Dietary lipid sources modulate the intestinal transport of fatty acids in the red swamp crayfish Procambarus clarkii

Journal Pre-proof Dietary lipid sources modulate the intestinal transport of fatty acids in the red swamp crayfish Procambarus clarkii

Fan Gao, Jie Liu, Aimin Wang, Bo Liu, Hongyan Tian, Xiaochuan Zheng, Xiaoyan Jia, Chang He, Xiangfei Li, Guangzhen Jiang, Cheng Chi, Wenbin Liu, Dingdong Zhang PII:

S0044-8486(19)32751-6

DOI:

https://doi.org/10.1016/j.aquaculture.2020.735091

Reference:

AQUA 735091

To appear in:

aquaculture

Received date:

16 October 2019

Revised date:

6 February 2020

Accepted date:

6 February 2020

Please cite this article as: F. Gao, J. Liu, A. Wang, et al., Dietary lipid sources modulate the intestinal transport of fatty acids in the red swamp crayfish Procambarus clarkii, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2020.735091

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© 2019 Published by Elsevier.

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Dietary lipid sources modulate the intestinal transport of fatty acids in the red swamp crayfish Procambarus clarkii Fan Gaoa; Jie Liua; Aimin Wangb; Bo Liuc; Hongyan Tianb; Xiaochuan Zhenga; Xiaoyan Jiaa; Chang Hea; Xiangfei Lia; Guangzhen Jianga; Cheng Chia; Wenbin Liua;

a

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Dingdong Zhanga*

Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College

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of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095,

College of Marine and Biology engineering, Yancheng institute of Technology,

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b

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China.

Yancheng 224051, China.

214081, China.

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Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi

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c

Dr. D. Zhang

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*Corresponding author:

Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College of Animal Science and Technology Nanjing Agricultural University Nanjing 210095 China Tel.: +86-25-84395382; Fax: +86-25-84395382 E-mail: [email protected] 1

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Abstract: The optimization of feed formulations is an effective strategy to increase the growth of aquatic animals, but the effects of dietary lipid sources other than fish oil are not well established in most of crustacean. In this study, we investigated how different dietary lipid sources influence fatty acid absorption, synthesis, and transport in the red swamp crayfish (Procambarus clarkii). Six isonitrogenous and isocaloric

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diets were formulated with the following lipid sources: fish oil (FO), corn oil (CO), rapeseed oil (RO), soybean oil (SO), palm oil (PaO), and beef tallow (BT). The

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crayfish fed FO, SO, or BT exhibited a significantly higher weight gain rate (WGR)

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and specific growth rate (SGR) and a lower feed conversion ratio (FCR) than those of

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crayfish fed PaO. Polyunsaturated fatty acid (PUFA) profiles in the hepatopancreas and muscle tissues closely mirrored those in the diets. Low total triglyceride (TG) and

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total cholesterol (TC) contents were observed in the hemolymph and hepatopancreas

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of crayfish fed FO or BT diet. In the lipid anabolism pathway, fatty acid synthase

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(fas), sterol regulatory element-binding protein 1 (srebp1), and retinoid X receptor (rxr) were down-regulated by dietary FO but up-regulated by some vegetable oils. A gene involved in fatty acid (FA) uptake and transport, fatty acid binding protein (fabp), was up-regulated by CO in the hepatopancreas and by BT in muscle tissues. With respect to intestinal fatty acid transporters, fatty acid transport protein 4 (FATP4) was up-regulated in the BT diet, diacylglycerol transferase1 (DGAT1) was down-regulated in the FO or BT diet, and Niemann-Pick C1- like 1 (NPC1L1) was down-regulated in the SO diet. Apolipoprotein B-48 (Apo B-48) and fatty acid translocase (FAT/CD36) were not influenced by dietary lipid sources. According to 2

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the observed results, BT is the more suitable candidate for FO replacement in crayfish diets, whereas PaO is not recommended. Keywords: Procambarus clarkii; lipid source; fatty acid; intestinal lipid transporter 1. Introduction The red swamp crayfish (Procambarus clarkii) was regarded as a bio- invasion

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species immigrated from North America to China in the early stage, whereas it is now widely cultured in inland areas of China. Its taste and high nutritional value make it

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very popular with consumers (Tan, et al., 2018). Although artificial feed is used for

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production, nutritional studies of this species have mainly focused on the appropriate

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levels of macronutrients (Jover, et al., 1999; Xu, et al., 2013), alternatives to fish meal (Hua, et al., 2015; Wan, et al., 2017), and the optimal ratio of dietary protein and lipid

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(Carmona-Osalde, et al., 2005; Hubbard, et al., 1986). However, little is known about

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appropriate dietary lipid sources for fish oil substitution in red swamp crayfish.

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Lipids are important nutrients for energy uptake and have key nutritional functions, including maintaining the integrity of cell membranes and providing essential fatty acids (Ma, et al., 2017). Lipids added to compound feed are usually derived from plant-origin or animal-origin oils. Alterations in the amount and composition of dietary lipids can influence the growth and health of aquatic animals. Fish oil (FO), rich in n-3 highly unsaturated fatty acids, is believed to be the more suitable lipid source for crustacean diets (Gonzalez-Felix, et al., 2009). However, FO demand is increasing, with limited production, emphasizing the need for vegetable and terrestrial animal fats as alternatives in aquafeed (Naylor, et al., 2009). Some 3

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studies have proven that canola oil (Kim, et al., 2013), linseed oil (Thompson, et al., 2010), and a mixture of vegetable and animal oils (Chen, et al., 2015) have no detrimental effects on the growth performance of shrimp compared with FO. With respect to fat bio- utilization, intestinal absorption and the delivery of dietary fat play a central role in lipid homeostasis (Abumrad, Davidson, 2012). However, the

during exposure to diverse lipid sources is unclear.

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modulation of the transintestinal transport of fatty acids and cholesterol in crustacean

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The crustacean gastrointestinal tract, a relatively simple straight tube, includes a

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foregut (mouth, oesophagus, and stomach), midgut, and hindgut (Poljaroen, et al.,

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2018; To, et al., 2004), and is mainly involved in the catabolism of ingested food and absorption of nutrients (Brown, 1995). Normally, lipid digestion and absorption occur

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by a complex, multistep process that begins in the stomach and ends in the intestine in

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mammals (Mcclements, Decker, 2009). Most dietary fat consumed by animals enters

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the intestine in the form of complex lipids rather than free fatty acids. Before the components can pass through the enterocyte membrane, they must be broken down into fatty acids (FA) and 2-monoacylglycerols (2-MG) by intestinal lipases (Quinlivan, Farber, 2017). Then, FA and 2-MG cross the intestinal epithelial membrane into cells in the presence of fatty acid transport protein 4 (FATP4) and bind to specific fatty acid binding proteins (Ii, Abumrad, 2012) . After being transported to the endoplasmic reticulum, FA and 2-MG are re-esterified to synthesize triglycerides (TG) under the action

of

monoacylglycerol

acyltransferase

(MGAT)

and

diacylglycerol

acyltransferase (DGAT) (Kindel, et al., 2010). With the help of microsomal 4

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triglyceride transporter (MTP), the re-synthesized TG enters the endoplasmic reticulum and combines with apolipoproteinB-48 (ApoB-48) to form a primordial chylomicron, which then further acquires apoA-Ⅰ to form mature chylomicrons and finally releases into the lymphatic system (Buttet et al., 2014; Julve et al., 2016). Thus, the aims of this study were to determine (1) whether the red swamp crayfish

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has these crucial intestinal transport proteins, (2) how various dietary lipid sources coordinate the intestinal transport of fatty acids and lipid metabolism, and (3) the

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more suitable lipid source for the replacement of FO.

