Fattening culture improves the gonadal development and nutritional quality of male Chinese mitten crab Eriocheir sinensis

Journal Pre-proof Fattening culture improves the gonadal development and nutritional quality of male Chinese mitten crab Eriocheir sinensis

Xugan Wu, Shaicheng Zhu, Hongcai Zhang, Meimei Liu, Na Wu, Jie Pan, Min Luo, Xichang Wang, Yongxu Cheng PII:

S0044-8486(19)31502-9

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734865

Reference:

AQUA 734865

To appear in:

aquaculture

Received date:

13 June 2019

Revised date:

13 December 2019

Accepted date:

13 December 2019

Please cite this article as: X. Wu, S. Zhu, H. Zhang, et al., Fattening culture improves the gonadal development and nutritional quality of male Chinese mitten crab Eriocheir sinensis, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734865

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© 2019 Published by Elsevier.

Journal Pre-proof

Fattening culture improves the gonadal development and nutritional quality of male Chinese mitten crab Eriocheir sinensis

Xugan Wu a, 1 , Shaicheng Zhu a, 1 , Hongcai Zhang b , Meimei Liu a, Na Wu b , Jie Pan a, Min Luo c, Xichang Wang b , Yongxu Cheng a, d, e*

Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean

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a

College of Food Science and Technology, Shanghai Ocean University, Lingang New City, Shanghai

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b

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University, Lingang New City, Shanghai 201306, China

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201306, China;

Jintan Fisheries Technical Extension Station of Changzhou City, Jintan 213200, China;

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Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai

201306, China

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c

Collaborative Innovation Center of Aquatic Animal Breeding Center certificated by Shanghai municipal education commission, Shanghai Ocean University, Shanghai, 201306, China;

1

These authors contributed equally to this work.

*Corresponding author:

Tel.: +86 21 61900417; fax: +86 21 61900405.

E-mail addresses: [email protected] (X.Wu); [email protected] (Y.Cheng)

Journal Pre-proof Abstract The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China, and more than 80% of aquaculture production comes from outdoor earth-ponds. Fattening immature E. sinensis with a high-quality diet has been proven to effectively improve male gonadal development and nutritional value. This study aimed to investigate the effects of different fattening periods on male gonadal development and nutritional quality of pond-reared male E. sinensis post pubertal molting. The gonadosomatic index (GSI) increased significantly during the 60-day fattening period, while significant increases in meat yield and total edible yield were only found between Day 0 and

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Day 20. The gonadal system, consisting of testis, deferens and accessory gland, had the highest increase during the fattening period. The fattening period did not significantly affect the proximate

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composition of hepatopancreas; however, the protein contents of male gonad and muscle increased significantly. The moisture and carbohydrate contents of the gonad, as well as muscular moisture,

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decreased significantly during the 60-day fattening period. A significant decrease in gonadal lipid

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contents was found between Day 40 and Day 60, while the trend of “low-high- low” was found in muscular carbohydrate levels during the whole fattening period. Significant increases were found in

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the percentage of triglyceride (TG) and free fatty acids (FFA) in male gonad, while decreases were found in the percentages of cholesterol and phospholipid in male gonads, FFA in hepatopancreas and

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TG in meat during the 60-day-fattening period. With respect to fatty acid composition, a trend of

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"high- low- high" was found for the percentages of mono-unsaturated fatty acids (MUFA), poly- unsaturated fatty acids (PUFA) and long chain poly- unsaturated fatty acids (LC-PUFA) in male gonad and hepatopancreas. The percentages of saturated fatty acid (SFA) and MUFA in muscle decreased significantly, while the significant increases were found in muscular PUFA and LC-PUFA during the fattening period. Total amino acid (TAA) levels of male gonads increased dramatically during the fattening period, and essential amino acid scores of male gonad and muscle were greater than 100 for all essential amino acids (EAA) after the 60-day fattening period. In conclusion, these results suggest that an appropriate fattening period is approximately 40 days for pond-reared male E. sinensis post pubertal molting. Keywords: Male Eriocheir sinensis; Nutritional composition; Male gonadal development; Accessory gland

Journal Pre-proof 1.

Introduction The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China, with

annual production of 758, 000 metric ton in 2017 (Bureau of Fisheries and Fishery Management, 2018). Meat, hepatopancreas and gonads are three major edible parts of adult E. sinensis; therefore, the status of gonadal development can not only determine supply time for market, but also affect edible yield and nutritional value (Wu et al., 2015; Shao et al., 2013). Previous reports regarding the

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fattening of adult E. sinensis have mainly concentrated on females, with no available information on male crabs to the best of our knowledge (Wu et al., 2004; Wu et al., 2007a; Sui et al., 2008, 2010).

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The gonadal system of male E. sinensis consists of testis, deferens and accessory gland, with the

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deferens and accessory gland accounting for the largest proportion (Hu and Hu, 1997; Xu et al.,

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2016). Male E. sinensis generally complete their pubertal molting between the end of the August and the middle of September in China, after which time they develop their gonadal system rapidly (He et

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al, 2014). Previous studies have suggested that feeding immature males with a high-quality diet

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during the process of gonadal development (defined as “fattening”) is an effective practice to

2016).

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accelerate gonadal development and to improve nutritional value of male E. sinensis (Xu et al.,

The developmental status of the gonad, total edible yields and nutritional quality are three important indicators used to evaluate the effectiveness of crab fattening (Wu et al., 2007b, 2010b; 2014). Among these indicators, the composition and contents of essential fatty acids (EFA) and essential amino acids (EAA) in edible components are important criteria for the evaluation of the nutritional quality of mature market crabs, which may be related to the fattening period and stages of gonadal maturity (Wu et al, 2014). Therefore, identification of the appropriate fattening period is very

Journal Pre-proof important for the optimization of fattening techniques for crab species. To date, there is no available information on the optimal fattening period for adult male E. sinensis.

Since outdoor pond-culture is the major production method for E. sinensis, and more than 80% of aquaculture production E. sinensis comes from outdoor ponds (Cheng et al, 2018). The current study was therefore designed to investigate the effects of a fattening period on male gonadal

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development, edible yields, proximate compositions, lipid classes, fatty acid composition and amino acid contents in the edible tissues of male E. sinensis post pubertal molting, which was reared in the

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outdoor earth-ponds. The results of this study could provide useful information for the fattening and

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the improvement of nutritional quality for male E. sinensis.

2. Materials and methods

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2.1 Crab source, culture conditions and management

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The culture experiment was conducted in small experimental ponds at the mitten crab research

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station of Shanghai Ocean University, Shanghai, China. In mid-September 2015, 220 pond-reared male E. sinensis with body weights of 150–180 g were obtained from a local mitten crab farm, and the males that underwent pubertal molting were further selected for the experiment. The claws of adult males that have completed pubertal molting are covered by thick long hairs and the petasma is hard (Xu et al, 2016). After the pubertal molt, male E. sinensis start to develop their gonadal system rapidly and there is no molting during this period (He et al, 2014; Xu et al, 2016). Prior to the start of the experiment, the crabs were acclimated to the outdoor culture conditions for 3–4 d and they were fed a commercial diet (Xinxin 3# diets for the adult crabs, Zhejiang Xinxin Feed Co., Ltd, Jiaxing, Zhengjiang Province, China). Crabs with immature gonads (Gonadosomatic index: 0.7–1.5%) were

