The growth performance, body composition and nonspecific immunity of Tilapia (Oreochromis niloticus) affected by chitosan

The growth performance, body composition and nonspecific immunity of Tilapia (Oreochromis niloticus) affected by chitosan

International Journal of Biological Macromolecules 145 (2020) 682–685 Contents lists available at ScienceDirect International Journal of Biological ...

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International Journal of Biological Macromolecules 145 (2020) 682–685

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

The growth performance, body composition and nonspecific immunity of Tilapia (Oreochromis niloticus) affected by chitosan Shengjun Wu ⁎ Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, 59 Cangwu Road, Haizhou 222005, China Jiangsu Key Laboratory of Marine Biotechnology, 59 Cangwu Road, Haizhou 222005, China Co-Innovation Center of Jiangsu Marine Bio-industry Technology, 59 Cangwu Road, Haizhou 222005, China

a r t i c l e

i n f o

Article history: Received 21 November 2019 Received in revised form 14 December 2019 Accepted 24 December 2019 Available online 27 December 2019 Keywords: Chitosan Growth performance Immunity

a b s t r a c t The aim of this study was to investigate the effects of dietary chitosan on the growth performance, body composition and non-specific immunity of tilapia (Oreochromis niloticus). Chitosan were added to the basic diet to formulate five kinds of test feeds (0, 2, 4, 6 and 8 g kg−1). The diets containing 4 g kg−1 chitosan increased body weight gain, feed conversion rate, specific growth rate, body protein, superoxide dismutase activity, catalase activity, lysozyme, disease resistance ability against Aeromonas hydrophila and decreased hepatopancrease lipid levels, plasma total cholesterol, plasma triacylglycerol, aspartate aminotransferase and alanine aminotransferase of tilapias compared with those of the control group. However, a high level of chitosan (8 g kg−1) decreased its efficiency compared to moderate level of chitosan (4 g kg−1). The results demonstrated that chitosan could promote the growth of tilapias and improve their disease resistance against A. hydrophila.

1. Introduction Tilapia (Oreochromis niloticus) is one of the most important freshwater fish cultures in the world. The fish is delicate, delicious, rich in protein and a variety of unsaturated fatty acids. It has been recommended by FAO as a breeding species to solve the problem of human protein sources in poor countries [1]. The rapid growth of tilapia production leads to huge demand for feeds and the outbreaks of infections resulting in substantial harm to the yield [1]. Thus, it is important to develop effective native compounds with immunomodulatory activity to fulfill the requirement for the manufacture of commercial diets [1]. Chitosan is a linear copolymer of 2-amino-2-deoxy-D-glucopyranose and traces of 2-acetamido-2-deoxy-D-glucopyranose linked by β-1,4glychitosanidic bond linked. Chitosan have many biological activities, such as antioxidant, antibacterial, hypolipidemic and immunomodulatory activities [2–5]. Chitosan-containing diets stimulated the growth performance of caspian kutum (Rutilus frisii kutum Kamenskii, 1901) fingerlings, shrimp (Penaeus monodon) and loach (Misgurnus anguillicadatus) [6–9]. Nevertheless, data regarding the effects of chitosan on the growth performance and immunity of tilapia are limited. This study explored the effects of dietary chitosan on the growth, body composition, nonspecific immunity and disease resistance ability

against Aeromonas hydrophila of tilapia. The results may provide a theoretical basis for the dietary supplementation of chitosan for tilapia. 2. Materials and methods 2.1. Materials Chitosan were prepared from shrimp shells Penaeus monodon and according to the methods described by Srinivasan, Velayutham and Ravichandran [10]. The molecular weight (MW) and degree of deacetylation were 32 × 104 Da and 93.7%, respectively. All other chemicals used in this study were of reagent grade. 2.2. Diet preparation The composition of the basal diet is provided in Table 1. Chitosan were added to the basal diet at five levels (0, 2, 4, 6 and 8 g kg−1 dry diets). All ingredients were fully mixed with appropriate amount of tap water, coldly extruded using an extruder (Polylab, Thermo Fisher Scientific Company, USA), cut into particles, air dried and stored at room temperature. 2.3. Tilapia culture

