Animal ethics
All the experimental procedures applied in this study were reviewed and approved by the Henan Society of Experimental Zoology. All procedures involving live fish handling, management, and health care followed the regulations of laboratory animals used for scientific purposes and were implemented within the perform experiments within the scope of the laboratory of the Henan University of Technology.
Preparation of B. Subtilis peptidoglycan
PG was derived from B. subtilis-02. The fermentation broth of B. subtilis-02 was centrifuged at 3000 rpm for 10 min, and then the precipitate was washed three times with 0.9% saline until the sediment was milky white. The precipitate (10 g) was dissolved with 10% trichloroacetic acid at a ratio of 10:1 (w/v), placed in a 75°C water bath for 30 min, and stirred to fully crack the bacteria. The solution was gradually cooled to room temperature and then centrifuged at 8000 for 10 min. The supernatant was discarded, the precipitate was washed with sterilized distilled water thrice, and the precipitate was collected. The precipitate was added with trypsin–phosphate buffer (3 mg/mL trypsin) at a ratio of 1:5 (w/v) and shaken on a shaker at 120 rpm for 3 h at 37°C. The solution was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The supernatant was centrifuged at 12000 rpm for 15 min, and the precipitate was collected. The precipitate was washed with sterilized distilled water thrice, suspended in ether for 30 min, and then centrifuged at 12000 rpm g for 15 min. The obtained precipitate was washed and dehydrated with absolute ethanol and dried at 70°C to obtain PG (Chen et al., 2019).
Selenated B. subtilis peptidoglycan preparation
The PG was hydrolyzed with lysozyme, freeze-dried, and stored. A certain amount of PG after enzymatic hydrolysis was weighed, added to 0.5% nitric acid (v/v) solution, and mixed well. Then, 2 mL of 0.05 g/mL sodium selenite solution was added dropwise. The reaction was carried out at 80°C for 8 h and then cooled. After the reaction was completed, the mixed solution was adjusted to pH 7 with saturated Na2CO3 solution and dialyzed with 1 kDa ultrafiltration membrane until no sodium selenium was detected. The dialysate was concentrated, added with 95% ethanol, and reacted overnight. After centrifugation, the precipitate was washed twice with absolute ethanol and freeze-dried to obtain Se-PG (He et al., 2019; Yang et al., 2020).
Feed preparation
Carp feed was prepared according to the feed nutrition standard (SC/T1026-1998). Basic feed was prepared with soybean meal, fish meal, and corn gluten meal as the protein sources and corn oil as the fat source (Table 1). The basic diet was supplemented with 0 (control group), 50, 100, 200, and 400 mg/kg Se-PG and mixed, extruded. Pellet feed with a 2.0 mm diameter was made by an SLKL-120B granulator, dried at 40°C for 24 h, and stored in a refrigerator at 4°C.
Table 1
Composition and nutrient levels of experimental diets (air-dry basis) %
Ingredients | Content | Nutrient levels | Content |
Fishmeal | 16.00 | Moisture | 4.59 |
Soybean meal Wheat bran Wheat flour Corn oil Dextrin Corn gluten Monocalcium phosphate Premix Lysine Methionine Choline chloride | 34.00 9.00 11.00 4.00 7.00 14.00 3.00 1.00 0.20 0.50 0.30 | Crude protein Crude fat Crude ash Crude fiber Calcium Selenium Total phosphorus Gross energy, kJ/g | 35.27 5.13 10.40 2.19 1.93 0.10 1.36 17.65 |
Note: 1 Compound premix: vitamin A, 3,600 IU/kg; vitamin D3, 1,200 IU/kg; vitamin E, 20 mg/kg, vitamin K3, 5 mg/kg; vitamin B1, 5 mg/kg; vitamin B2, 7 mg/kg; vitamin B6, 6 mg/kg; vitamin B12, 20 µg/kg; calcium pantothenate, 20 mg/kg; nicotinic acid, 30 mg/kg; folic acid, 1.7 mg/kg; biotin, 0.05 mg/kg; inositol, 90 mg/kg; magnesium, 150 mg/kg; iron, 120 mg/kg; manganese, 30 mg/kg; copper, 4 mg/kg; cobalt, 0.5 mg/kg; iodine, 1 mg/kg. 2 Each gram of selenized peptidoglycan contains 369.32 µg selenium. 3 Nutrient levels were measured. |
Fish and treatment
Juvenile carps were purchased from Henan Academy of Fishery Sciences (Yellow River Carp Breeding Farm). The juvenile carps weighing 39.76 ± 0.14 g were randomly distributed into fifteen cylindrical fiberglass tanks with a volume of length (100 cm) × width (60 cm) × height (70 cm), with 30 fish in each tank. Each dietary treatment was assayed in triplicate. The fish were subjected to an adaptation experiment for 2 weeks before the start of the formal experiment, during which they were fed through a basic diet without selenium supplementation. At the end of domestication, the control group was fed with basic diet, and the experimental groups were fed with experimental feed containing 50, 100, 200, and 400 mg/kg Se-PG. The fish were fed two times a day at 8:30 and 16:30 with the diets for 6 weeks. Weighed and recorded the weight of the fish in batches every 2 weeks. The daily feeding rate was about 3% of the body weight. One hour after feeding, uneaten food was siphoned out, dried, and weighed to determine feed intake. Once every 3 days, 30% of the rearing water was replaced. The mean water quality features were kept as follows: temperature, 26 ± 0.5°C; dissolved oxygen, 6.0 mg/mL; pH, 6.8 ± 0.2; and photoperiod, 12 h light, and 12 h darkness.