Journal Pre-proof randomly selected and placed into four outdoor small earth ponds (Length×Width×Depth = 7.8 m × 7.8 m × 0.8 m) at a density of 40 males per pond. Plastic boards (thickness: 0.5 mm) surrounded each pond to prevent escape, while the water inlet and outlet of each pond had plastic nets (mesh size: 0.17 mm) to exclude indigenous fishes and other predators from the water source and drainage channel. In order to provide shelter for the crabs and to maintain water quality (remove nitrogen and phosphorus), aquatic plants (Elodea canadensis and Alternanthera philoxeroides) were transplanted into the

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experimental ponds. During the experiment, the crabs were fed a commercial fattening diet (Aohua 4# for crab

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fattening, Zhejiang Aohua Feed Co., Ltd, Jiaxing, Zhengjiang Province, China) once daily at 17:30

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with a ratio of approximately 1–3% total biomass depending on residual feed, water temperature, and

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the weather. The nutrition composition of the diet was shown in Supplementary Table 1. During the culture trail, the mortality was checked and recorded very morning; if any dead crab was found, it

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was removed from the experimental ponds. Over the period of the experiment, a water depth of 70

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cm was maintained with 30–50% of the water in each pond exchanged every 3–5 days. The water

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temperature was recorded by online temperature logger (OnSolution Pty Ltd, Baulkham Hills, NSW, Australia), and the changes of water temperature during the experiment was shown in supplementary Fig 1. The pH, dissolved oxygen (DO), ammonia and nitrite concentrations were monitored regularly and maintained at pH: 7.0–9.0; DO: > 5 mgL-1 ; ammonia: < 0.5 mgL-1 and nitrite: < 0.2 mgL-1 , respectively. These water parameters were within the suitable levels for adult E. sinensis (He et al., 2014). The culture experiment lasted 60 days from September 28 to November 27, 2015.

2.2 Sampling procedure Four crabs from each experimental pond were randomly sampled prior to the experiment (on day

Journal Pre-proof 0) as well as on days 20, 40 and 60. Prior to dissection, the wet weights of the crabs were measured. The crabs were then dissected to obtain hepatopancreas and male gonad system tissues (including testis, deferens and accessory gland), while the meat from all other body parts was carefully removed. The meat, hepatopancreas, accessory gland and whole gonad system from each crab were subsequently weighed and stored separately at -40°C for later biochemical analysis. The meat yield (MY), hepatosomatic index (HSI), gonadsomatic index (GSI), accessory gland index (AGI), the

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percentage of accessory gland to male gonad system (AG/MGS) and total edible yield (TEY) of the crab was calculated using the following formulas:

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MY (%) = 100 × Meat wet weight/Body wet weight.

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HSI (%) = 100 × Hepatopancreas wet weight/Body wet weight.

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GSI (%) = 100 × Gonad system wet weight/Body wet weight. AGI (%) = 100 × accessory gland wet weight/Body wet weight.

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AG/MGS = 100 × accessory gland wet weight/male gonad system wet weight.

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Total edible yield (%) = MY + HSI + GSI

2.3 Proximate composition analysis

Prior to biochemical analysis, the same tissues of crabs from each experimental pond were pooled and homogenized. The moisture content of each sample was obtained by drying tissue in a 70°C oven for 48 h and calculating the weight differences prior and post drying. Each tissue sample was then freeze-dried for later analyses, separately. Crude protein levels (Kjeldahl method, using a 6.25 N to protein conversion factor) were analyzed according to AOAC procedures (AOAC, 1995), while the total lipid (TL) content was extracted with chloroform- methanol (2:1, V/V) and analyzed based on the method described by Folch et al. (1957). Total carbohydrate was extracted with 1M

Journal Pre-proof sodium hydrate solution and the content was determined using the phenol-sulfuric acid method (Kochert, 1978).

2.4 Lipid class and fatty acid analysis Lipid fractions were separated and quantified using an Iatroscan MK-6s TLC-FID analyzer (Iatron Laboratries Inc., Tokyo, Japan). The developing solvent system was hexane/diethyl

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ether/formic acid (42/28/0.3, v/v/v) (Wu et al., 2010a). Lipid classes were quantified for total phoshpholipids (PL), triacylglycerol (TG), free fatty acids (FFA) and cholesterol (CHO). The levels

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of each lipid fraction were expressed as a percentage of both larval dry weight (% dry weight) and

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total lipids (% total lipid). For fatty acid analysis, fatty acid methyl esters (FAME) were prepared by

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transesterification with boiling 14% borontrifluoride/methanol (w/w) (Morrison & Smith, 1964). FAME was analytically verified by flame ionization detection (FID) after injecting a sample into an

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Agilent 6890 gas chromatograph fitted with an Omegawax 320 fused silica capillary column (30 m ×

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0.32 mm; Supelco, Billefonte, PA, USA). Peaks were identified by comparing retention times with

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known standards (Sigma-Aldrich Co, St. Louis. MO, USA). For further details on methodology of analysis, please refer to Wu et al. (2010a). Fatty acid compositions are expressed as a percentage of each fatty acid to the total fatty acids (% total fatty acids).

2.5 Amino acid analysis and essential acid score The amino acid content was analyzed according to the method described by Chen et al. (2007). Prior to the amino acid analysis, each tissue sample was freeze-dried and homogenized separately. Each sample (approximately 0.1 g dry weight) was then weighed and placed in a 40- ml hydrolyzation tube. Eight milliliters of 6.0 M HCl solution was subsequently added. The hydrolyzation tube was

Journal Pre-proof then vacuumed and filled with nitrogen at 110°C for 24 h. The resulting reaction mixture was diluted with distilled water to a volume of 50 ml, centrifuged at 2659 ×g, and the hydrolyzate was then filtered by filter paper. One milliliter of hydrolyzate was subsequently sampled and vacuum dried at 50°C to remove HCl. Depending on the content, the hydrolyzate was then dissolved in 2–5 ml of 0.02 M HCl and 1 µl of supernatant sample was used for amino acid analysis on a Sykam S-433D automatic amino acid analyzer (Sykam GmbH, Eresing, Germany). For tryptophan analysis, the

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pre-weighed samples were hydrolyzed in 5 M NaOH containing 5% SnCl2 (w/v) for 20 h at 110°C. After hydrolysis, the hydrolyzate was neutralized with 6 M HCl, centrifuged at 2659 ×g, and filtered

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using filter paper. The subsequent analysis was similar to the methods described above. The

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and hydrolysis (Spindler et al., 1984).

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determination of methionine and cystine was based on the methods of oxidation with performic acid

The identity and quantity of the amino acids were assessed by comparing the retention times

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and peak areas of the standard amino acids (Sigma-Aldrich, St. Louis. MO, USA). The amino acid

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content was expressed as a percentage of a particular amino acid to the tissue wet weight (%). The

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essential amino acid score (EAAS), gauged on a WHO/FAO/UNU reference to the amino acid requirement for adult maintenance (Millward, 2012), was calculated for each of the individual essential amino acids using the following formula (Wu et al., 2014): EAAS = 100 × essential amino acid content of the sample/FAO reference for essential amino acid content Where the amino acid content was expressed as mg individual amino acid per g total protein (mg/g).

2.6 Statistical analysis

Journal Pre-proof Data are presented as mean ± standard deviation (SD). The experimental pond was considered as the experimental replicate for all measurements, so the replicates of this study were 4. Homogeneity of variance was tested with Levene’s test. When necessary, arcsine-square root or logarithmic transformations were performed prior to analysis. The fattening period was considered as only experimental factor in this study, and one-way ANOVA was used to determine any significant differences for the parameters (tissue indices, total edible yield and biochemical composition in

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edible tissues) among the different fattening periods. If any significant difference was detected, Tukey's multiple range test was used as the means separation procedure. When a normal distribution

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and/or homogeneity of the variances was not achieved, data were subjected to the Kruskal-Wallis H

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nonparametric test, followed by the Games-Howell nonparametric multiple comparison test. P<0.05

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was regarded as statistically significant. All statistics were performed using SPSS package (version

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3. Results

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18.0).

