.00 ± 5.29a 41.33 ± 3.51a 35.33 ± 4.16a 50.67 ± 3.21b

Notes

  • Values (Means ± SD, n = 3) in the same row with different superscript letters are significantly different (p < .05).
  • Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GLU, glucose; TCHO, total cholesterol; TG, triacylglycerol; TP, total protein.

3.4 Digestion related enzyme activities

The effect of dietary lipid source on the digestive enzyme activities of intestine and hepatopancreas is shown in Table 7. No differences were observed for the activities of protease and amylase in intestine and hepatopancreas among the six treatments. The lipase activity in the intestine of group SO was significantly higher than that of other five groups (p < .05). The lipase activity in the hepatopancreas of the crayfish fed diet FO, SO, CO and LO was significantly higher than that of the crayfish fed diet PO and PL (p < .05).

TABLE 7. Digestive enzyme activities in intestine and hepatopancreas of red swamp crayfish fed diets with different lipid source for 8 weeks
Items Dietary lipid sources
FO SO CO PO LO PL
Intestine
Protease (U/g protein) 25.98 ± 2.33 25.59 ± 2.05 24.75 ± 1.94 23.81 ± 1.30 24.17 ± 0.97 23.40 ± 2.09
Lipase (U/g protein) 35.60 ± 2.82c 41.64 ± 3.16d 32.81 ± 2.69bc 28.50 ± 3.92ab 29.04 ± 2.90ab 23.98 ± 1.84a
Amylase (U/mg protein) 0.47 ± 0.03 0.46 ± 0.03 0.45 ± 0.04 0.43 ± 0.03 0.45 ± 0.02 0.48 ± 0.05
Hepatopancreas
Protease (U/g protein) 56.96 ± 4.28 56.45 ± 2.72 52.36 ± 2.89 55.83 ± 4.50 56.82 ± 4.15 54.18 ± 3.51
Lipase (U/g protein) 24.78 ± 1.25b 25.34 ± 0.93b 24.43 ± 1.12b 18.57 ± 1.51a 26.71 ± 2.52b 17.52 ± 1.28a
Amylase (U/mg protein) 0.39 ± 0.05 0.41 ± 0.02 0.42 ± 0.04 0.42 ± 0.04 0.43 ± 0.03 0.40 ± 0.04

Notes

  • Values (Means ± SD, n = 3) in the same row with different superscript letters are significantly different (p < .05).

4 DISCUSSION

In this study, the growth performance and feed utilization efficiency of crayfish could be influenced by different dietary lipid sources. Crayfish fed diet SO exhibited the best growth performance among all treatments. In addition, crayfish fed the diet FO and LO showed no significant effects on growth performance compared with diet SO. Gao et al. (2020) found similar beneficial effects of FO and SO diets on crayfish growth. Although diet SO and LO lacked EPA and DHA compared with diet FO, substitute fatty acids, such as 18:2 and 18:3, may satisfy physiological function and provide energy to sustain high growth rates of crayfish. Ying et al. (2006) and Li et al. (2011) also reported the similar results in other freshwater crustacean species.

Several studies have reported that PO can be used as a lipid source for aquatic animals without incurring significant negative effects on the growth (Fonseca-Madrigal et al., 2015; Zhang et al., 2016). However, compared with FO, SO and LO groups, crayfish fed diet PO showed a lower final weight and SR in this study. Similar results were observed in juvenile swimming crab (Portunus trituberculatus) (Yuan et al., 2019) and red swamp crayfish (Liu et al., 2020). Gao et al. (2020) also reported that crayfish fed dietary PO had worse growth performance than those in the FO, SO and beef tallow groups. This maybe due to the high content of SFA, low content of PUFA and lack of EFA, could not satisfied with the lipid nutritional requirements of crayfish. Moreover, high 16:0 content, as found in the PO diet, might induce inflammatory response (Li et al., 2019). These could explain why crayfish fed PO obtained a slightly worse growth.