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2. Materials and methods

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2.1. Ethics statement

The experimental animal procedures were guided by the Care and Use of

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Laboratory Animals in China. The Animal Care and Use Committee of Nanjing

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Agricultural University (Nanjing, China) approved all experimental protocols in this

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study (No. IACUC2018613).

2.2. Crayfish and Experimental conditions Red swamp crayfish were purchased from a farm in Pukou, Jiangsu province, China. Crayfish were initially fed with a commercial feed (Haipurui Feed Co., Ltd., Taizhou Jiangsu) twice a day (6:00 and 18:00). After 9 days of acclimation to the experimental conditions, 336 crayfish (average initial weight: 11.73 ± 0.05 g) were randomly assigned to 24 cement pools (1.0 × 1.0 × 1.0 m, L: W: H; six treatments with four replicates) with a stocking rate of 14 individuals per pool. The crayfish were fed twice a day with a ration of 3–5% of the body weight. Every 2 weeks, crayfish 5

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were reweighed to adjust the daily feeding volume. During the trial, one-third of the pool water was changed every 3 days and water quality parameters were determined periodically. Water temperature, pH, dissolved oxygen and ammonia nitrogen were maintained as follows: 28 ± 2°C, 7.39 ± 0.13, 5.72 ± 0.36 mg/L, and <0.05 mg/L, respectively.

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2.3. Experimental diets The formulation and proximate composition are presented for six isonitrogenous

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and isocaloric diets (Table 1). Fish meal, soybean meal, rapeseed meal, and

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cottonseed meal served as the main protein sources, wheat flour and α-starch were

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used as the main carbohydrate sources, and fish oil (FO), corn oil (CO), rapeseed oil (RO), soybean oil (SO), palm oil (PaO), or beef tallow (BT) was added as the lipid

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source. The fatty acid profiles of diets with different lipid sources are shown in Table

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2. All ingredients were sieved through a 0.25- mm mesh before production and were

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weighed and mixed thoroughly. Pelleting was carried out through a single-screw meat grinder (JR-32, Baicheng Co., Guangzhou, China). The pellets were dried in a ventilation room for 24 h, broken up to an appropriate size for crayfish, and stored in sealed plastic bags at −20°C until use. 2.4. Sample collection Crayfish were fasted for 24 h after the last meal in the trial to ensure that the intestines were empty (Davis, Robinson, 2010). Before sample collection, crayfish were anesthetized on ice. Then, six hemolymph samples from each replicate were drawn from the pericardial cavity using a 2-mL syringe preloaded with 1 mL of 6

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anticoagulant buffer (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, and 10 mM EDTA, pH 4.6) (Liao, et al., 2018). The supernatant was aspirated immediately from the mixture after centrifugation and stored at −80°C for subsequent analyses. The crayfish were then dissected to obtain the hepatopancreas, muscle, and midgut (a sector of the intestine near the stomach). One part of these

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tissues was frozen at −80°C and another part was stored at −20°C. 2.5. Analytical procedures

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2.5.1. Growth parameters

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To evaluate the growth performance of crayfish, the weight gain rate (WGR),

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specific growth rate (SGR), feed conversion ratio (FCR), and hepatopancreas index (HI) were calculated according to the following formulas:

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WGR = (Wt –W0 ) × 100/W0 ,

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SGR = (LnWt −LnW0 ) × 100/T,

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FCR = Feed intake/total wet weight gain, HI = Hepatopancreas weight × 100/body weight, where W0 and Wt are the initial and final body weight, respectively, T is the duration of the rearing period (days), and Feed intake was estimated using the feed ration given as a fixed percentage of fish's weight. 2.5.2. Proximate composition analysis According to the standard methods of AOAC (Cunniff, 1995), both diets and crayfish were evaluated with respect to various parameters, including moisture, crude protein, crude lipid, ash, and gross energy. Moisture was estimated by oven-drying 7

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twice at 105°C until achieving a constant weight. The micro-Kjeldahl method was adopted to determine the crude protein content (nitrogen × 6.25) using an Auto Kjeldahl System (KT260; FOSS, Zürich, Switzerland). Crude lipids were analyzed using Soxtec System HT6 (Tecator, Hoganas, Sweden) by solvent extraction. Ash was measured using a muffle furnace at 550°C for 4 h. Gross energy was analyzed

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using a Bomb Calorimeter (PARR 1281; Parr Instrument Company, Moline, IL, USA).

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2.5.3. Analysis of physiological and biochemical properties

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Physiological and biochemical properties, including total TG (Cat. No. A110-1,

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Jiancheng Co., Nanjing, China), total cholesterol (TC) (Cat. No. A111-1, Jiancheng Co.), high density lipoprotein cholesterol (HDL-C) (Cat. No. RJ-25215, Renjie Co.,

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Shanghai, China), and low-density lipoprotein cholesterol (LDL-C) (Cat. No.

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RJ-25216, Renjie Co.) were detected using commercial kits in the hemolymph and

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hepatopancreas. All processes were performed according to the manufacturers’ recommendations.

2.5.4. Fatty acid composition analysis Lipids in the diets, hepatopancreas, and muscle tissues were extracted with a chloroform: methanol (2:1 V:V) mixture (Folch, et al., 1957), then methylated using 0.5 mol/l NaOH in methanol for 30 min at 60 °C and then esterified in 14% boron trifluoride in methanol (Park, Goins, 1994). Fatty acid methyl esters were quantitatively analyzed by gas chromatography (GC-2030; Shimadzu Co. Ltd., Kyoto, Japan), according to a previously described method (Bondia, et al., 1994) and made 8

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some adjustment. 2.5.5. Quantitative RT-PCR Total RNA was extracted from the hepatopancreas, muscle, and intestine using the RNAiso Plus Kit (Cat. No. 9109, TaKaRa, Co. Ltd., Dalian, China), following the manufacturer’s protocols. The concentration and quality of RNA were analyzed by

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spectrophotometry using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The purity of total RNA was measured by absorbance at 260 and 280 nm,

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and integrity was evaluated by 1.0% formaldehyde denaturing agarose gel

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electrophoresis. cDNA was prepared from 500 ng of DNase-treated RNA using a

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PrimeScript™ RT Master Mix Kit (Cat. No. RR036A, TaKaRa, Co. Ltd., Dalian, China), following the manufacturer's recommendations. The resulting cDNA was

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diluted with DEPC-treated water and used as a template for quantitative PCR using a

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TB GreenT M Premix Ex TaqT M (Tli RNaseH Plus) Kit (Cat. No. RR420A, TaKaRa,

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Co. Ltd.) and a real-time PCR detection system (ABI7300; Applied Biosystems, Foster City, CA, USA). The total volume of PCRs was 20 μL, consisting of 10 μL of TB GreenT M Premix Ex TaqT M (Tli RNaseH Plus) (2×), 0.4 μL of PCR forward primer (10 µM), 0.4 μL of PCR reverse primer (10 μM), 0.4 μL of ROX reference dye (50×), 2.0 μL of cDNA template, and 6.8 μL of dH2 O. The reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The specificity of PCR products was determined by melting curves and electrophoresis. Ct values were obtained and the relative gene expression levels were calculated by the 2−ΔΔCt method as described previously (Livak, Schmittgen, 2001). Eukaryotic 9

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translation initiation factor (EIF) (Jiang, et al., 2015) was used as an endogenous control for normalization of the gene expression levels of target genes. Gene-specific primers for fatty acid binding protein (fabp), fatty acid synthase (fas), and sterol regulatory element-binding protein 1 (srebp1) were designed using Primer Premier 5, and the primer pairs for retinoid X receptor (rxr) were described previously (Dai, et

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al., 2016). All primers are presented in Table 3. 2.5.6. Western blot analysis

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Total protein was extracted from the intestine utilizing RIPA lysis buffer (Cat. No.

