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Effect of Antibacterial Peptides on the Growth and Immunity of Freshwater Mud Eels
News sources:【管理員】    Release time:2019-04-10   Browse the number:666Time



The Aquatic Economic Animal Research Center of Yangtze University was cooperated with Shenzhen Sunsmile Biotechnology Co., Ltd. to explore the effect of antibacterial peptides produced by Sunsmile on the growth and immunity of mud eels in 2018. The test methods and results are as follows.


 1. Test materials and culture management  


1.1 Antimicrobial peptides (AMPs)

AMPs was provided by Shenzhen Sunsmile Biotechnology Co., Ltd.

1.2 Mud eels

Mud eels were bred in the pond at the Aquatic Economic Animal Research Center of Yangtze University. Fishes with uniform size (30±5 g/head) and health were placed in the pond cage (2 x 2 m) for testing. Three replications for each treatment were made and each replication was placed in the same cage.

1.3 Study area

The experiment was carried out over a period of 68 days during July 20, 2018 to September 28 in laboratory aquarium, belonging to the Aquatic Economic Animal Research Center of Yangtze University.

1.4 Supplied feed

Five different AMPs concentrations were separately mixed in feed including: control (no added AMPs in feed), AMPs 200 mg/kg (I), AMPs 400 mg/kg (II), AMPs 600 mg/kg (III) and AMPs 800 mg/kg (IV). The composition of feed in each group is shown in Table 1. The feeds were supplied to each cage one a day at 6.00 P.M. at the rate of 7 to 11% of the body weight. During the experiment, the water quality, feed intake and the residue of feed were recorded. Sampling was performed after fish no feeding for 24 hours at the end of the experiment. 

Table 1 The composition of basal diet




1.5 Indicator determination method

1.5.1 Growth performance

At the end of the experiment, the final body weight of each group was determined and the survival rate, relative weight gain rate and feed coefficient were calculated.

Relative weight gain rate = (final weight / g - initial weight / g) + [initial weight / g] x 100%

Feed coefficient = (feeding amount / g) + (weight gain / g).


1.6 Determination of biochemical indicators and immune indicators

1.6.1 Sample Collection

12 fishes in each group were randomly selected and each two fishes were combined to one sample. Fish blood was collected and centrifuged at 4000 r/min and the supernatant was aspirated and stored at -80 °C.

1.6.2 Determination of biochemical indicators and immune indicators

In this experiment, a total of 8 biochemical and immunological indicators in serum were detected: total protein TP, triglycerides (TG), glutamate pyruvic transaminase (GPT), superoxide dismutase (Superoxide dismutase, SOD), catalase (CAT), glutathione peroxidase (GSH-Px), acid phosphatase (ACP), alkaline phosphatase (alkaline phosphatase, AKP) was measured using a kit.

1.7 Determination of the survival rate of challenge

At the end of the feeding trial, 20 eels with similar weights were selected and placed in pre-numbered 60L plastic drums. After a 7-day acclimation period, fish was intraperitoneally inject of Aeromonas hydrophila (A. hydrophila strain from the Aquatic Laboratory of Huazhong Agricultural University) at 0.4 ml/head (1 x 109 CFU/ml). Then, fish swimming and death were observed every two hours. After 7 days, the death fish and death time were counted, and the survival rate (SR) and immune protection (RPS) after the challenge were calculated.

Survival rate (%) = 100 × survival number / initial total;

Immunoprotective power (%) = 100 × (test group survival rate - control group survival rate) / control group survival rate

1.8 Data Statistics and Analysis

The test data were analyzed by one-way ANOVA and Duncan's multiple comparisons in the SPSS 20.0 software package to examine the differences between the groups. Different lowercase letters indicated significant differences between the groups (p < 0.05). All results are expressed as mean standard error (mean ± SEM).


  2
Test results  

2.1 The effect of antimicrobial peptides on the growth and survival rate of mud eels

The growth and survival rate of mud eels are shown in Table 2. The growth rate of mud eels was increased with increasing AMPs rate in bait. The growth rate of the mud eels can be effectively promoted, the feed coefficient was lowered, and the survival rate was improved. Mud eels fed with AMPs 600 mg/kg showed significantly (p<0.05) promoted the growth rate, decreased the feed coefficient, and increased the survival rate.

Table 2 Effect of AMPs on the growth and survival rate of mud eels

Note: The data in the table are mean ± standard deviation (n = 6), and the lowercase letters of the same column data indicate significant difference (p< 0.05).


2.2 Effect of AMPs on serum biochemical parameters of mud eels

The addition of AMPs in bait could increase (p<0.05) the TP level in the serum of eels and the TP content was increased with increasing the AMPs concentrations. The highest TP content was found in AMPs 600 mg/kg group. It was significantly increased by 15.99% compared with the control group (p<0.05). AMPS was significantly (p<0.05) decreased TG level in mud eels and the TG content was the lowest in AMPs 600 mg/kg, which was significantly lower than that of the control group by 32.10%. The serum alanine aminotransferase activity of each group with AMPs was increased to some extent and the activity was significantly (p<0.05) difference when used AMPs at 800 mg/kg.

Table 3 Effect of antimicrobial peptides on serum biochemical parameters of mud eels

Note: The data in the table is the mean ± standard deviation (n = 6), the lower column of the same column data indicates that the difference is significant (p <0.05).


2.3 Effect of AMPs on serum phosphatase of mud eels

AMPs could significantly increase the activity of serum phosphatase (Table 4). Alkaline phosphatase (AKP) activity was the highest when the addition ratio was 800 mg/kg and was significantly increased by 39.36% compared with the control group. The activity of acid phosphatase (ACP) was the highest when AMPs 600 mg/kg was added, which was significantly increased by 65.47% compared with the control group, and the activity was decreased when AMPs added to 800 mg/kg.

Table 4 Effect of AMPs on serum phosphatase activity of mud eels

Note: The data in the table is the mean ± standard deviation (n = 6), the lower column of the same column data indicates that the difference is significant (p<0.05).



2.4 Effect of AMPs on serum oxidase of mud eels

The results showed that (Table5), AMPs had significant effects on serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) (p<0.05). All three enzymes showed the highest activity in AMPs 600 mg/kg in the bait. Compared with the control group, the SOD was significantly increased by 25.36% (p<0.05). GSH-Px was significantly improved by 22.10% (p<0.05) and CAT significantly increased by 34.66% (p<0.05). The oxidase activity was decreased with increasing AMPs concentrations.
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Table 5 Effect of AMPS on serum antioxidant enzyme activity of mud eels 

Note: The data in the table is the mean ± standard deviation (n = 6), the lower column of the same column data indicates that the difference is significant (p <0.05).


2.5 Effect of AMPs on the survival rate of mud eels after A. hydrophila bacteria challenge

The pathological changes of dead fish are mainly manifested by congestion, hemorrhage, and extensive tissue degeneration and necrosis of liver and kidney. Feeding with AMPs could increase the survival rate of mud eels after the infection of A. hydrophila and the survival rate showed the highest when the feeding at 800 mg/kg (Table 6). The survival rate was significantly increased by 15% when compared with the control group (p<0.05) and the immune protective capacity was 85.71%.

Table 6 Results of the attack test

Note: Different lowercase letters in the same column of data indicate significant differences (P < 0.05).

  Summary  

The effect of AMPs on the growth and immunity of freshwater mud eels showed that the addition of AMPs in feed can effectively promote the growth and improve the immunity of mud eels. Addition at 600 mg/kg in feed is optimal.



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