Feed pretreatment
G. lemaneiformis was collected from the same batch of artificial cultivation in Xiamen, Fujian Province, China. Nano-Se was purchased from Guangdong Jichuang Selenium Nano Research Institute Co.Ltd. G. lemaneiformis were cleaned with natural seawater to remove the epiphytes, and acclimated in the aquarium filled with natural seawater at 25℃ for three days. 2000 g G. lemaneiformis were randomly divided into two equal portions and then placed in two 100 L fish tanks at 25℃. Nano-Se was introduced into one tank at a final concentration ofof 1500 mg/L for Se enrichment, while the other tank without any supplementation was supplied as the control. After three days of culture, G. lemaneiformis were cleaned with natural seawater, and then subjected to drying, followed by grinding. The resultant G. lemaneiformis powder was stored at room temperature until diet preparation.
The diet was purchased from Guangdong Yuequn Marine Biological Research and Development Co. Ltd. 0.7mg Nano-Se/kg-diet (group N), 1% dried G. lemaneiformis powder (group G) and 1% Nano-Se-enriched dried G. lemaneiformis powder (group NG) were separately supplemented into the basal diet, and the group without any addition was supplied as the control (Table 1) [14, 17]. The prepared diet was stored at 4℃ until further applications.
Fish maintenance and treatment
Grouper were collected from Guangdong Marine Fishery Experimental Center and allowed to acclimate for one week at 25.2 ± 0.1 °C in natural seawater. Groupers were fed with basic feed twice daily under a photoperiod of 12 hours of light: 12 hours of dark. The seawater for farmed grouper was renewed every other day. After the preliminary acclimation, 240 fish (80.89 ± 0.23 g in average weight) were randomly picked and evenly transferred to 12 filter circulation aquariums (810×365×700 mm, 150 L, 20 fish per aquarium). Fish were fed one of the four diets (Table 1) in triplicate groups at 8:30 and 18:30 every day, with a daily feed equivalent to 3% of their body weight. The body weight of grouper was measured once a week, and then the daily feed amount was updated. The remains of bait and feces were separately collected 1-2 hours post feeding and weighed. Throughout the experimental period of 45 days, the natural seawater was maintained at 25.2±0.1°C, pH 7.1±0.05, with dissolved oxygen of 5.8±0.5mg/L. 12-hour light: 12-hour dark photoperiod was used for breeding. One half of the seawater in each aquarium was renewed every 3 days to ensure the water quality.
Samples collection
Five fish from each aquarium were sampled on the 0th, 15th, 30th and 45th day during fish rearing, and subjected to the analysis of weight gain rate (WG), specific growth rate (SGR), feed conversion ratio (FCR) and condition factor (CF). The tail vein blood of grouper was collected with a sterile syringe for later determination of serum antioxidant enzyme activities. The liver and total viscera of the groupers were separated with a sterile scalpel, and then weighed to calculate the hepatosomatic index (HSI) and viscerosomatic index (VSI). The gills, spleen, kidney and intestines of grouper were stripped and frozen immediately in liquid nitrogen for the analysis of immune-related genes. At the 45th day, the muscle and liver was stripped for analyzing Se concentration and nutrient composition.
Analytical methods
The Se level in the diets, muscle and liver was measured by inductively coupled plasma mass spectrometry (ICP-OES, thermo iCAP 7400 series, USA). The calculation formula of growth performance is as follows [9]:
WG (%) = [final body weight (g) − initial body weight (g)]/initial body weight (g) × 100;
SGR (% day-1) = [(ln final body weight (g) − ln initial body weight (g)]/ days × 100;
CF (%) = final body weight (g) / total length3 (cm) × 100;
FCR= [final body weight (g) − initial body weight (g)]/ dry feed consumed (g)
HSI (%) = liver weight (g)/whole body weight (g) ×100;
VSI (%) = visceral weight (g)/ whole body weight (g) ×100.
The superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were detected by Total SOD Assay Kit with NBT, CAT Assay Kit, Cellular GPx Assay Kit with NADPH, respectively, as described in previous studies [18]. All assay kits were purchased from Beyotime Biotechnology. Total RNA of gills, spleen, kidney and intestines were collected by Trizol reagent (Beyotime, China) according to the manufacturer's instructions. The mRNA levels of immunoglobulin M (IgM), tumour necrosis factor-α (TNF-α), interleukin-8 (IL-8) were analyzed using a BeyoRT™II First Strand cDNA Synthesis Kit and a Real Time PCR System. Primers specific for these genes were listed in Table 2. The moisture (GB 5009.3-2016, China), ash (GB 5009.4-2016, China), lipid (GB 5009.5-2016, China), protein (GB 5009.6-2016, China) and amino acid of muscle (GB 5009.9-2016, China) were determined by Guangzhou Kingmed Testing Science & Technology Co., Ltd, (Guangzhou, China).
Statistical analysis
All the data are displayed in the form of mean values ± SD. Excel 2010 and SPSS 16.0 were used to process the experimental data. One-way ANOVA and the least Significant Difference (LSD) multiple comparison tests were used to reveal the significance between different treatment groups at the same sampling time, and the significance of different sampling time in the same treatment group. p < 0.05 represents a significant difference.