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P0013B; Beyotime Institute of Biotechnology, Shanghai, China) and centrifuged at

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12,000 rpm and 4°C for 10 min. The supernatant was used to quantify protein contents using a commercial BCA protein kit (Cat. No. P0012; Beyotime Institute of

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Biotechnology). A total of 30 μg of heat-denatured protein was loaded into each well

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and separated by SDS-PAGE using the Mini-Protean Tetra Electrophoresis System

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(BioRad, Hercules, CA, USA), followed by transfer to polyvinylidene fluoride membranes (IPVH00010; Millipore Co., Billerica, MA, USA). After blocking non-specific binding, the membrane was incubated with the primary antibody before adding a horseradish peroxidase-conjugated secondary antibody. Finally, the BeyoECL Plus Kit (Cat. No. P0018M; Beyotime Institute of Biotechnology) was used to visualize protein expression. Antibodies against FATP4 (Cat No. SC-393309; Santa Cruz Biotechnology, Santa Cruz, CA, USA), APO B-48 (Cat. No. SC-13538; Santa Cruz), FAT/CD36 (Cat. No. SC-7309; Santa Cruz), DGAT1 (Cat. No. SC-271934; Santa Cruz), NPC1L1 (Cat. No. SC-166802; Santa Cruz), and GAPDH 10

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(Cat. No. SC-47724; Santa Cruz) were used. 2.6. Statistical analyses Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was employed to determine the effects of different lipid sources on crayfish. Tukey's HSD multiple range test was applied for comparisons between dietary treatments.

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Differences were considered significant at P < 0.05. The results were analyzed using

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SPSS version 24.0 (SPSS Inc., Chicago, IL, USA).

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3. Results

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3.1. Growth performance

The final weight of crayfish was significantly higher in the FO group than in the

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PaO group (Table 4). Crayfish fed dietary PaO had a lower WGR than those in the FO,

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SO, and BT groups (Table 4). The PaO group had a significantly lower SGR than

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those in the other groups, except the RO group. Compared to other groups, BT group got very close WGR and SGR to FO group. FCR was highest in the PaO group (Table 4), and there were no significant differences in HI among groups. 3.2 Whole body composition The proximate composition of the whole body (% wet basis) is presented in Table 5. The moisture, crude protein, crude lipid, and ash contents as well as gross energy did not differ significantly among groups treated with various dietary lipid sources. 3.3. Biochemical parameters in the hemolymph and hepatopancreas

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In the hemolymph, the RO group had the highest TG and TC contents, while the BT and FO groups had the lowest TG and TC levels. (Table 6). However, no significant differences were observed in HDL-C and LDL-C among groups. In the hepatopancreas, the SO group had the lowest TG level, which was significantly lower than the levels in the CO, RO, and PaO groups. The hepatopancreas TC content was

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lower in the FO group than in the other groups, except the BT group. Crayfish fed the BT diet exhibited the lowest LDL-C, which was significantly lower than that in the

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CO group. However, there were no differences in HDL-C among groups.

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3.4. Hepatopancreas, and muscle FA profiles

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The FA profiles of hepatopancreas are shown in Tables 7. The saturated fatty acid (SFA) contents in the FO, BT, and PaO groups were significantly higher than those in

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the SO, CO, and RO groups. The MUFA contents were highest in the RO group,

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while crayfish fed PaO and BT diets were higher than FO, CO and SO diets, and FO

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group was higher than CO or SO groups. The hepatopancreatic PUFA contents were significantly higher in SO and CO groups than others, mealwhile FO and RO groups were higher than PaO and BT groups. N-6 PUFA accounted for the majority of PUFA (especially C18:2n-6), and crayfish fed CO and SO diets had higher hepatopancreatic n-6 PUFA contents than others, while RO group was higher than BT group. The EPA and DHA contents were highest in crayfish fed the FO diet, whereas the C18:3n-3 contents were significantly higher in RO and SO groups than others even though FO group was higher than PaO and BT groups.

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In the FA profiles of muscle tissue, there were no significant differences in C17:0, C18:0, SFA, C16:1, and C20:4n-6 among groups (Table 8). MUFA contents were significantly higher in the groups fed the BT, PaO, and RO diets than FO, CO and SO diets. The EPA contents were higher in FO group than CO, RO and SO groups while DHA contents were higher in FO group just than SO group. Similar to the FA

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composition in the hepatopancreas, the CO and SO groups had significantly higher n-6 PUFA contents (mostly C18:2n-6) than other experimental groups. The higher

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total PUFA levels were observed in CO and SO groups than others (except FO

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group).

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3.5 Gene expression levels in the hepatopancreas, muscle, and intestine of crayfish In the hepatopancreas, fabp mRNA levels were significantly higher in the CO

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group than in the PaO and BT groups (Fig. 1A). Crayfish fed the RO and BT diets had

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significantly higher levels of fas than those in the FO, SO, and PaO groups (Fig. 1B).

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The gene expression levels of srebp1 were remarkably lower in the FO and BT groups than in the CO, RO, SO, and PaO groups (Fig. 1C). There were no significant differences in the gene expression of rxr among groups (Fig. 1D). In muscle tissues, crayfish in the BT group had the highest levels of fabp, which were significantly higher than those in the RO group (Fig. 1E). Compared with the FO, CO, and RO treatments, the BT group had higher gene expression levels of fas (Fig. 1F). Crayfish fed the SO diet exhibited the highest gene expression levels of srebp1 which were significantly higher than those in the FO and BT groups, while SO,

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RO, CO and PaO groups showed no significant difference (Fig. 1G). There were no significant differences in rxr expression among groups (Fig. 1H). With respect to the intestinal expression of fatty acid metabolism-related genes, fas gene expression was significantly higher in the BT group than in the FO, CO, or SO groups (Fig. 1J). The srebp1 mRNA level in the SO group was significantly

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higher than those in the FO and PaO groups (Fig. 1K). The transcript expression of rxr was significantly higher in the RO group than in the SO, PaO, and BT groups (Fig.

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1L). No significant differences in fabp gene expression among groups were observed

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3.6 Protein expression in the intestine

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(Fig. 1I).

Protein expression patterns were determined to evaluate intestinal transport in fish

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fed different oil sources (Fig. 2). The BT diet significantly increased the expression of

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FATP4 in the intestine while the SO diet significantly reduced the expression of

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FATP4 compared to levels in the other groups. DGAT1 expression levels were significantly lower in fish fed the FO and BT diets in fish fed the CO, RO, and SO diets. There was a significant difference in NPC1L1 expression between the RO and SO diets. No significant differences in APOB-48 and FAT/CD36 expression were observed among groups.