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3.1 Effects of fattening period on gonadal development and edible yields During the 60-day fattening period, GSI increased significantly from 1.33% to 3.31% (Fig. 1, P<0.05). Within the fattening period, the greatest increase was found from Day 0 to Day 20, with the GSI being approximately 70% higher at Day 20 than that at Day 0. Only 13% of the GSI increase was detected between Day 40 and Day 60, and the increase was not significant (P >0.05). Male gonadal system was consisted of testis, deferens and accessory gland (Fig. 2), and accessory gland within the male gonadal system had the highest increase during the fattening period, which could be clearly observed in Fig. 2. As a result, accessory gland index (AGI, %) increase significantly during the 60-day fattening period (Fig. 3). However, the highest increase of the percentage of accessory

Journal Pre-proof gland to male gonad system (AG/MGS, %) was found on the period between Day 0 and Day 20, then the percentage of AG/MGS increased gradually between Day 20 and Day 60 (Fig. 3). During the fattening process, there were no significant changes for HSI (P >0.05, Table 2). Although increasing trends were found in MY and TEY, the only significant increase was detected between Day 0 and Day 20 (P <0.05).

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3.2 Effects of fattening period on proximate composition and lipid classes of different edible tissues The proximate compositions of gonad, hepatopancreas and muscle of male E. sinensis were

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shown in Table 1. As the length of the fattening period increased, the proportions of moisture content,

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lipid and total carbohydrate decreased significantly while gonadal protein levels increased

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significantly, particularly between Day 0 and Day 40 (Table 1). No significant differences were detected in moisture and carbohydrate contents in the hepatopancreas during the 60-day fattening

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period, while hepatopancreatic protein levels increased significantly (P<0.05), and hepatopancreatic

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lipid levels increased significantly between Day 0 and Day 40 (P<0.05), then decreased slowly from

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Day 40 to Day 60 (P>0.05). For muscle tissue, no significant differences were detected in moisture content during the 60-day fattening period, while the levels of protein and lipid increased significantly from Day 0 to Day 40 (P<0.05). The lowest carbohydrate content in muscle was detected at Day 60, while the highest level was found at Day 20 (P<0.05).

Table 2 showed the changes of lipid classes (% total lipids) of different edible tissues during the 60-day fattening period, with different tissues exhibiting different lipid class profiles. PL was the most dominant lipid fraction in the gonad, and a decreasing trend was found during the whole fattening period. An increasing trend was found in the percentages of TG and FFA during the

Journal Pre-proof fattening period, but no clear changing pattern was found in the percentage of CHO. For lipid class profiles of hepatopancreas, the percentage of TG increased significantly from Day 20 to Day 40, while the opposite trend was found for the percentages of FFA and CHO. The percentage of PL decreased significantly from Day 0 (6.34%) to Day 40 (3.63%), then increased significantly to Day 60 (5.50%). The lipid class profiles of muscle revealed significant increases in the percentage of CHO and PL from Day 0 to Day 40 and Day 20 to Day 40, respectively; however, a decreasing trend

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was found in the percentage of TG from Day 20 to Day 60 (P<0.05). A relatively stable and low

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percentage of FFA (2.3–3.2% total lipids) in the muscle was found during the fattening period.

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3.3 Effect of fattening period on fatty acid composition of different edible tissues

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Fatty acid composition of gonad changed significantly during the fattening period (Table 3). Among saturated fatty acids (SFA), 16:0 and 18:0 were the major constituents in male gonads, and

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the percentage of 18:0 decreased significantly during the fattening p eriod (Table 3). For

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mono- unsaturated fatty acids (MUFA), 14:1n-7, 16:1n-7 and 18:1n-9 were the three most dominant

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fatty acids. During the fattening period, a decreasing trend was detected for the percentages of 14:1n-7 and 18:1n-9, while the percentages of 18:1n-7, 20:1n-7 and 20:1n-9 had an increasing trend; the lowest percentages of 16:1n-7 and 18:1n-9 were found on Day 20 and Day 40, respectively. For poly- unsaturated fatty acids (PUFA), during the fattening period, no clear trend was found for 18:2n-6 and 22:6n-3, which were at the lowest percentage at Day 40. Both 20:4n-6 and 22:6n-3 decreased initially, then increased significantly (P<0.05). Overall, the ratios of n-3PUFA/n-6PUFA decreased significantly, while 22:6n-3/20:5n-3 increased significantly (Table 5).

The fatty acid compositions (% of total fatty acids) of hepatopancreas are presented in Table 4.

Journal Pre-proof For SFAs, significant decreasing trends were found in the percentages of C14:0, C16:0 and C18:0 during the fattening period (Table 4). Among SFAs, 16:0 was the major fatty acid in hepatopancreas, accounting for 70% of the total SFA content. For the MUFA composition, the major fatty acids were 16:1n-7 and 18:1n-9, with combined percentages accounting for more than 10% of total fatty acids. The percentages of 18:1n-9 and 20:1n-9 increased significantly during the fattening period, which led to increasing percentages of ∑MUFA. For the PUFAs, 18:2n6, 18:3n3, 20:4n6(ARA), 20:5n3(EPA)

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and 22:6n3(DHA) were the five dominant fatty acids. An increasing trend was found in

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decreased significantly during the fattening period.

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20:4n6(ARA), 20:5n3(EPA) and 22:6n3(DHA), while the percentages of 18:2n6 and 18:3n3

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Fatty acid composition of muscle changed significantly during the fattening period (Table 5). During the fattening period, the percentages of 14:0, 16:0, 17:0 and ∑SFA decreased significantly,

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and no clear trend was found in the other SFAs (Table 5). For the MUFA composition, the dominated

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fatty acid was 18:1n-9, with percentages of more than 17% of total fatty acids. The percentage of

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18:1n-9 decreased significantly during the fattening period, while muscle tissue at Day 20 had significantly higher 14:1n7. For the PUFAs, 18:2n6, 20:4n6(ARA), 20:5n3(EPA) and 22:6n3(DHA) were the four dominant fatty acids. An increasing trend was found in 22:6n3(DHA) during the fattening period, while the lowest percentages of 20:4n6(ARA) and 20:5n3(EPA) were found at Day 20, and these fatty acids increased significantly. Overall, the ratios of n-3PUFA/n-6PUFA increased significantly, while the highest percentages of ∑PUFA, ∑n-3PUFA and ∑LC-PUFA were found at Day 20 (Table 5).

3.4 Effect of fattening period on amino acid contents and EAA scores of different edible tissues

Journal Pre-proof There were 19 detected amino acids in the gonad, and the changes in amino acid contents are given in Table 6. During the fattening period, both EAAs and non-essential amino acids (NEAAs) increased significantly, particularly from Day 0 to Day 40. Among EAAs, the greatest increase of 81% was found in isoleucine during the 60-day fattening period. There were also greater than 60% increases in leucine, cysteine, phenylalanine, tyrosine, and threonine, while the increase in other EAAs was less than 25%. For NEAAs, aspartic acid, glutamic acid and proline were the three major

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constituents with more than 10 mg/g wet weight in male gonads for all sampling times. The highest increase (121%) was detected in proline during the 60-day fattening period, and aspartic acid and

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alanine had the second highest increases at 50% and 65%, respectively. Moreover, a significant

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decrease (approximately 68%) was found in taurine during the fattening period, from 1.45 mg/g wet

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weight at Day 0 to 0.46 mg/g wet weight at Day 60. Overall, the TAA content increased significantly from 126.79 mg/g wet weight at Day 0 to 187.44 mg/g wet weight at Day 60, representing a 48%

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increase over the whole fattening period.