⁎ Corresponding author at: Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, 59 Cangwu Road, Haizhou 222005, China. E-mail address: [email protected]

https://doi.org/10.1016/j.ijbiomac.2019.12.235 0141-8130/

The aquaculture equipment was a 240 L glass tank equipped with recirculating aquaculture system. Tilapias with an average body weight of 50.13 ± 4.13 g were temporarily cultured for 2 weeks and then

S. Wu / International Journal of Biological Macromolecules 145 (2020) 682–685

supernatant was then collected and stored at −60 °C until use. The hepatopancrease was collected.

Table 1 Composition of basal diets of Tilapia (Oreochromis niloticus). Ingredient

%

Fish meal Soybean meal Cottonseed meal Middling Flour DDGS Soybean oil Vitamin mixturea Mineralb

25 10 10 15 13 21 2 2.4 1.6

Proximate analysis (% dry matters) Water Crude protein Crude lipid Carbohydrate Ash

683

9.51 36.74 3.65 30.08 8.81

a Vitamin mixture provided per kg of diet: VA 12,500,000 IU; VD 2,000,000 IU; VE 7000 IU; VK 2000 mg; VB1 800 mg; VB2 2500 mg; VB6800 mg; VB1210 mg; niacin 3000 mg; pantothenic acid 10, 000 mg; folic acid 300 mg; biotin 20 g; VC 20,000 mg. b Mineral provided per kg of diet: Mn 19 mg; Mg 230 mg; Co 0.1 mg; I 0.25 mg; Fe 140 mg; Cu 2.5 mg; Zn 65 mg; Se 0.2 mg.

placed in clean aquaculture water without feeding for 2 days to adequately defecate and adapt to the aquaculture environment. During the temporary feeding period, the tilapias were fed with basal diet three times a day (08:00, 12:30, and 17:00), and the total feeding weight was 4% (w/w) of the body weight. A total of 450 tilapias were randomly assigned to 15 glass tanks, resulting in 3 tanks per group and 30 tilapias per tank. During the feeding period, the tilapias were fed three times a day (08:00, 12:30, and 17:00), and the total feeding weight was 3%–4% (w/w) of the body weight (Adjusting feeding amount according to fish body size). During the feeding period, continuous aeration was applied, and natural cycle lighting was used. The culture period was 56 days.

2.6. Biochemical assays Moisture, protein and lipid contents were determined in accordance with standard methods [11]. Plasma total cholesterol (TC), triacylglycerol (TG) and gluchitosane were assayed by using commercial assay kits. Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase (SOD) activity, catalase (CAT) activity, and Lysozyme (LZM) were determined using ELISA kits in accordance with the methods of Gao et al. [12]. A unit of enzyme activity was defined as the amount of enzyme that decreased the absorbance by 0.001 min−1. 2.7. Pathogen challenge test At the end of the 56-day feeding experiments, 15 tilapias were randomly selected from each tank and raised in tanks. After 5-day acclimation, the tilapias were subjected to intraperitoneal injection of 1.5 × 105 cfu/g body weight of A. hydrophila based on our previous results. The challenge test period was carried out for 2 weeks. The culture conditions were identical to the aforementioned culture conditions. During the challenge test, the behaviour of the tilapias was observed and the mantissa was recorded. 2.8. Statistical analysis All experiments were conducted in triplicate. All data were all expressed by mean ± standard deviation (SD). SPSS19.0 was used for one-way ANOVA analysis of data. Duncan's method was used for multiple comparisons between groups and p b .05 indicates significant differences. 3. Results and discussion

2.4. Growth performance

3.1. Growth performance and survival rate

At the end of the aquaculture experiment, the body weight of tilapias in each group was weighed. Body weight gain rate (BWGR), feed conversion ratio (FCR), specific growth rate (SGR) and survival rate were calculated according to the following equations:

With the increase of chitosan doses, BVGR, FCR and SGR increased first and then decreased (Table 2). The tilapias fed with the diet containing 4 g kg−1 chitosan exhibited higher BVGR, FCR and SGR compared with control group (p b .05). Chitosan have antibacterial and immunomodulatory activities and inhibited the growth and infections the bacterial and viral, thus simulating growth performance of tilapias [3,5]. However, a high dose of chitosan (N6 g kg−1) did not further increase BVGR, FCR and SGR. High dose of chitosan presumably exhibited hypolipidemic activity and thus decreased the growth performance of the tilapias [4].