4. Discussion We evaluated the effects of lipid sources on growth performance in crayfish. We found similar beneficial effects of FO, BT, and SO diets on crayfish growth. A case in 14

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Chinese mitten crab also demonstrated that BT and SO are good FO substitutions in terms of growth (Chen, et al., 2016). EPA, DHA, arachidonic acid (ARA), linoleic acid and α- linolenic acid have been shown benefical values to the growth and other functions for aquatic animal (Glencross, 2009). FO contains a balanced fatty acid profile and high EPA and DHA contents, while the SO diet has high linoleic acid

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(C18:2n-6) and α- linolenic acid (C18:3n-3) contents, which contribute to metabolic processes and provide energy to sustain high growth rates (Li, et al., 2011; Wen, et al.,

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2003). Generally, high SFA and low n-6 PUFA contents, as found in the BT diet,

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might promote n-3/ n-6 PUFA ratio in muscle and result in a final product quality

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(Emery, et al., 2014; Peng, et al., 2016). However, in some other research, EPA and DHA were more bioavailable in SFA/MUFA-rich BT-based diets (Bowzer, et al.,

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2016; Rombenso, et al., 2017), thereby improving normal growth and physiological

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function. In contrast, although PaO diet is also rich in SFA/MUFA as BT, BT has

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higher level of C18:0 while PaO is rich in C16:0. Excess C16:0 or high percentage of dietary palm oil could induce inflammatory reponse (Li, et al., 2019). The surplus of C16 leading to possible inflamatory responses might shift the preferred substrate/pathway for β-oxidation towards the availability of C18 (Loften, et al., 2014). This could explain why crayfish fed PaO obtained a slightly worse growth. The fatty acid profiles in the hepatopancreas and muscle tissues were similar to the fatty acid profiles of the diets, consistent with previous results for crustacean, such as Macrobrachium nipponense (Ding, et al., 2014), Sagmariasus verreauxi (Shu-Chien, et al., 2017), and Litopenaeus vannamei (Gonzalez-Felix, et al., 2010). 15

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The dietary fatty acid composition has a crucial effect on the lipid digestibility. It was concluded that fatty acid digestion in crustacea improves as the level of unsaturation increases, and also as the length of fatty acid chain decreases (Glencross, 2009). Our results indicated fatty acids, such as DHA, EPA and ARA, appeared to be retained in the muscle, as evidenced by their higher concentrations in muscle tissues than in

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experiment diets. In contrast, the content of linoleic acid accumulated in muscle was less than that in the corresponding experiment diet, and this might be that crayfish

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synthesize highly unsaturated fatty acid from linoleic acid, as shown in Litopenaeus

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vannamei (Chen, et al., 2017). Interestingly, studies of terrestrial oils indicated that

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their inclusion in aquafeeds may minimise the utilisation of n-3 LC-PUFA for β-oxidation and thereby maximise their retention/deposition in muscle (Francis, et al.,

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2014). Some FAs, particularly SFA and MUFA, appear to enhance the retention of

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n-3 LC-PUFA in the muscle (Trushenski, 2009), and this occurrence was later termed

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“n-3 LC-PUFA sparing effect” (Rombenso, et al., 2018). The preferential reservation of certain n-3 PUFAs at the cost of SFA or MUFA has been demonstrated in some other shrimp (Ouraji, et al., 2011; Vasagam, et al., 2005). By contrast, DHA and EPA levels were lower in the hepatopancreas than in the diets, indicating an enhanced utilization of n-3 PUFAs in the crayfish hepatopancreas, as supported by some other studies (Sanchez, et al., 2014; Yuan, et al., 2019). This consequence was rational since most of the n-3 PUFA (especially DHA and EPA) are dietary essential FAs for crustacean (Glencross, 2009), and are specifically designed to satisfy the growth requirements (Velu, Munuswamy, 2004). Hence, differential utilization of various 16

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fatty acids in the hepatopancreas and muscle tissues might depend on tissue-specific functions and mainly experimental conditions. Biochemical parameters in the hemolymph reflect the nutritional status, in vivo metabolism, and health in aquatic animals (Zhou, et al., 2015). We found that hemolymph TG and TC, but not HDL-C and LDL-C, were affected by the lipid

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source. n-3 PUFAs are more likely to attenuate TG and TC levels by impairing very low density lipoprotein assembly or secretion (Kjaer, et al., 2008). Similar results

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have been obtained for Trachinotus ovatus (Liu, et al., 2018) and Acipenser sinensis

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(Wu, et al., 2014). DGAT, an enzyme involved in TG biosynthesis, is positively

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correlated with plasma TG (Sachdev, et al., 2016a). In this study, DGAT1 expressions in FO, PaO and BT were lower than those in other groups, and this might explain the

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low TG levels in crayfish fed BT, PaO and FO in our study. Besides DGAT, oleic acid

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may contribute plasma TG. Several studies in vitro indicated that oleic acid (C18:1)

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decreased the expression of PPARα and increased the expression of PPARγ (Wu, et al., 2012). Lower PPARα can inhibit fatty acid catabolism while higher PPARγ can promote triacylglycerol synthesis and lipid accumulation (Cui, et al., 2010; Edvardsson, et al., 2006). This may explain why RO diet could stimulate the synthesis or secretion of TG for the high C18:1 content, as observed in Atlantic salmon hepatocytes (Kjaer, et al., 2008). We further found that hepatopancreatic fabp, which is involved in FA uptake and transport (Lappas, 2014), is upregulated in the CO group. The levels of fabp in the hepatopancreas may reflect levels of hepatopancreatic and dietary PUFA, which can 17

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induce fabp expression (Bass, 1988; Kaikaus, et al., 1993). Compared with linoleic acid (LA), high contents of α- linolenic acid (ALA) appeared to be more conducive to suppress fabp expression (Xu, et al., 2019), supporting why a little more PUFA in SO group can not induce more fabp expression as CO group. Levels of hepatopancreatic fas, which is related to FA synthesis (Lappas, 2014), were lowest in crayfish fed FO

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due to the high levels of EPA and DHA, which suppress lipid synthesis. Similarly, srebp1 regulates lipid biosynthesis and is decreased by EPA+DHA (Jin, et al., 2017).

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We detected low levels of srebp1 in animals fed the FO diet, rich in EPA and DHA.

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However, in Salmo salar, srebp levels increase after cholesterol treatment (Minghetti,

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et al., 2011). In accordance with the high cholesterol level in the hepatopancreas of

study.