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Table 7 shows the changes of amino acid contents in male muscle during the 60-day fattening period. Leucine and lysine were the two major EAAs with more than 10 mg/g wet weight in male muscle. Among EAAs, the highest increase was found in histidine and threonine contents with increases of more than 20% during the fattening period, while lysine had the lowest increase at 3.01%. The increase in the other EAAs ranged from 11% to 18% during the fattening period. For NEAAs in muscle tissue, aspartic acid, glutamic acid, alanine and arginine were the four major components, contributing more than 10 mg/g wet weight at all sampling times. During the 60-day fattening period, taurine, aspartic acid and glycine had the highest increase (more than 21%), while the lowest increases were found in alanine and proline (less than 7.5%). Overall, there was an 18%

Journal Pre-proof increase in TAA in the muscle during the whole fattening period, particularly from Day 0 to Day 40.

Table 8 showed the changes in EAAS of male gonad and muscle based on the reference protein for adult maintenance (WHO/FAO/UNU, 2007). If an EAAS was more than 100, this amino acid meets the requirements of adults and is not the limiting amino acid in the food. For male gonads, EAAS of leucine was less than 100 at Day 0 and Day 40, but the EAAS of the other EAAs were

lysine,

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more than 100. During the fattening period, a decreasing trend was found in the EAAS of histidine, valine, and tryptophan, while an increasing trend was detected

in isoleucine,

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Methionine+Cysteine and Phenylalanine +Tyrosine. For male gonad, the EAAS of all EAAs were

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more than 100, but isoleucine, leucine and tryptophan had relatively lower EAAS during the

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fattening period. Overall, male gonads had higher mean EAAS than the muscle for each sampling

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4. Discussion

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point/time.

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Gonad, muscle and hepatopancreas are three major edible tissues of E. sinensis, and their ratios to body weight represent important indices to evaluate the market product quality (He et al., 2014). After pubertal molting, male E. sinensis require a large amount of exogenous nutrients to develop their gonadal system rapidly and increase the accumulation of nutrients in the hepatopancreas (Xu et al., 2016). Therefore, feeding males following the completion of their pubertal molting with good quality diets (defined as fattening) is vital to improve product quality for the consumer. Fattening males in this way allows for the development of the gonadal system. Males with well-developed gonadal systems are generally sold at a higher price than less developed males, and therefore the status of male gonadal development directly affects the nutritional value and price of market E.

Journal Pre-proof sinensis (He et al., 2014). The present study showed that GSI increased significantly between Day 0 and Day 20 of the fattening period, while no significant increase was found between Day 40 and Day 60. This suggests that the male gonadal system was nearly mature after 40 days fattening (unt il early November). In mature crabs, the accessory gland is the largest component of the male gonadal system for the mature E. sinensis, normally accounting for approximately 70% of the total weight of male gonadal system, and corresponding to approximately 2.5% of the total body weight (Xu et al.,

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2016). This study clearly showed the percentage of accessory gland to male gonad system as well as GSI reached the peak at Day 40 sampling, which did not increase significantly after the 40-day

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fattening period. Therefore, increases in male GSI are mainly dependent on an increase in male

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accessory gland. The significant increase size of accessory gland during the fattening period is highly

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related to the development of male gonad system, which serve many important functions during the male reproduction and copulation, including conservation and transportation of spermatophore,

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al, 2010; He et al, 2013)

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digestion of spermatophore wall, capacitation and potentially acrosome activation of sperm (Hou et

The major aim of fattening is to improve total edible yields of mature male E. sinensis, including the gonadal system, muscle and hepatopancreas (Wu et al., 2015). The only increase (2.37%) in meat yield was detected during the early fattening period (Day 0 to Day 20), which was probably due to a rapid accumulation of nutrients in the muscle fo llowing molting (Ma et al., 2014). The hepatopancreas is the major organ for nutrient digestion and storage in crustaceans, and a negative correlation has been found between GSI and HSI during the ovarian development of female E. sinensis, as a result of hepatopancreatic nutrients being transported to the developing ovaries (Wen et al., 2001; He et al., 2014). However, the correlation between GSI and HSI is not clear for male E.

Journal Pre-proof sinensis during the process of gonadal development, and inconsistent results have been found by different studies (He et al., 2014; Xu et al., 2016). The present study found no significant change in HSI during the male fattening process or gonadal development of E. sinensis, which may be ascribed to the good culture conditions in this study, including adequate diet supply and suitable water temperature. Therefore, male crabs were able to feed normally, resulting in a relatively stable HSI during periods of male gonadal development. On the whole, after 40 days of fattening, the total

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edible yield of male E. sinensis did not increase significantly.

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During the early- and mid-periods of fattening (Day 0 to Day 40), the decrease in moisture and

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carbohydrate contents in the gonad was associated with significant increases in the levels of protein

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and total lipids. During the early fattening period of Day 0 to Da y 20, the increasing contents of hepatopancreatic lipids led to a significant decrease in the moisture content in hepatopancreas. This is

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because the hepatopancreas is the most important lipid metabolism center for crustaceans, and the

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storage lipids in the hepatopancreas play an important role during overwintering and reproductive

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migration (Lai et al., 1994; Wang et al., 2004). During the fattening period between Day 0 and Day 40, the contents of protein, lipid and carbohydrate in the muscle increased significantly, but no significant difference was found in the proximate composition in the muscle after Day 40. Generally, the proximate composition in the muscle is relatively conservative in order to maintain their normal basic functions, such as moving and protection of the internal organs (Wu et al., 2014; Tian et al, 2017).

In the process of fattening, the percentage of TG and FFA in the gonad increased significantly, which could be explained by the fact that spermatogenesis and formation of the spermatophore

Journal Pre-proof require a large number of lipid substances (Wang et al., 2004; Ma et al., 2017). The contents and composition of total lipids in the hepatopancreas are common indices to evaluate the nutritional status of crustaceans. In general, a high ratio of TG to CHO is an indicator of good nutritional status for animals (Fraser, 1989; Wouters et al., 2001; Cavalli et al., 2001). The major lipid class in the hepatopancreas is TG (more than 85%), which is the main energy storage for crustaceans (Wu et al., 2010a). The percentage of hepatopancreatic TG increased significantly, which indicated male E.

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sinensis had good nutritional conditions during the fattening process in the present study. Generally, male gonad and muscle contained high levels of protein and PL, but low lipid and CHO contents

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following the fattening period, which suggests these tissues are of high nutritional value for human

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consumption.

The percentage of ∑MUFA in the gonads decreased significantly during the fattening period

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between Day 0 and Day 20, which was mainly due to the decline in 14:1n7 and 18:1n9 content.

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Between Day 20 and Day 60, the increasing levels of 20:4n6 and 22:6n3 and decreasing levels of

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18:2n6 and 20:5n3 in gonad tissue suggested that the males had a high requirement for dietary 20:4n6 and 22:6n3, but a low demand for 18:2n6 and 20:5n3 during gonadal development. In the process o f male crab fattening, the ∑SFA content in the hepatopancreas decreased significantly, while the contents of ∑MUFA and ∑PUFA increased significantly, in particular the percentages of 18:1n9, 20:1n9, EPA and DHA, which indicates the fatty acid nutritional value in the hepatopancreas improved during the fattening process. During the fattening period between Day 20 and Day 60, the percentages of 20:4n6 (ARA), 20:5n3 (EPA) and 22:6n3 (DHA) in the muscle increased significantly. This might be related to the descending water temperature because the high levels of muscular EPA could enhance the resistance ability of crabs to low temperature (Kong et al., 2006).