BWGR ð%Þ ¼ ðW1 –W0 Þ=W0  100% FCR ð%Þ ¼ W3 =W2  100%

  −1 ¼ ½Ln ðW1 Þ–Ln ðW0 Þ=t ðdaysÞ  100% SGR %day

Survival rate ð%Þ ¼ ðN1 =N0 Þ  100%: where W0 is initial body weight; W1 is final body weight; W2 is total weight of dried diet consumption; W3 is net body weight gain; N1 is final number of tilapias; t is time; N1 is initial number of tilapias. 2.5. Sampling At the end of the culture experiment, three tilapias from each tank were randomly collected and killed with MS222. Blood samples were collected from tail fin and allowed to clot at 3 °C for 24 h. The resulted

3.2. Proximate composition of whole body and hepatopancrease Effects of dietary chitosan on proximate composition of whole body and hepatopancrease of tilapia are presented in Table 3. There were no differences in moisture contents of whole body and hepatopancrease (p N .05). The increases in chitosan doses increased whole body protein content and decreased lipid content in whole body and hepatopancrease. The diets containing 4, 6 and 8 g kg−1 chitosan exhibited higher protein contents in whole body and hepatopancrease and lower lipid contents in hepatopancrease compared with control group (p b .05). Chitosan play important roles in the modulation of many receptors, e.g. olfactory receptor, epidermal growth factor receptor, Tolllike receptor 4, TLR4/MD-2 receptor, scavenger receptor BI and CYP7A1 as well as Calcium-Sensing Receptor (CaSR) [13–19]. Therefore, chitosan presumably were mediated by some receptors and subsequently stimulated protein biosynthesis. In addition, high dose of

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Table 2 Growth performance and survival rate of Tilapia (Oreochromis niloticus) after 56 days of feeding different diets. Parameters

Chitosan level (g kg−1) 0

2

4

6

8

Initial weight (g) BVGR (%) FCR (%) SGR (%) Survival rate (%)

50.13 ± 4.13a 251.62 ± 3.46ab 1.21 ± 0.02a 2.16 ± 0.13ab 100

50.11 ± 4.09a 271.14 ± 4.35bc 1.25 ± 0.01ab 2.34 ± 0.17bc 100

50.11 ± 4.12a 291.12 ± 5.49c 1.34 ± 0.02bc 2.45 ± 0.19c 100

50.15 ± 4.17a 268.07 ± 4.68bc 1.33 ± 0.03bc 2.15 ± 0.14ab 100

50.14 ± 4.13a 239.53 ± 3.16a 1.22 ± 0.02a 2.11 ± 0.11a 100

Different superscript letters indicate significant differences between the same row (p b .05). Values are the mean ± SD (n = 3).

chitosan presumably exhibited hypolipidemic activity and thus inhibited lipid biosynthesis of the tilapias [4].

in tilapia serum, and this could be due to the antioxidant and immunomodulatory activities of chitosan [2,5].