Growth parameters
At the end of the 6-week feeding trial, all the fish were anesthetized with MS-222. The weights of fish and liver were measured. Standard formulae were used to assess growth performance, feed utilization, and other parameters:
Weight gain rate (WGR, %) = [(final weight − initial weight)/initial weight] × 100;
Specific growth rate (SGR, %) = [ln(final weight) − ln(initial weight)/t] × 100;
Feed conversion rate (FCR, %) = (feed intake/weight gain) ×100;
Hepatosomatic index (HI, %) = (liver weight/whole body weight) × 100;
Condition factor (CF, %) = [body weight (g)/body length3] × 100.
Tissue homogenate
Blood was collected randomly from the caudal vein of six fish per tank. The extracted blood was poured into an Eppendorf tube for hematological analyses. Then, the blood was allowed to clot at 4°C for 1 h and then centrifuged at 4000 rpm, 20min. Serum was separated and frozen at − 80°C until used. The liver of each fish was packed in a sealed sample bag, immediately frozen with liquid nitrogen, and frozen at − 80°C. One gram carp liver sample was accurately weighed, placed in a 10 mL centrifuge tube, added with 9 g PBS solution, and fully homogenized on ice with an automatic homogenizer to obtain a tissue homogenate. The tissue homogenate was centrifuged at 4°C and 4,000 rpm for 20 min, and the supernatant was transferred in a 1.5 mL Eppendorf tube and immediately separated and frozen at − 80°C until used.
Determination of immune index in crap
The contents of serum immunoglobulin (IgE, IgG, and IgM), complement 3 (C3), interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 10 (IL-10), and tumor necrosis factor α (TNF-α) were determined according to enzyme-linked immunosorbent assay per the instructions of the kits (Shanghai Pyram Bioengineering Institute). The operation process of the determination was as follows. The standard was diluted first; then the number of plates required was determined based on the number of samples to be tested plus the number of standards. Each standard product and blank hole were duplicated. Finally, the sample 50 µL was accurately added to the standard sample on the enzyme coated plate, and the sample diluent 40 µL was added to the sample hole, and then the sample 10 µL was added. Add the sample to the bottom of the hole of the enzyme plate. After sealing the plate with the membrane, 30 min was incubated at 37°C. Remove the sealing film, discard the liquid, shake dry, fill each hole with detergent, rest for 30 seconds, then discard the detergent, repeat for 5 times, pat dry. Then 50 µL enzyme-labeled reagents were added to each hole except the blank hole. After sealing the plate with the sealing plate membrane, the 30 min was incubated again and the plate was washed. Then add chromogenic agent A 50 µL to each hole, and then add chromogenic agent B 50 µL, slightly shake and mix, and avoid light at 37°C to develop 10 min. After that, the terminating solution of 50 µL was added to each hole, and the blue in the enzyme plate hole immediately turned to yellow, and the blank hole was adjusted to zero. The absorbance value of each hole was measured sequentially at 450 nm wavelength, and the determination should be carried out in 15 min. The standard regression equation is calculated according to the concentration of the standard sample and the corresponding absorbance value, and then the corresponding sample concentration is calculated on the regression equation according to the absorbance value of the sample.
Determination of antioxidant activities in crap
Total antioxidant capacity (T-AOC) and GPx, catalase (CAT), and superoxide dismutase (SOD) activities were determined by colorimetry per the instructions of the kits. (Wuhan Meimian Bioengineering Institute).
Statistical analyses
Each sample was tested three times. The main statistical characteristics of the studied parameters were evaluated (mean ± standard deviation), and SPSS 17.0 statistical software was used for one-way ANOVA. The significance level of the difference was P < 0.05.