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ARA, EPA and DHA are three important long chain polyunsaturated fatty acids (LC-PUFAs) for the health of human and animals (Innis, 2000). Because humans do not have an adequate ability to biosynthesize these LC-PUFAs under conditions of rapid growth or augmented loss, especially for fetuses, infants, adolescents and pregnant or lactating women, these LC-PUFAs are considered “conditionally essential fatty acids” (Muskiet et al., 2006). EPA and DHA can prevent hypertension,

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inhibit the proliferation of tumor cells and arthritis (Harper and Jacobson, 2005; Roynette, 2004), while ARA can promote the development of the central nervous syste m of fetuses and infants (Innis,

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2000; Muskiet et al., 2006). However, excessive levels of ARA have also been associated with

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hypertension (Zheng et al., 1999) and Crohn’s disease (Gil, 2002). Therefore, the composition and

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content of LC-PUFAs in aquatic products is an important index to evaluate the nutritional value of fatty acids (Wu et al., 2010b, 2014). Overall, the muscle had the highest percentages of LC-PUFAs,

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and the lowest levels were found in the hepatopancreas. As a result, the fatty acid value of muscle is

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better than that of gonad and hepatopancreas in male E. sinensis. N-3PUFA/n-6PUFA is also an

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important index to measure the nutritional value of fatty acids in food (FAO/WHO, 1994). FAO/WHO (1994) recommended that the appropriate ratio of n-3/n-6 in the human diet was 0.1–0.2, and the higher ratio of n-3/n-6 is more beneficial to human health. The results of the present study showed that the n-3/n-6 ratio of various edible tissues in male E. sinensis were greater than 0.58, and therefore this species has high nutritional value following a fattening process. In addition, the composition and contents of volatile flavor substances were affected by body fatty acid composition in E. sinensis, and MUFAs and PUFAs can degrade into aldehydes and ketones with unique aromas after cooking (Zhuang et al., 2016; Wang et al., 2016).

Journal Pre-proof During the process of fattening, most of the amino acids in the gonad increased significantly, and the increases in isoleucine, leucine, cysteine, phenylalanine, tyrosine, and threonine were more than 60%, which suggested that these six EAAs were particularly important in gonadal development of male E. sinensis. For the NEAAs, the proline content increased more prominently than the other NEAAs during the fattening period. Proline is the major constituent of collagen, which suggests that male E. sinensis gonad contains a high collagen content (Shao et al., 2013). The contents of threonine,

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aspartate, glutamic acid and proline in male gonads were more than 14 mg/g wet weight, which means male crabs have a high demand for these four EAAs during the process of fattening as well as

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during gonadal development. Therefore, it would be beneficial to optimize the contents and

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proportion of these amino acids in commercial fattening diets (Wu et al., 2014). Glutamic acid and

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aspartic acid are two umami tasting amino acids (Shao et al., 2013), and their high contents in gonad

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and muscle tissue indicates these tissues have high taste value for consumers.

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All EAAS of male gonad and muscle were greater than 100 for all EAAs after the 60-day

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fattening period, which suggests that male E. sinensis represents a good protein source with high nutritional value. FAO/WHO/UNU has recommended the ideal ratio of EAA/TAA is approximately 0.4, and considers higher ratios (> 0.4) to be more beneficial to human health (FAO/WHO/UNU, 1985). The results of this study show that the EAA/TAA ratios in E. sinensis gonad and muscle were 0.46 and 0.45, respectively, after fattening, which are higher than those of the FAO/WHO/UNU (1985) recommended standards. This suggests that the male gonad and muscle have high nutritional value for amino acid composition after fattening.

5. Conclusion

Journal Pre-proof The fattening significantly improved the GSI, MY and TEY of male E. Sinensis, with the highest TEYs and the majority of proximate compositions in edible tissues occurring after 40 days fattening. After fattening, all edible parts of E. sinensis had good nutritional values, in terms of high protein contents and EAAS, low lipid levels as well as the composition of fatty acids and amino acids. These indices suggest that an appropriate fattening period for pond-reared male E. sinensis is

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approximately 40 days following their pubertal molting.

Acknowledgements

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This study was funded by two projects (No.31471608 and No. 31572630) from the Natural

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Science Foundation of China, the Special Fund (CARS-48) of Chinese Agriculture Research System

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from Ministry of Agriculture of China, and the R&D project (D-8006-15-0054) from the Shenzhen Alpha Feed Co. Ltd. Infrastructure costs were supported by the construction and improvement project

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(No. A1-2801-18-1003) for high level university in Shanghai from Shanghai Education Commission,

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Shanghai talents development fund for the young scientists (No. 2018100) from Shanghai Municipal

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Human Resources and Social Security Bureau and the innovation and extension project (Y2017-4) of Science and technology from Jiangsu ocean and Fisheries Bureau. Conflict of interest: None References AOAC, 1995. Official methods of analysis of the association of official analytical chemists,16th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Bureau of Fisheries and Fishery M anagement, M inistry of Agriculture and Rural affairs of China, 2018. China fisheries statistical yearbook in 2017. Chinese Agricultural Press, Beijing, China. (in Chinese) Cavalli, R. O., Tamtin, M ., Lavens, P., Sorgeloos, P., 2001. Variations in lipid classes and fatty acid content in tissues of wild Macrobrachium rosenbergii (de M an) females during maturation. Aquaculture 193(3-4), 311–324. Chen, D.W., Zhang, M ., Shrestha, S., 2007. Compositional characteristics and nutritional quality of Chinese mitten crab ( Eriocheir sinensis). Food Chem. 103(4), 1343-1349. Cheng, Y., Wu, X., & Li, J. (2018). Chinese mitten Crab Culture: Current Status and Recent Progress Towards Sustainable Development. In: Aquaculture in China: Success Stories and M odern Trends (Edit by: Gui, Tang, Liu, Li and De Silva),

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on the gonadal development, tissue proximate comp osition, lipid class and fatty acid composition of precocious Chinese mitten

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Wu, X.G., Cheng, Y.X., Sui, L.Y., Zeng, C.S., Southgate, P. C., Yang, X.Z., 2007a. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H.

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M ilne-Edwards), female broodstock. Aquaculture 273(4), 602–613. Wu, X., Cheng, Y., Sui, L., Yang, X., Nan, T., Wang, J., 2007b. Biochemical composition of pond-reared and lake-stocked Chinese

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mitten crab Eriocheir sinensis (H. M ilne-Edwards) broodstock. Aquac. Res. 38(14), 1459–1467. Wu, X.G., Long, X.W., Liu, N.G., He, J., Wang, W., Cheng, Y.X., 2015. Comparative study on gonadal development and biochemical composition among three population mitten crabs: Eriocheir sinensis, E. japonica and their hybrids. Freshwater Fisheries 45(3): 3-8. (in Chinese with English abstract)

Wu, X.G., Wang, Q., Lou, B., Liu, Z.J., Cheng, Y.X., 2014. Effects of fattening period on ovarian development and nutritional quality of female swimming crab Portunus trituberculatus. J. Fish. China, 38(2), 170-182. (in Chinese with English abstract) Wu, X., Zhou, B., Cheng, Y., Zeng, C., Wang, C., Feng, L., 2010b. Comparison of gender differences in biochemical composition and nutritional value of various edible parts of the blue swimmer crab. J. Food Compost. Anal. 23(2), 154–159. Xu, J.Q., Wu, X.G., Zhang, P.C., He, J., Fan, Y.W., Liu, M .M ., Cheng, Y.X., 2016. Growth, gonadal development and secondary sexual characteristics of pond-reared male Chinese mitten crab (Eriocheir sinensis) during the second year culture. Chinese J. Zool. 51(3), 19-26. (in Chinese with English abstract). Zheng, Z.J., Folsom, A.R., M a, J., Arnett, D.K., M cGovern, P.G., Eckfeldt, J.H., 1999. Plasma fatty acid composition and 6-year incidence of hypertension in middle-aged adults: The atherosclerosis risk in communities (ARIC) study. Am. J. Epidemiol., 150(5), 492–500. Zhuang, K.J., Wu, N., Wang, X.C., Wu, X.G., Wang, S., Long, X.W., Wei, X., 2016. Effects of three feeding modes on the volatile and non-volatile compounds in the edible tissues of female Chinese mitten crab (Eriocheir sinensis). J. Food Sci. 81(4): 968-981.