3.3. Serum biochemical indicators

3.4. Immunological indicators

Effects of dietary chitosan on serum biochemical indicators are shown in Table 4. The diets containing 4, 6 and 8 g kg−1 chitosan showed lower serum TC, TG, gluchitosane, ASP and ALP levels compare with control group (p b .05). The decreased serum TC, TG and gluchitosane levels could be due to the hypolipidemic and hypoglycemic activities of chitosan [4,21]. Both ASP and ALP are vital aminotransferases, and they involve in protein metabolism. The changes in activities of ASP and ALP are important indicators that reflect the healthy status of hepatocyte. A relatively low activity of aminotransferase reflects a normal serum condition. When the hepatocytes are impaired, cell membrane permeability could increase, and large amounts of ASP and ALP infiltrate into the blood, thus increasing the activities of ASP and ALP in the blood. The results showed that the diets containing 4, 6 and 8 g kg −1 chitosan increased the activities of ASP and ALP

Table 5 shows effects of dietary chitosan on immunological indicators. The diets containing chitosan exhibited higher serum SOD, CAT and LYZ activities compare with control group (p b .05). An antioxidant system can scavenge excess free radicals and thus protect itself from oxidative damage. Stress increases free radical production and results in the accumulation of free radicals in the body, whereas excessive free radicals can cause lipid peroxidation damage. Both SOD and CAT can scavenge free radicals and decrease lipid peroxidation damage. LYZ is an important non-specific immune indicator in aquatic animals. This enzyme can effectively kill Gram-positive bacteria [21]. LYZ activity in aquatic animals directly reflects their immune and healthy status, and the immunity of aquatic animals is positively related to LYZ activity [22]. The results showed that serum SOD, CAT and LYZ activities of tilapias fed with diets containing chitosan considerably increased.

3.5. Challenge test Table 3 Proximate composition of whole body and hepatopancrease of Tilapia (Oreochromis niloticus) after 56 days of feeding different diets. Parameters Chitosan level (g kg−1) 0

2

4

6

8

Whole body Moisture 73.12 (%) ± 1.14a Protein (%) 14.75 ± 0.45a Lipids (%) 7.13 ± 0.21d

73.04 ± 1.17a 15.08 ± 0.47ab 6.84 ± 0.19cd

72.93 ± 1.08a 15.67 ± 0.51bc 6.31 ± 0.17bc

73.11 ± 1.12a 16.13 ± 0.62cd 6.02 ± 0.15ab

73.15 ± 1.17a 16.46 ± 0.71de 5.48 ± 0.13a

Hepatopancrease Moisture 70.08 (%) ± 0.34a Lipids (%) 10.73 ± 0.27d

70.12 ± 0.37a 10.38 ± 0.24cd

70.27 ± 0.41a 10.14 ± 0.21bc

70.21 ± 0.32a 9.91 ± 0.18ab

70.05 ± 0.31a 9.16 ± 0.14a

Different superscript letters indicate significant differences between the same row (p b .05. Values are the mean ± SD (n = 3).

Effects of dietary chitosan on survival rate of tilapias after being challenged by A. hydrophila are presented in Table 6. Dietary chitosan increased survival rate of tilapias after being challenged by A. hydrophila, and this result could be attributed to the antibacterial and immunomodulatory activities of chitosan [3,5]. Similarly, a previous study reported that chitosan also increased the disease resistance ability of loach fish against A. hydrophila [9].

4. Conclusions In conclusion, oral administration of chitosan diets improved the growth performance, body composition and immunity status of tilapias. However, high dose of chitosan (N 6 g kg−1) decreased growth performance. Therefore, 6 g kg−1 was optimal for the growth of tilapias. The results indicated that chitosan may be used as a diet supplement to improve the growth, body composition and immunity of tilapias.

Table 4 Serum biochemical indicators after 56 days of feeding different diets. TC: plasma total cholesterol; TG: plasma triacylglycerol; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Parameters

Chitosan level (g kg−1) 0

TG (mmol/L) TC (mmol/L) AST (mmol/L) ALT (mmol/L)

2 a

4.06 ± 1.02 3.04 ± 0.07a 16.13 ± 1.32a 6.71 ± 0.61a

4 ab

3.72 ± 0.91 2.82 ± 0.06ab 15.58 ± 1.09ab 6.47 ± 0.58ab

6 bc

3.27 ± 0.85 2.62 ± 0.05bc 15.27 ± 0.93bc 6.13 ± 0.51bc

Different superscript letters indicate significant differences between the same row (p b .05). Values are the mean ± SD (n = 3).