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crayfish fed vegetable oil, srebp1 was up-regulated by vegetable oil-based diets in this

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Fatty acids contribute the majority of the energy required in muscle (Storch,

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Thumser, 2010), one of the main site of lipid deposition (Corraze, Kaushik, 2009). Fabp has tissue-specific functions in lipid and fatty acid metabolism and enhances uptake of FAs into the cell and facilitates LCFA transport to maintain efficient mitochondrial β-oxidation in the muscle (Haunerland, Spener, 2004; Storch, Thumser, 2010). The high levels of fabp in the BT group suggested that BT promotes fatty acid oxidation in the muscle (Figure 1). Usually in animal there is a FA substrate preference during β-oxidation, with SFA being the primary oxidization targets, followed by MUFA and PUFA (Araujo, et al., 2018). Fas catalyzes SFA biosynthesis from simple precursors, and higher dietary PUFA inhibit de novo fatty acid synthesis 18

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than diets containing SFA and MUFA (Khatun, et al., 2017). In present results, BT up-regulated fas expression to facilitate lipid synthesis, and an equilibrium between synthesis and catabolism might be suitable for growth. Furthermore, FO down-regulated srebp1 in muscle tissues, while RO and SO up-regulated srebp1 (Figure 1). The results of Sagmariasus verreauxi also indicated that vegetable oil

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blend enriched in linolenic acid (18:3n-3) significantly upregulated srebp1 expression (Shu-Chien, et al., 2017), as might explain why RO and SO could up-regulate srebp1

pr

in our study.

e-

The intestine is responsible for the assimilation of dietary lipid (Storch, Thumser,

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2010). It was reported that dietary n-3 PUFA (especially DHA and EPA) suppresses the proteolytic processing of srebp1, and down-regulates the expression of target

al

genes involved in fatty acid synthesis including fas (Lin, et al., 2019; Peng, et al.,

rn

2017). However, SO diet increased srebp expression indicated a need for higher

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cholesterol biosynthesis (Carmona-Antonanzas, et al., 2014). In our results, FO could reduce fas and srebp1 to restrain lipogenesis while BT and SO could accelerate this process, consistent with previous results for Litopenaeus vannamei (Chen, et al., 2015) and Sagmariasus verreauxi (Shu-Chien, et al., 2017). Rxr regulates adipogenesis and fatty acid homeostasis (Lappas, 2014). In the hepatopancreas and muscle tissues, the lipid source had no effect on rxr levels, unlike in the intestine, where RO- fed crayfish displayed an increase in rxr. According to previous studies, n-3 PUFAs are likely to activate rxr as a specific ligand (Fan, et al., 2003; Hiebl, et al., 2018); crayfish fed FO in the present study exhibited a similar trend, but the differences among diets were not 19

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significant. Although RO was not rich in n-3 PUFA, the high oleic acid content might induce the acativation of rxr, as demonstrated in a previous study (Lengqvist, et al., 2004). The

intestinal digestion of dietary

lipids produces

free

fatty acids,

monoacylglycerols, and lysophospholipids, and these products are absorbed by (Beguin, et al.,

2014). FA absorption involves

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intestinal epithelial cells

protein- mediated uptake mechanisms (Hussain, 2014); therefore, we analyzed five

pr

representative fatty acid transporters. FATP4 is the main fatty acid transporter in the

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intestine and mediates effective absorption (Stahl, et al., 1999). We found that

Pr

intestinal FATP4 was significantly up-regulated by the BT diet but was lower in the SO group than in the FO, CO, RO and PaO groups. In the intestine, DGAT1 is

al

involved in TG biosynthesis via dietary fatty acids (Sachdev, et al., 2017). In rats,

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DGAT1 expression is positively correlated with plasma TG and TC (Chandak, et al.,

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2011; Hung, Buhman, 2019; Sachdev, et al., 2016b). Similarly, our results suggested that FO or BT reduce DGAT1 and inhibit TG synthesis. In addition, Niemann-PickC1 like1 (NPC1L1) is a reliable marker of cholesterol transport and absorption and is important for the control of plasma cholesterol (Alqahtani, et al., 2015). In our study, NPC1L1 expression was lower in crayfish fed the SO diet than RO diet. High PUFA levels in SO can explain this finding, since in-vitro experiments have verified that PUFA decreases NPC1L1 expression, thereby limiting cholesterol absorption and increasing cholesterol secretion from the body (Park, Carr, 2013). CO and SO groups had the similar levels of PUFA, but CO group could not decrease NPC1L1 expression 20

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as SO group. This might be explained that CO group had lower ALA level than SO group, supporting by a previous study (Deng, et al., 2016). However, we did not detect differences in FAT/CD36 or APOB-48 in crayfish, consistent with previous results for Salmo salar fed FO or vegetable oil (Morais, et al., 2011; Torstensen, et al., 2009).

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In conclusion, our results demonstrated that crayfish fed SO or BT diets exhibit similar growth performance to that of crayfish fed the FO diet, while dietary PaO

pr

clearly suppresses grow compared to FO. Furthermore, the SO and BT diets

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up-regulated some lipid anabolism genes to promote lipid synthesis, while the FO diet

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had the opposite effects. FATP4, DGAT1, and NPC1L1 in the lipid transport pathway were influenced by the lipid source. Based on the expression of these transport

al

proteins, we speculate that BT could inhibit TG synthesis and promote FA transport.

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Taken together, our findings suggested that BT is the more suitable alternative to FO

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with respect to growth performance or lipid synthesis and transport, and PaO is not a suitable lipid source for red swamp crayfish.

Acknowledgements This work was supported by Jiangsu Agricultural Industry Technology System (Red Swamp Crayfish) (JFRS-03), the earmarked fund for China Agriculture Research System (CARS-48), and Jiangsu Agriculture Science and Technology Innovation Fund (CX (17) 2007-01). We would also like to thank Qian, H., Liu, J., Zheng, X. C., Jia, X. Y., He, C., Wang, C. C., and Shi, H. J. for their help during the 21

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feeding trial and experimental process.

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digestibility, nitrogen and phosphorus excretion in red swamp crayfish, Procambarus clarkii. Aquacult Int. 25, 543-554. Wen, X.B., Yao-Mei, K.U., Zhou, K.Y., 2003. Growth Response and Fatty Acid Composition of Juvenile Procambarus clarkii Fed Different Sources of Dietary Lipid. Agricultural Sciences in China. 2, 583-590. Wu, F., Liu, W., Wei, Q.W., Wen, H., Jiang, M., Yang, C.G., Tian, J., 2014. Effects of 32

Journal Pre-proof dietary lipid sources on growth performance, carcass composition, and blood parameters of juvenile Chinese sturgeon (Acipenser sinensis Gray, 1835). J Appl Ichthyol. 30, 1620-1625. Wu, H.T., Chen, W., Cheng, K.C., Ku, P.M., Yeh, C.H., Cheng, J.T., 2012. Oleic acid activates peroxisome proliferator-activated receptor delta to compensate insulin resistance in steatotic cells. J Nutr Biochem. 23, 1264-1270. Xu, H.G., Liao, Z.B., Wang, C.Q., Wei, Y.L., Liang, M.Q., 2019. Hepatic

oo f

transcriptome of the euryhaline teleost Japanese seabass (Lateolabrax japonicus) fed diets characterized by alpha- linolenic acid or linoleic acid. Comp Biochem Phys D. 29, 106-116.

pr

Xu, W.N., Liu, W.B., Shen, M.F., Li, G.F., Wang, Y., Zhang, W.W., 2013. Effect of

e-

different dietary protein and lipid levels on growth performance, body

Pr

composition of juvenile red swamp crayfish (Procambarus clarkii). Aquacult Int. 21, 687-697.

al

Yuan, Y., Wang, X.X., Jin, M., Sun, P., Zhou, Q.C., 2019. Influence of different lipid sources on growth performance, oxidation resistance and fatty acid profiles of

rn

juvenile swimming crab, Portunus trituberculatus. Aquaculture. 508, 147-158.

Jo u

Zhou, Q.C., Jin, M., Elmada, Z.C., Liang, X.P., Mai, K.S., 2015. Growth, immune response and resistance to Aeromonas hydrophila of juvenile yellow catfish, Pelteobagrus fulvidraco, fed diets with different arginine levels. Aquaculture. 437, 84-91.