Journal Pre-proof Figure legends

Figure 1 Effects of a 60-day fattening period on gonadsomatic index (GSI), hepatosomatic index (HSI), meat yield (M Y) and total edible y ield (TEY) in adult male Eriocheir sinensis. The colu mns with d ifferent superscripts are significantly different (P <0.05).

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Figure 2 Changes of gonadal system components of adult male Eriocheir sinensis during the 60-day fattening period. A. Male gonadal system at Day 0; B. Male gonadal system at Day 20; C. Male gonadal system at Day 40; D.

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Male gonadal system at Day 60. T: Testis; D: Deferens; AG: Accessory sex gland.

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Figure 3 Changes of accessory gland index (AGI, %) and the percentage of accessory gland to male

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gonad system (AG/MGS, %)

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Fig. 1

Fig. 2

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Fig. 3

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Table 1 Effect of a 60-day fattening period on proximate co mposition of different edible tissues in male Eriocheir sinensis (% wet weight). Day 0

Day 20

Day 40

Day 60

moisture

77.78±1.71c

74.32±1.05b

71.30±1.71a

70.25±1.51a

protein

13.31±0.46a

16.11±0.91b

19.05±1.25c

19.81±0.98c

lipid

1.53±0.05b

1.62±0.25b

1.66±0.13b

1.27±0.15a

carbohydrate

1.12±0.15b

0.80±0.13a

0.73±0.09a

0.78±0.08a

moisture

50.77±8.52

42.40±5.15

44.16±1.98

48.64±5.08

protein

7.40±1.24a

8.46±0.38ab

8.28±0.65ab

9.57±0.77b

lipid

35.57±3.71a

41.15±2.02b

42.69±1.61b

38.40±4.36ab

1.46±0.39

1.43±0.21

1.36±0.19

1.71±0.45

Tissue Gonad

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carbohydrate

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Hepatopancreas

78.69±2.53ab

protein

17.24±1.83a

lipid

1.14±0.16a

carbohydrate

0.34±0.15a

77.38±1.10b

75.73±0.75a

76.13±1.70ab

18.23±0.78ab

19.73±0.70bc

20.39±1.28c

1.48±0.10b

1.62±0.23c

1.46±0.13bc

0.55±0.10b

0.42±0.16ab

0.28±0.03a

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moisture

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Muscle

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(P<0.05).

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Data are presented as mean ± SD (n =4). Values in the same ro w with different superscripts are significantly different

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Table 2 Effect of a 60-day fattening period on main lipid classes of different edible tissues in male Eriocheir sinensis (%

Day 0

Day 40

Day 60

10.94±3.12a

18.75±2.41b

18.89±3.18b

23.29±1.36c

FFA

1.62±0.44a

1.99±0.36a

2.65±0.11b

3.66±0.61c

CHO

5.52±0.79bc

6.57±0.39c

3.01±0.98a

4.10±0.88ab

PL

81.92±1.89c

72.87±2.38b

75.45±2.31b

68.95±1.10a

86.27±4.66a

85.11±3.61a

92.98±0.87b

91.03±1.91ab

4.50±1.79b

4.59±0.42b

2.21±0.49a

2.44±0.23a

2.16±0.93b

3.79±0.64c

0.94±0.27a

0.96±0.14a

6.34±1.63b

4.91±1.29ab

3.63±0.82a

5.50±1.83b

9.49±1.02b

9.72±1.34b

2.39±0.40a

2.28±0.88a

3.20±0.30

2.97±0.48

3.10±0.43

2.31±0.51

2.16±0.48a

2.19±0.87ab

2.87±0.53b

2.80±0.15b

85.15±0.84a

85.12±1.93a

91.64±0.50b

92.60±1.54b

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TG

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Gonad

Day 20

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Tissue

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total lipids).

FFA CHO PL Muscle TG FFA CHO PL

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TG

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Hepatopancreas

Data are presented as mean ± SD (n =4). Values in the same ro w with different superscripts are significantly different (P<0.05). TG: triglyceride; FFA: free fatty acids; CHO: cholesterol; PL: phoshpholipids.

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Table 3 Effect of a 60-day fattening period on fatty acid profile in the gonad of male Eriocheir sinensis (% total fatty acids) Day 20

Day 40

Day 60

14:0

0.76±0.04a

0.80±0.22ab

0.96±0.12b

0.70±0.06a

15:0

0.38±0.03ab

0.31±0.04a

0.50±0.00c

0.38±0.01b

16:0

11.02±0.35

11.55±1.73

11.52±0.08

11.14±0.35

17:0

0.68±0.05a

0.53±0.13a

0.91±0.05b

0.91±0.12b

18:0

6.78±0.03c

7.39±1.38bc

5.94±0.45b

5.41±0.03a

∑ S FA

19.63±0.37b

20.59±0.74b

19.83±0.60b

18.54±0.26a

14:1n7

6.55±0.44b

5.49±0.52a

5.91±1.16ab

5.17±1.08a

16:1n7

5.40±0.33

5.64±1.10

5.37±0.22

5.24±0.30

16:1n5

0.93±0.01b

0.66±0.14a

1.41±0.18c

1.42±0.05c

17:1

0.44±0.05a

0.41±0.23ab

0.52±0.04b

0.53±0.03b

18:1n9

18.69±0.47b

16.95±1.80ab

16.46±1.01a

16.86±0.14a

18:1n7

2.78±0.13b

2.09±0.31a

2.99±0.49b

3.14±0.49b

20:1n9

1.37±0.38ab

1.30±0.03a

1.84±0.11bc

1.94±0.05c

20:1n7

0.38±0.04a

0.37±0.04a

0.62±0.16b

0.55±0.08b

∑ MUFA

36.54±1.84

32.90±3.04

35.13±1.03

34.86±2.02

18:2n6

6.39±0.13b

7.61±0.13c

4.93±0.16a

5.61±0.86a

18:3n3

1.95±0.33

2.00±0.80

1.84±0.21

1.84±0.55

18:4n3

0.35±0.08

0.27±0.06

0.31±0.06

0.29±0.05

20:2n6

1.78±0.27

1.57±0.29

1.69±0.02

1.75±0.14

20:3n3

0.48±0.05b

0.47±0.13ab

0.38±0.09a

0.34±0.08a

20:4n6

10.33±0.28b

8.10±0.69a

12.20±0.82c

12.39±0.76c

20:5n3

8.53±0.33c

7.50±1.12bc

6.21±0.04a

7.04±0.20b

0.52±0.05b

0.50±0.12ab

0.44±0.04a

0.42±0.07a

0.61±0.00a

0.52±0.14ab

0.74±0.00b

0.75±0.08b

3.69±0.11b

3.80±0.01c

3.56±0.01a

4.02±0.14d

22:2n6 22:5n3 22:6n3

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Day 0

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Fatty acid

∑PUFA

34.78±1.64b

32.74±0.96a

32.70±0.61a

34.57±2.29a

∑n-3PUFA

13.74±0.46c

12.76±0.85bc

11.34±0.20a

12.44±0.24b

∑n-6PUFA

19.10±0.85

17.98±0.91

19.52±1.02

20.29±1.98

n-3/n-6

0.72±0.01b

0.71±0.01b

0.58±0.04a

0.62±0.07a

∑LC-PUFA

23.80±0.78b

20.79±1.43a

23.49±0.66b

24.65±0.74b

DHA/EPA

0.43±0.03a

0.51±0.08ab

0.57±0.00b

0.57±0.00b

Data are presented as mean ± SD (n=4). Values in the same row with different superscripts are significantly different (P<0.05); the fatty acid with more than 0.3% of total fatty acids is shown in the table.