Animal oils, such as beef tallow and pork lard, rich in SFA, are frequently used in crustaceans (Chen et al., 2015; Deering et al., 1997; Guo et al., 2010). Gao et al. (2020) and Chen et al. (2016) found similar beneficial effects of FO, SO and beef tallow diets on growth of crayfish and Chinese mitten crab (Eriocheir sinensis). However, Thompson et al. (2010) reported that red claw crayfish fed a diet with beef tallow performed poorly compared with linseed oil and corn oil in regards to weight gain. In this experiment, the lowest WGR and SR values were observed in group PL, possibly reflecting a lack of unsaturated long-chain polyunsaturated fatty acid (LC-PUFA) and EFA in PL that lead to poor growth performance (Deering et al., 1997). A similar result was reported for red claw crayfish (Li et al., 2011) and shrimp (Zhou et al., 2007). The FCR value of crayfish fed the diet PL was significantly higher than that of other groups, which indicated that crayfish of PL group showed poor feed utilization. Further studies are required to evaluate the effect of animal oils on crayfish due to inconsistencies of the review studies.

In this study, results indicate that muscle moisture, protein and ash contents were not influenced by the different dietary lipid sources. However, crayfish with those fed with the diet PO and PL had higher levels of lipid than those fed other diets. This may be explained by the high contents of SFA in the PO (48.11%) and PL (51.53%) diet. These results were consistent with the report of bullfrog (Lithobates catesbeiana) (Zhang et al., 2016) and red swamp crayfish (Liu et al., 2020). Clarke et al. (1990) have reported that saturated fatty acids are easier to deposit in tissues than monounsaturated fatty acids and polyunsaturated fatty acids. However, several studies about shrimp have shown that carcass lipid of fish oil group is increased when compared with that of plant oil groups (Catacutan, 1991; Lim et al., 1997; Zhou et al., 2007). These differences are probably due to the difference of dietary oil source, aquatic animal species and experimental conditions.

The fatty acid composition of tissues was correlated with the fatty acid composition of the experimental diets. This has been demonstrated in several studies with different aquatic species (Chen et al., 2015; Li et al., 2011; Peng et al., 2017; Thompson et al., 2010; Yang et al., 2020). In general, muscle is an important edible part of crayfish. Thus, increasing of n-3 HUFA content in muscle will provide consumers better access to these health-promoting fatty acids (Calder, 2001; Naylor et al., 2009). FO possesses higher nutritional value than other oils because of rich n-3 HUFA concentrations (Catacutan, 1991; Lim et al., 1997). In agreement with this observation, the crayfish fed FO diet had highest contents of EPA and DHA in muscle than those fed the other diets. Moreover, despite low levels of EPA and DHA (<2.07% and 3.61% respectively) in diet SO, CO, PO, LO and PL, crayfish fed these diets showed a certain level of accumulation of EPA and DHA in the muscle. These results demonstrated that crayfish preferred retention of LC-PUFAs, especially EPA and DHA. In addition, relatively higher EPA and DHA contents of muscle were detected in groups LO and SO compared with groups CO, PO and PL. Similar result was observed in the same species (Liu et al., 2020). Therefore, the accumulation of EPA and DHA in tissue of crayfish not only can be affected by dietary fatty acid composition, but also be perhaps related to the ability of convert linolenic acid to LC-PUFAs of crayfish. However, further research is needed before drawing firm conclusions.

Serum or plasma parameters are often regarded as suitable monitoring tools of the physiological metabolic state of fish, which is closely related to their nutritional status (Bowyer et al., 2012; Coz-Rakovac et al., 2008; Monge-Ortiz et al., 2018; Richard et al., 2006). However, few information on the influences of different dietary lipid sources on haemolymph biochemical parameters of crayfish was reported. The levels of TCHO and TG are generally affected by the metabolism of lipids (Andersen et al., 1991). In the present study, higher haemolymph TG content was observed in group PL than in the other groups. This result might be partly attribute to the low content of Σn-3 PUFA in the diet PL. Peng et al. (2017) reported that n-3 LC-PUFA decreases plasma TG level in turbot (Scophthalmus maximus L.) by reducing hepatic very low-density lipoprotein/TG production. In crustaceans, haemolymph glucose is a major component of circulating carbohydrates (Chang & O'Connor, 1983). The high levels of haemolymph GLU were found in the groups CO, PO and PL. Further studies were needed to explore associations between glucose metabolism and dietary lipid sources in crayfish.