33

Journal Pre-proof

Tables and figures Table 1: Ingredients and proximal composition of experimental diets. Table 2: Fatty acid profile (% of total fatty acids) of each diet. Table 3: Primer sequences used to assay gene expression by RT-qPCR. Table 4: Growth performance of crayfish fed diets with six different dietary lipid sources. Table 5: Whole body composition (% wet basis) of crayfish fed diets with six

oo f

different dietary lipid sources. Table 6: Biochemical indicators of crayfish fed diets with six different dietary lipid

pr

sources in the hemolymph and hepatopancreas.

Table 7: Hepatopancreas fatty acid profiles of crayfish fed diets with six different

e-

dietary lipid sources.

Pr

Table 8: Muscle fatty acid profiles of crayfish fed diets with six different dietary lipid

al

sources.

rn

Fig. 1. Effects of crayfish fed diets contianing six different dietary lipid types on lipid metabolism-related gene expression. gene expression levels of fabp (A), fas (B),

Jo u

srebp1 (C), and rxr (D) were determined in the hepatopancreas, gene expression levels of fabp (E), fas (F), srebp1 (G), and rxr (H) were detected in the muscle, and gene expression levels of fabp (I), fas (J), srebp1 (K), and rxr (L) were determined in the intestine. Target genes were normalized to the gene expression of EIF. Values are presented as means ± SEM (n = 8). Bars with different letters are significantly different (P < 0.05). FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow.

Fig. 2. Effects of crayfish fed diets containing six dietary lipid types on the expression levels of FATP4 (A), APOB-48 (B), FAT/CD36 (C), DGAT1 (D), and NPC1L1 (E) in the intestine. Target protein levels were normalized to the expression of GAPDH. 34

Journal Pre-proof Values are presented as means ± SEM (n = 3). Bars with different letters are significantly different (P < 0.05). FO, fish oil; CO, corn oil; RO, rapeseed oil; SO,

Jo u

rn

al

Pr

e-

pr

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soybean oil; PaO, palm oil; BT, beef tallow.

35

Journal Pre-proof Table1: Ingredients and proximal composition of experimental diets. FO

CO

RO

SO

PaO

BT

Fish meal

12.00

12.00

12.00

12.00

12.00

12.00

Soybean meal

21.58

21.58

21.58

21.58

21.58

21.58

Rapeseed meal

13.46

13.46

13.46

13.46

13.46

13.46

Cottonseed meal

8.00

8.00

8.00

8.00

8.00

8.00

Wheat flour

30.00

30.00

30.00

30.00

30.00

30.00

α- starch

4.00

4.00

4.00

4.00

4.00

4.00

Fish oil

6.21

0.00

0.00

Corn oil

0.00

6.21

0.00

Rapeseed oil

0.00

0.00

6.21

Soybean oil

0.00

0.00

0.00

Palm oil

0.00

0.00

0.00

Beef tallow

0.00

0.00

Ca(H2 PO4 )2

2.20

2.20

NaCl

0.40

0.40

Chitin

0.15

Carboxymethyl cellulose

0.50

Ethoxyquinoline

0.50

0.00

0.00

0.00

0.00

0.00

0.00

6.21

0.00

0.00

e-

0.00

6.21

0.00

0.00

0.00

6.21

2.20

2.20

2.20

2.20

0.40

0.40

0.40

0.40

0.15

0.15

0.15

0.15

0.15

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

1.00

1.00

1.00

1.00

1.00

pr

0.00

al

1.00

0.00

rn

a

0.00

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Premix

oo f

Ingredients (% dry matter)

Pr

0.00

Proximate analysis (measured value, % dry weight) Moisture

9.37

12.25

11.38

14.06

12.85

11.19

90.63

87.75

88.62

85.94

87.15

88.81

Crude protein

31.83

30.52

31.14

30.55

30.74

31.06

Crude lipids

8.04

7.86

7.74

8.17

8.00

7.90

Ash

7.99

7.71

7.71

7.49

7.67

7.79

Gross energy (MJ/kg)

20.05

19.37

19.71

18.62

19.19

19.57

Dry matter

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. Note: a Premix supplied the following minerals (g/kg) and vitamins (IU or mg/kg ): CuSO4 ·5H2 O, 2 g; FeSO4 ·7H2 O, 25 g; ZnSO4 ·7H2 O, 22 g; MnSO4 ·4H2 O, 7 g; Na2 SeO3 , 0.04 g; KI, 0.026 g; Co Cl2 ·6H2 O, 0.1 g; Vitamin A, 1,500,000 IU; Vitamin D, 200,000 IU; Vitamin E, 5,000 mg; Vitamin K 3 , 220 mg; Vitamin B1 , 320 mg; Vitamin B2 , 1090 mg; Vitamin B5 , 2,000 mg; Vitamin B6 , 500 mg; Vitamin B12 , 36

Journal Pre-proof 15 mg; Vitamin C, 14,000 mg; Pantothenate, 1,000 mg; Folic acid, 230 mg; Choline, 60,000 mg;

Jo u

rn

al

Pr

e-

pr

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Biotin, 130 mg; Myoinositol 45,000 mg; Niacin, 3,000 mg.

37

Journal Pre-proof Table 2: Fatty acid profile (% of total fatty acids) of the trail diets. FO

CO

RO

SO

PaO

BT

C12:0

0.08

0.01

0.02

0.01

0.16

0.05

C14:0

5.48

0.74

0.76

0.80

1.41

2.70

C15:0

0.58

0.07

0.08

0.08

0.09

0.37

C16:0

19.05

13.98

8.14

12.94

34.13

22.66

C17:0

0.52

0.13

0.11

0.16

0.14

0.79

C18:0

3.65

2.03

2.31

4.32

3.76

21.70

C20:0

0.51

0.40

0.59

0.37

0.383

0.267

C22:0

0.20

0.16

0.36

ΣSFA

30.07

17.53

12.37

C16:1

6.13

0.88

0.95

C18:1

19.46

26.16

43.57

C20:1

3.59

0.37

3.52

C22:1

4.48

0.12

ΣMUFA

33.66

27.54

C18:3n-3

1.69

1.40

C20:3n-3

0.10

C20:5n-3 (EPA)

8.91

C22:5n-3

1.33

C22:6n-3(DHA)

11.37

oo f

FA

0.14

0.12

19.07

40.23

48.68

0.87

0.94

2.20

22.80

37.20

32.05

0.34

0.24

0.36

0.17

0.12

0.20

55.04

24.18

38.50

34.81

5.89

5.53

1.01

1.20

0.02

0.01

0.01

0.01

0.02

1.55

1.55

1.54

1.53

1.63

0.19

0.20

0.19

0.19

0.21

1.52

1.51

1.50

1.53

1.61

11.25

50.01

23.08

47.72

16.77

11.48

0.08

0.03

0.04

0.03

0.03

0.09

0.19

0.03

0.14

0.05

0.02

0.04

C20:4n-6

0.86

0.13

0.13

0.13

0.13

0.16

C22:3n-6

0.12

0.01

0.03

0.01

0.01

0.03

C22:4n-6

0.37

0.04

0.04

0.03

0.04

0.04

ΣPUFA

36.27

54.93

32.60

56.75

21.27

16.51

Σn-3

23.40

4.68

9.15

8.77

4.27

4.67

Σn-6

12.86

50.25

23.45

47.98

17.00

11.83

C18:3n-6 C20:2n-6

ePr

7.00

al rn

Jo u

C18:2n-6

pr

0.38

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. FA, fatty acid; ΣSFA, saturated fatty acids; ΣMUFA, monounsaturated fatty acids; ΣPUFA, polyunsaturated fatty acids; Σn−3, n−3 long-chain PUFA; Σn−6, n−6 long-chain PUFA. 38