Journal Pre-proof

Table 4 Effect of a 60-day fattening period on fatty acid profile in the hepatopancreas of male Eriocheir sinensis (% total total fatty acids) Fatty acid 14:0

Day 0

Day 20

Day 40

1.89±0.15c

1.37±0.08b

1.28±0.07ab

Day 60 1.19±0.06a

0.95±0.09

0.91±0.09

0.87±0.13

0.81±0.05

16:0

19.50±1.70c

17.57±0.26b

17.31±0.47ab

16.67±0.56a

17:0

0.94±0.07

0.84±0.10

0.98±0.17

0.89±0.13

18:0

3.41±0.35

3.36±0.63

3.09±0.14

2.90±0.34

∑ S FA

27.06±2.23c

24.48±0.34b

23.99±0.49ab

23.02±0.73a

14:1n7

3.83±0.32b

2.82±0.35a

3.01±0.84ab

2.51±0.68a

16:1n7

12.18±1.09

13.04±0.99

12.43±0.72

12.72±0.83

16:1n5

2.08±0.45

2.24±0.26

2.49±0.32

2.76±0.31

17:1

0.84±0.12ab

0.80±0.04a

0.83±0.10ab

0.90±0.02b

18:1n9

20.93±0.80a

21.22±1.36a

22.16±1.42ab

23.13±0.70b

18:1n7

2.68±0.33b

2.17±0.19a

2.23±0.07a

2.44±0.16ab

20:1n9

2.59±0.38a

2.74±0.38a

3.25±0.29b

3.50±0.34b

20:1n7

0.57±0.05

0.51±0.05

0.54±0.04

0.61±0.08

45.53±1.94a

46.94±0.54ab

48.57±1.13b

10.28±0.53b

9.41±1.62ab

8.54±0.39a

4.29±1.14b

3.69±0.24ab

3.07±0.38a

0.38±0.07

0.40±0.03

0.36±0.03

Pr

e-

pr

oo

f

15:0

45.68±0.95a

18:2n6

8.70±0.92a

18:3n3

4.31±0.62b

18:4n3

0.41±0.07

20:2n6

1.28±0.13a

1.46±0.15a

1.55±0.18ab

1.84±0.31b

20:4n6

2.90±0.47

3.26±0.22

3.32±0.41

3.38±0.40

2.42±0.31ab

2.61±0.31b

2.54±0.12b

22:2n6

rn

Jo u

20:5n3

al

∑ MUFA

2.07±0.18a 0.21±0.14

0.16±0.14

0.36±0.28

--

0.45±0.07a

0.51±0.07ab

0.60±0.06b

0.64±0.08b

1.32±0.11a

2.02±0.13b

2.14±0.29bc

2.26±0.09c

22.29±1.73a

25.63±1.69ab

24.79±0.69b

23.79±0.38a

∑n-3PUFA

8.81±0.90

9.99±1.63

9.79±0.93

9.47±0.48

∑n-6PUFA

13.33±1.00a

15.40±0.52b

14.92±0.87ab

14.06±0.31a

n-3/n-6

0.66±0.02

0.65±0.10

0.66±0.09

0.67±0.04

∑LC-PUFA

7.22±0.83a

8.83±0.98ab

9.29±0.91b

9.72±0.52b

DHA/EPA

0.64±0.03a

0.84±0.08b

0.82±0.11b

0.89±0.06b

22:5n3 22:6n3 ∑PUFA

Data are presented as mean ± SD (n=4). Values in the same row with different superscripts are significantly different (P<0.05); the fatty acid with more than 0.3% of total fatty acids is shown in the table.

Journal Pre-proof

Table 5 Effect of a 60-day fattening period on fatty acid profile in the muscle of male Eriocheir sinensis (% total total fatty acids) Day 0

Day 20

Day 40

Day 60

14:0

0.71±0.02b

0.82±0.09c

0.64±0.06a

0.60±0.01a

15:0

0.43±0.01

0.48±0.03

0.38±0.02

0.37±0.01

16:0

14.17±0.04c

12.81±0.36b

11.21±0.31ab

10.59±0.53a

17:0

1.04±0.02c

0.94±0.08ab

0.93±0.03b

0.83±0.02a

18:0

8.30±0.13

7.41±0.88

8.62±1.19

7.17±0.58

∑ SFA

24.92±0.01c

22.65±0.96bc

21.89±1.47b

19.56±1.06a

14:1n7

3.95±0.92a

7.12±1.51b

16:1n7

4.52±0.20

4.47±0.11

16:1n5

1.11±0.13

1.03±0.14

17:1

0.51±0.03

0.54±0.05

18:1n-9

19.23±0.58b

18.87±1.25ab

18:1n-7

2.46±0.03

20:1n-9

4.07±1.09a

4.49±0.36

4.10±0.41

1.18±0.00

1.18±0.00

0.59±0.08

0.54±0.01

18.01±1.04a

17.42±0.40a

2.36±0.33

2.09±0.19

2.30±0.03

0.92±0.06

1.01±0.12

1.21±0.15

1.07±0.10

20:1n-7

0.16±0.02

0.15±0.13

0.21±0.14

0.14±0.20

∑ M UFA

32.85±1.96ab

35.56±1.80b

31.10±1.06a

30.82±0.11a

18:2n-6

6.92±0.47

7.14±0.86

7.52±0.34

7.26±0.55

18:3n-3

1.10±0.35

1.13±0.12

1.15±0.00

1.19±0.02

20:2n6

1.59±0.24

1.33±0.06

1.43±0.13

1.58±0.09

20:3n3

0.44±0.11

0.33±0.05

0.37±0.05

0.39±0.01

20:4n-6

8.17±0.59ab

7.09±0.70a

8.86±0.10b

9.50±0.24c

9.63±0.20a

9.58±1.02a

12.22±0.28b

13.91±0.98b

0.94±0.00

0.73±0.04

0.88±0.01

0.77±0.07

6.39±0.01a

6.75±0.16b

8.89±0.03c

9.04±0.02d

∑PUFA

37.71±2.43a

36.49±0.90b

43.62±0.06c

45.36±0.14d

∑n-3PUFA

18.93±1.01a

17.83±0.95a

22.85±0.38b

24.44±0.70b

∑n-6PUFA

17.04±1.46ab

15.83±0.12a

18.09±0.17b

18.59±0.43b

n-3/n-6

1.12±0.16ab

1.13±0.06a

1.26±0.03b

1.32±0.07c

∑LC-PUFA

25.94±0.84a

24.58±1.72a

31.49±0.40b

33.75±0.93b

DHA/EPA

0.66±0.01a

0.71±0.09ab

0.73±0.01b

0.65±0.05a

20:5n-3 22:5n3 22:6n3

rn

al

Pr

e-

pr

3.31±0.56a

Jo u

oo

f

Fatty acid

Data are presented as mean ± SD (n=4). Values in the same row with different superscripts are significantly different (P<0.05); the fatty acid with more than 0.3% of total fatty acids is shown in the table.