8 cd

3.08 ± 0.76 2.51 ± 0.05cd 15.23 ± 0.87cd 5.86 ± 0.47cd

2.67 ± 0.72de 2.39 ± 0.04de 14.81 ± 0.81de 5.51 ± 0.42de

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Table 5 Immunological indicators after 56 days of feeding different diets. SOD: superoxide dismutase; CAT: catalase; LYZ: lysozyme. Parameters

SOD (U/ml) CAT (U/ml) LYZ (U/ml)

Chitosan level (g kg−1) 0

2

4

6

8

28.17 ± 2.14a 2.46 ± 0.23a 196.24 ± 10.21a

32.61 ± 3.42b 3.51 ± 0.38b 214.46 ± 12.34b

37.49 ± 3.61c 4.61 ± 0.44c 225.62 ± 15.72bc

42.37 ± 4.06d 5.74 ± 0.53d 234.24 ± 16.08cd

43.72 ± 4.17de 5.86 ± 0.56de 237.09 ± 16.11d

Different superscript letters indicate significant differences between the same row (p b .05). Values are the mean ± SD (n = 3).

Table 6 Survival rate (%) of Tilapia (Oreochromis niloticus) after being challenged by Aeromonas hydrophila. Parameters

Survival rate (%, 1 week) Survival rate (%, 2 week)

Chitosan level (g kg − 1) 0

2

4

6

8

65.21 ± 5.71a 53.22 ± 6.12a

68.42 ± 5.53b 64.36 ± 7.64b

73.46 ± 7.31c 70.72 ± 8.79c

78.27 ± 8.17d 75.82 ± 9.35d

82.38 ± 8.89e 80.83 ± 10.56e

Different superscript letters indicate significant differences between the same row (p b .05). Values are the mean ± SD (n = 3).

Ethics statement This study was approved by the ethics committee of Jiangsu Ocean University, China. All procedures were conducted in compliance with relevant laws and institutional guidelines. CRediT authorship contribution statement Shengjun Wu: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Visualization, Investigation, Supervision, Software, Validation, Writing - review & editing. Acknowledgements This research was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and preparation and properties of Porphyra yezoensis polysaccharide (CXKT20180215). References [1] FAO, The State of World Fisheries and Aquaculture, Contributing to Food Security and Nutrition for all, 200 pp. Rome, Italy. 978-92-5-109185-2 2016. [2] S.H. Chang, C.H. Wu, G.J. Tsai, Effects of chitosan molecular weight on its antioxidant and antimutagenic properties, Carbohydr. Polym. 181 (2018) 1026–1032. [3] S. Sharma, Enhanced antibacterial efficacy of silver nanoparticles immobilized in a chitosan nanocarrier, Int. J. Biol. Macromol. 104 (2017) 1740–1745. [4] W. Zhang, J. Zhang, Q. Jiang, W. Xia, A comparative study on hypolipidemic activities of high and low molecular weight chitosan in rats, Int. J. Biol. Macromol. 51 (2012) 504–508. [5] R. Harikrishnan, J.S. Kim, C. Balasundaram, M.S. Heo, Immunomodulatory effects of chitin and chitosan enriched diets in Epinephelus bruneus against Vibrio alginolyticus infection, Aquaculture 326-329 (2012) 46–52. [6] M. Kamali Najafabad, M.R. Imanpoor, V. Taghizadeh, A. Alishahi, Effect of dietary chitosan on growth performance, hematological parameters, intestinal histology and stress resistance of Caspian kutum (Rutilus frisii kutum Kamenskii, 1901) fingerlings, Fish Physiol. Biochem. 42 (2016) 1063–1071. [7] J. Niu, C.H. Li, L.X. Tian, Y.J. Liu, X. Chen, K.C. Wu, W. Jun, Z. Huang, Y. Wang, H.Z. Lin, Suitable dietary chitosan improves the growth performance, survival and immune function of tiger shrimp, Penaeus monodon, Aquac. Res. 46 (2015) 1668–1678.

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