Jo u

rn

al

Pr

e-

pr

oo f

Journal Pre-proof

39

Journal Pre-proof Table 3: Nucleotide sequences of the primers used to assay gene expression by RT-qPCR

Target fabp

fas

srebp1

rxr

eif

GU937432.1

MF062033.1

MF279131.1

KX673813.1

KR135170.1

Forward

CCACTGCTGA

GAAATGGCCC

GTTTTTCGGC

CCTTCACCAT

GGAATAAGGG

(5’-3’)

TGGCCGAAAT

GTCTTCTGGA

TCTTGGCTGG

TGGGTCGA GT

GACGAAGACC

Reverse

CGTTTGTA GA

GTCCTGGGCC

CAGGGTTCAC

AGCTGTAGA C

GCAAACACAC

(5’-3’)

CCCTCTTGCAC

TCGTTGTTTA

CAGGGTTGTT

GCCATAGTGC

GCTGGGAT

184

165

192

171

126

R2

0.9971

0.9993

0.9951

0.9971

0.9991

E (%)

87.16

109.04

87.09

93.57

90.08

gene Accession

oo f

number

Amplicon

Pr

e-

pr

size (bp)

Jo u

rn

al

E: Amplification efficiency; R2 : Pearson's coefficients of determination.

40

Journal Pre-proof Table 4: Growth performance of crayfish fed diets with six kinds of dietary lipid sources . FO

CO

RO

SO

PaO

BT

P-value

11.61±0.13

11.89±0.17

11.97±0.04

11.64±0.09

11.61±0.09

11.64±0.04

0.086

32.68±0.87b

31.84±0.96ab

31.93±0.59ab

31.48±0.74ab

28.43±0.81a

31.91±0.68ab

0.019

181.39±5.11b

167.68±6.51ab

166.87±4.53ab

170.25±4.48b

144.82±5.65a

174.03±5.08b

0.003

1.85±0.03b

1.76±0.04b

1.75±0.03ab

1.78±0.03b

1.60±0.04a

1.80±0.03b

0.002

2.01±0.06a

2.05±0.08a

2.03±0.06a

2.07±0.07a

2.44±0.12b

1.91±0.03a

0.003

5.42±0.13

5.66±0.20

5.78±0.17

5.26±0.27

5.27±0.09

0.094

IW a (g) FW b (g) WGR c (%) d

FCR

e

pr

(%)

HI f

5.11±0.09

e-

(%)

oo f

SGR

Pr

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. Means in the same colu mn with different superscripts are significantly different (P < 0.05). a IW: init ial weight;

b

FW : final weight;

c

W GR: weight gain rate; SGR d : specific growth rate; e FCR: feed

Jo u

rn

al

conversion ratio; f HI: hepatopancreas index.

41

Journal Pre-proof Table 5: Whole body composition (% wet basis) of crayfish fed diets with six kinds of dietary lipid sources. FO

CO

RO

SO

PaO

BT

P-value

65.92±0.96

66.39±0.58

65.80±0.47

65.86±1.42

66.83±1.08

67.12±0.61

0.862

13.30±0.17

12.84±0.20

13.19±0.20

12.35±0.54

13.13±0.18

12.69±0.21

0.203

2.97±0.31

3.03±0.21

2.66±0.30

2.73±0.48

2.99±0.31

2.76±0.32

0.946

12.63±0.58

12.40±0.34

12.66±0.47

13.16±0.50

12.06±0.63

11.69±0.16

0.361

15.30±0.42

15.69±0.33

15.19±0.51

15.10±0.54

16.08±0.36

0.625

Moisture (%) Crude protein (%) Crude lipid

oo f

(%) Ash (%)

(MJ/kg)

pr

Gross energy

15.13±0.55

e-

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow.

Jo u

rn

al

Pr

Means in the same column with different superscripts are significantly different (P < 0.05).

42

Journal Pre-proof Table 6: Biochemical indicators of crayfish fed diets with six kinds of dietary lipid sources in hemolymph and hepatopancreas . FO

CO

RO

SO

PaO

BT

P-value

0.13±0.01ab

0.22±0.02bc

0.22±0.01c

0.20±0.03abc

0.14±0.03abc

0.13±0.02a

0.002

0.42±0.01a

0.56±0.03bc

0.66±0.04c

0.58±0.05bc

0.50±0.02ab

0.47±0.03ab

<0.001

0.90±0.05

0.93±0.05

0.93±0.04

0.80±0.01

0.86±0.05

0.95±0.03

0.131

1.84±0.16

1.62±0.14

1.88±0.17

1.71±0.15

1.67±0.06

1.83±0.04

0.650

0.27±0.03ab

0.35±0.03b

0.31±0.03b

0.20±0.02a

0.32±0.02b

0.30±0.02ab

0.004

0.05±0.00a

0.10±0.01b

0.11±0.01b

0.10±0.01b

0.09±0.00ab

<0.001

0.55±0.09

0.86±0.06

0.62±0.06

0.58±0.11

0.58±0.14

0.62±0.14

0.333

1.14±0.06ab

1.49±0.14b

1.10±0.12ab

1.04±0.06ab

1.07±0.11ab

0.92±0.11a

0.018

Hemolymph TG a (mmol/l) TC b (mmol/l)

oo f

c

HDL-C

(mg/dl) LDL-C

d

pr

(mg/dl)

TG

e-

Hepatopancreas

TC

Pr

(mg/g)

0.10±0.00b

(mg/g)

al

HDL-C

(μg/mg)

Jo u

LDL-C

rn

(μg/mg)

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. Means in the same column with different superscripts are significantly different (P < 0.05). a TG: total triglycerides;

b

TC: total cholesterol;

c

HDL-C: h igh density lipoprotein cholesterol;

d

LDL-C: lo w

density lipoprotein cholesterol.