Journal Pre-proof

Table 6 Effect of a 60-day fattening period on amino acids content in the gonad of male E. sinensis (mg/g wet weight) Day 20

Day 40

Day 60

Histidine

3.94±0.14a

4.89±0.20b

4.56±0.32b

4.95±0.53b

Isoleucine

4.32±0.12a

5.75±0.48b

7.04±0.34c

7.81±0.31d

Leucine

6.98±0.39a

9.69±0.36b

10.15±0.65c

11.21±0.80c

Lysine

9.09±0.15a

8.75±1.57ab

9.15±0.15ab

9.34±0.12b

M ethionine

1.49±0.20a

2.10±0.12c

1.77±0.23ab

1.86±0.14b

Cysteine

4.47±0.38a

5.43±0.35b

6.83±0.48c

7.42±0.29d

Phenylalanine

5.32±0.14a

6.74±1.11b

8.04±0.56b

9.02±0.42c

Tyrosine

4.55±0.42a

6.23±0.53b

7.36±0.06c

7.86±0.32d

Threonine

8.48±0.35a

9.93±0.38b

13.00±0.70c

14.06±0.84c

Valine

9.95±0.38a

11.65±0.83b

11.41±1.13b

10.58±0.36ab

Tryptophan

1.91±0.13

1.80±0.12

1.88±0.09

1.95±0.17

60.50±1.27a

72.96±2.38b

81.19±1.70c

86.05±3.52c

oo

pr

1.45±0.05c

1.13±0.22b

0.46±0.06a

Aspartic acid

12.15±0.32a

15.06±0.41b

17.40±1.20c

18.25±1.61c

Serine

4.94±0.15a

5.75±0.28b

7.03±0.12c

7.26±0.12c

Glutamic acid

16.72±0.91a

20.03±0.69b

21.20±0.69bc

22.62±1.16c

Glycine

5.60±0.30a

6.71±0.36b

6.70±0.60bc

7.36±0.36c

8.01±0.45a

9.69±0.30b

11.74±0.94c

13.24±0.54d

6.74±0.77a

9.22±0.65b

8.86±0.21b

8.66±0.48b

10.68±1.39a

14.27±1.13b

20.20±1.17c

23.55±3.07c

66.29±1.45a

82.47±2.26b

94.28±2.80c

101.40±3.96d

126.79±2.79a

155.40±4.38b

175.47±4.42c

187.44±6.82d

0.48±0.01b

0.47±0.01ab

0.46±0.00a

0.46±0.01a

Proline NEAA TAA EAA/TAA

rn

Arginine

Jo u

Alanine

1.64±0.30bc

Pr

Taurine

al

EAA

f

Day 0

e-

Amino acids

Data are presented as mean ± SD (n=4). Values in the same row with different superscripts are significantly different (P <0.05); EAA means essential fatty acids; NEAA means no-essential fatty acids; TAA means total amino acids.

Journal Pre-proof

Table 7 Effect of a 60-day fattening period on amino acid content in the muscle of male E. sinensis (mg/g wet

Day 0

Day 20

Day 40

4.54±0.05a 6.14±0.81a

5.06±0.32b 6.53±0.39a

Leucine Lysine

11.36±0.29a 12.45±0.67a

11.28±0.29a 15.33±0.37b

12.83±0.17b 15.85±0.73b

12.63±0.22b 15.46±1.09b

Methionine

3.17±0.36a

3.65±0.12b

3.54±0.23b

3.60±0.43b

Cysteine

4.35±0.27a

5.06±0.37b

5.03±0.63b

5.17±0.32b

Phenylalanine

7.09±0.50a

8.66±1.35ab

8.35±0.44b

8.35±0.34b

Tyrosine

6.34±0.22a

7.06±0.42b

7.96±0.23c

7.50±0.21b

Threonine

6.67±0.41a

6.86±0.43a

7.65±0.52ab

8.25±0.31b

Valine Tryptophan

9.43±0.35a 1.43±0.12

10.32±0.76b 1.63±0.18

11.11±0.48b 1.49±0.12

10.75±0.95b 1.64±0.16

72.97±0.96a

81.42±0.96b

86.65±1.04c

86.15±1.64c

0.34±0.10a

0.35±0.12ab

0.45±0.03b

0.41±0.15ab

Aspartic acid Serine

15.42±0.40a

16.30±0.41b

19.71±0.81c

19.90±0.47c

6.50±1.04ab

5.93±0.64a

6.08±0.54a

7.44±0.44b

Glutamic acid

20.67±0.29a 8.98±0.20a

22.67±0.52b 10.58±0.86b

24.60±1.20c 11.80±0.95b

23.99±0.87c 11.32±0.30b

Alanine

14.26±0.54a

13.67±0.54a

15.73±0.32b

15.30±0.53b

Arginine Proline

15.81±0.62a 8.01±0.24ab

17.28±0.95b 7.91±0.34ab

17.62±0.40b 7.85±0.29a

18.63±0.86b 8.49±0.27b

NEAA

89.99±0.28a

94.69±1.10b

103.83±1.45c

105.47±0.76c

TAA

162.96±1.25a 176.11±2.04b

190.48±2.49c

191.62±1.15c

0.45±0.01

0.45±0.01

Glycine

Pr

al

rn Jo u

Taurine

EAA/TAA

pr

Histidine Isoleucine

EAA

0.45±0.01

5.45±0.39bc 7.38±0.17b

Day 60

e-

Amino acids

oo

f

weight)

0.46±0.00

5.58±0.12c 7.22±0.38b

Data are presented as mean ± SD (n=4). Values in the same row with different superscripts are significantly different (P <0.05); EAA means essential fatty acids; NEAA means no-essential fatty acids; TAA means total amino acids.

Journal Pre-proof

Table 8 Effect of a 60-day fattening period on essential amino acids score (EAAS) in the gonad and muscle of male E. sinensis Adult EAA

Gonad

M uscle

requirements (mg/g protein)

Day 0

Day 20

Day 40

Day 60

Day 0

Day 20

Day 40

Day 60

15

207

210

173

176

186

191

191

194

Isoleucine

30

114

123

134

139

126

124

129

126

Leucine

59

93

106

98

101

118

109

114

112

Lysine

45

159

125

116

111

170

193

185

179

22

213

220

223

225

210

225

205

208

38

205

220

231

237

217

235

225

218

Threonine

23

291

278

322

Valine

39

201

192

167

Tryptophan

6

251

193

179

174

193

185

182

181

+Tyrosine

178

169

175

187

145

148

150

150

144

146

154

131

143

166

172

167

168

Pr

Mean

326

e-

Phenylalanine

pr

Cysteine

oo

M ethionine+

f

Histidine

EAAS=100 × one essential fatty acid content in sample/one essential fatty acid content in reference protein for adult maintenance

Jo u

rn

al

(WHO/FAO/UNU 2007).

Journal Pre-proof

Research highlights 

Fattening immature crab is important to improve gonadal development and product quality of male Eriocheir sinensis.



Gonadosomatic index and total edible yields increased significantly during the fattening.



Fattening period significantly affected nutritional composition of gonad, hepatopancreas and meat.

rn

al

Pr

e-

pr

oo

f

The appropriate fattening period is approximately 40 days for pond-reared male E. sinensis.

Jo u