Serum TP concentration and ALP activity were associated with the nutritional status and health status of fish (Tahmasebi-Kohyani et al., 2012; Yano, 1996). Haemolymph TP content in this study was significantly increased in crayfish fed with diet FO, SO and LO compared with that of crayfish fed with other diets, which indicates the superior nutritional status of crayfish fed with these three lipid sources. The lowest ALP activity was observed in the PL group, suggested that the immune function of crayfish perhaps was suppressed by diet PL. AST and ALT are important amino acid-metabolizing enzymes that are mainly present in the cardiomyocytes, hepatocytes and liver. When the hepatocytes are diseased or damaged, AST and ALT flow out in to the bloodstream. Therefore, the activities of these aminotransferases are directly related to the extent of tissue damage of the liver (Hegazi et al., 2010; Lemaire et al., 1991). In this study, the significantly increased AST activity was observed in groups CO and PL compared with other groups. The highest ALT activity was detected in PL group. These results suggest that PL might be harmful to hepatic function of crayfish. Similar results were observed in yellowcheek carp (Elopichthys bambusa) (Chen et al., 2013) and gibel carp (Carassius auratus) (Ye et al., 2012).

Several studies had demonstrated that the activities of digestive enzymes in crustaceans were regulated by the dietary nutrients (Li et al., 2012; Puello-Cruz et al., 2002). Therefore, the digestive enzymes activities can be used as an important indicator for the digestion, absorption and utilization of various nutrients in the feed of crustacean (Guo et al., 2010; Wei et al., 2018). In the current study, the protease and amylase activities were not markedly affected by dietary lipid source, similar to the result in red claw crayfish (Lu et al., 2019). These may be due to using the same kind and amount of protein source and carbohydrate source in six diets. Previous study has demonstrated that the lowest activities of lipase in intestinal and hepatopancreas were detected in gibel carp fed with PL treatment (Wang et al., 2011). Similarly, the present study highlighted a significant decrease in the activities of intestinal and hepatopancreas lipase of crayfish fed diet PL. In addition, the lipase activities in intestinal of crayfish fed diet PO were significantly lower than those of crayfish fed diet FO, SO, CO and LO. The lipase activities generally increased with increasing dietary lipid levels (Fountoulaki et al., 2005; Liao et al., 2017). In this study, the lipid content of six diets was about equal to 85 g/kg. The difference of lipase activities was might be related to the types and contents of the fatty acid (Mukhopadhyay & Rout, 1996). In some fish species, the source of dietary lipids, such as high levels of PUFA, or high content of phospholipids increased lipase activities (Cai et al., 2016; Morais et al., 2004, 2007). The lipase activity in hepatopancreas and intestine exhibited a similar tendency with growth performance, which indicates that digestive enzyme activities could influence digestive capacity and ultimately affect growth performance.

5 CONCLUSIONS

The results of this study demonstrated that fish oil could be replaced by soybean oil or lecithin oil in crayfish diets for an experimental period of 8 weeks without negatively affecting the growth performance and survival. These alternative lipid sources have effect on the fatty acid composition of muscle which was correlated to the fatty acid composition of diets. The present study suggested that pork lard might be harmful to hepatic function of crayfish. Moreover, the results showed that the pork lard could decrease hepatic and intestine lipase activity of crayfish. These findings may contribute to optimize diets formulation of red claw crayfish when considering selection of optimal lipid sources.

ACKNOWLEDGEMENTS

This work was supported by the Open Project of Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, and Key Laboratory of Freshwater Aquaculture genetic and breeding of Zhejiang Province, China (ZJK202005); Major projects of Hubei province technical innovation special program (2019ABA077); Key Research and Development Project of Sichuan Province (2018NZ0152); and Industry-university-research cooperation of Tongwei Co. Project (TW2018I001)

    DATA AVAILABILITY STATEMENT

    All data are available from the corresponding author by request.

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