43

Journal Pre-proof

CO

RO

SO

PaO

BT

P-value

C14:0

2.69±0.08c

0.86±0.10a

0.66±0.14a

0.68±0.08a

0.85±0.09a

1.35±0.03b

<0.001

C16:0

22.79±0.61cd

17.84±0.98ab

14.42±1.78a

16.39±0.49ab

26.54±0.70d

20.67±0.44bc

<0.001

C18:0

3.91±0.11ab

2.75±0.14a

3.15±0.20ab

4.23±0.12b

3.48±0.08ab

11.44±0.61c

<0.001

others a

1.97±0.06b

1.46±0.07a

1.37±0.08a

1.50±0.07a

1.31±0.09a

1.59±0.05a

<0.001

ΣSFA

31.36±0.68b

22.91±1.18a

19.59±2.05a

22.80±0.67a

32.19±0.84b

35.06±0.37b

<0.001

C16:1

7.72±0.97b

3.23±0.98ab

4.61±1.35ab

2.43±0.25a

5.27±0.68ab

5.11±1.05ab

0.027

C18:1

25.95±0.39a

29.09±0.70a

43.46±1.64b

27.17±0.40a

41.00±1.46b

41.48±0.75b

<0.001

C20:1

3.74±0.23b

1.11±0.07a

3.27±0.22b

0.90±0.08a

1.07±0.09a

0.81±0.05a

<0.001

C22:1

3.37±0.34b

0.68±0.06a

4.29±0.62b

0.36±0.15a

0.48±0.06a

0.34±0.01a

<0.001

ΣMUFA

40.78±0.66b

34.11±0.22a

55.63±1.08d

30.86±0.35a

47.82±1.23c

47.75±0.74c

<0.001

C18:3n-3

2.75±0.18b

2.13±0.25ab

3.76±0.25c

4.37±0.22c

1.66±0.13a

1.60±0.14a

<0.001

C20:5n-3

4.87±0.15b

1.24±0.01a

1.06±0.07a

1.24±0.13a

1.10±0.15a

1.26±0.10a

<0.001

C22:6n-3

3.51±0.36b

0.69±0.02a

0.57±0.07a

0.71±0.04a

0.52±0.14a

0.87±0.04a

<0.001

others b

0.59±0.05b

0.13±0.00a

0.12±0.01a

0.14±0.01a

0.12±0.02a

0.18±0.00a

<0.001

C18:2n-6

14.46±0.76ab

37.78±1.65c

18.47±0.81b

38.89±0.87c

15.40±0.06ab

12.41±0.43a

<0.001

C20:4n-6

0.84±0.04b

0.39±0.01a

0.27±0.02a

0.38±0.06a

0.50±0.08a

0.36±0.03a

<0.001

others c

0.83±0.04b

0.61±0.03ab

0.52±0.03a

0.62±0.05ab

0.68±0.12ab

0.52±0.03a

0.024

ΣPUFA

27.87±1.08b

42.98±1.37c

24.78±1.12b

46.34±0.50c

19.99±0.41a

17.19±0.42a

<0.001

Σn-3

11.73±0.39d

4.20±0.26ab

5.51±0.36bc

6.46±0.31c

3.40±0.38a

3.91±0.13a

<0.001

Σn-6

16.14±0.84ab

38.78±1.63c

19.27±0.85b

39.88±0.77c

16.59±0.18ab

13.28±0.42a

<0.001

pr

e-

al

rn

Jo u

oo f

FO

Pr

Table 7: Hepatopancreas fatty acid profiles of crayfish fed diets with six kinds of dietary lipid sources .

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. Means in the same column with different superscripts are significantly different (P < 0.05). othersa include C12:0, C15:0, C17:0, C20:0 and C22:0; ΣSFA, saturated fatty acids; ΣMUFA, monounsaturated fatty acids; othersb include C20:3n-3 and C22:5n-3; others c include C18:3n -6, C20:2n-6, C22:3n-6 and C22:4n-6; ΣPUFA, polyunsaturated fatty acids; Σn−3, n−3 long-chain PUFA; Σn−6, n−6 long-chain PUFA.

44

Journal Pre-proof

CO

RO

SO

PaO

BT

P-value

C14:0

1.58±0.09c

1.19±0.05bc

0.51±0.10a

0.75±0.09a

0.76±0.11ab

0.90±0.10ab

<0.001

C16:0

19.11±0.53b

17.80±0.06ab

17.09±0.70ab

16.80±0.22ab

18.81±0.73b

16.39±0.28a

0.009

C17:0

0.59±0.00

0.51±0.05

0.63±0.04

0.70±0.10

0.59±0.04

0.64±0.04

0.315

C18:0

8.02±0.30

6.69±0.27

7.94±0.95

8.47±0.35

7.25±0.32

8.56±0.19

0.101

others a

1.09±0.07a

0.97±0.01bc

0.8±0.04ab

0.87±0.08abc

0.66±0.09a

0.74±0.01ab

0.002

ΣSFA

30.39±0.68

27.17±0.35

26.97±1.68

27.58±0.61

28.08±0.68

27.23±0.41

0.118

C16:1

2.71±0.45

2.69±0.09

2.16±0.32

2.77±0.08

2.06±0.15

2.74±0.31

0.294

C18:1

22.89±0.75a

24.03±0.89a

31.33±1.25b

22.49±1.03a

30.55±1.40b

29.74±0.28b

<0.001

C20:1

1.99±0.04b

1.04±0.10a

1.79±0.14b

1.01±0.08a

0.91±0.01a

1.08±0.08a

<0.001

C22:1

0.39±0.00c

0.21±0.02ab

0.29±0.02bc

0.23±0.04ab

0.15±0.01a

0.22±0.02ab

<0.001

ΣMUFA

27.97±0.53a

27.97±0.85a

35.57±1.72b

26.49±0.96a

33.68±1.53b

33.77±0.19b

<0.001

C18:3n-3

1.09±0.04a

1.85±0.19ab

2.64±0.19bc

3.18±0.28c

1.53±0.20a

1.67±0.09a

<0.001

C20:5n-3

18.36±0.77b

12.44±0.58a

12.61±0.66a

12.22±1.54a

14.63±1.27ab

15.52±0.33ab

0.004

C22:6n-3

9.77±0.55b

7.40±0.65ab

7.11±0.31ab

6.67±0.90a

7.36±0.73ab

8.63±0.45ab

0.039

others b

0.33±0.02

0.26±0.01

0.26±0.02

0.28±0.05

0.35±0.05

0.29±0.01

0.276

C18:2n-6

8.05±0.55a

18.78±1.12b

10.59±0.53a

18.18±1.47b

8.57±0.19a

8.40±0.26a

<0.001

C20:2n-6

0.86±0.05a

1.11±0.01bc

0.89±0.04ab

1.17±0.10c

0.87±0.03ab

0.75±0.01a

<0.001

C20:4n-6

2.69±0.09

2.39±0.02

2.81±0.36

3.49±0.87

4.20±0.32

3.23±0.32

0.109

others c

0.49±0.03

0.63±0.05

0.54±0.05

0.74±0.17

0.74±0.16

0.53±0.04

0.400

ΣPUFA

41.64±0.92ab

44.86±0.69b

37.46±0.86a

45.92±0.35b

38.24±2.05a

39.01±0.47a

<0.001

Σn-3

29.55±1.29b

21.96±1.36a

22.62±0.84ab

22.35±2.17a

23.87±1.98ab

26.10±0.82ab

0.026

Σn-6

12.09±0.40a

22.91±1.09b

14.84±0.23a

23.57±2.10b

14.37±0.29a

12.90±0.46a

<0.001

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Table 8: Muscle fatty acid profiles of crayfish fed diets with six kinds of dietary lipid sources .

FO, fish oil; CO, corn oil; RO, rapeseed oil; SO, soybean oil; PaO, palm oil; BT, beef tallow. Means in the same colu mn with different superscripts are significantly different (P < 0.05). others a include C12:0, C15:0, C20:0 and C22:0; ΣSFA, saturated fatty acids; ΣMUFA, monounsaturated fatty acids; othersb include C20:3n-3 and C22:5n-3; others c include C18:3n-6, C22:3n-6 and C22:4n-6; ΣPUFA, polyunsaturated fatty acids; Σn−3, n−3 long-chain PUFA; Σn−6, n−6 long-chain PUFA.

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1. PUFA profiles in the hepatopancreas and muscle tissues closely mirrored those

in the diets.

2. FATP4, DGAT1, and NPC1L1 in intestinal transport were influenced by

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various lipid sources.

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3. BT is the optimal candidate for FO substitution, whereas PaO is not

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recommended

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Dear editors, No conflict of interest exits in the submission of this manuscript, and neither the entire manuscript nor any part of its content has been published or has been

Sincerely yours Fan Gao

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accepted elsewhere.

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Figure 1

Figure 2