2.1. Fish
All experiments were conducted in accordance with standard ethical guidelines and approved by the Animal Ethics Committee of Shahid Chamran University of Ahvaz (Approval number: EE/98.11.2.51020/scu.ac.ir). A total of 360 juvenile Siberian Sturgeon (mean weight: 34 ± 4.5 g) with no clinical signs of disease, were obtained from one of the Ahvaz fish farms and transferred to the aquatic animal health research laboratory of the faculty of veterinary medicine, Shahid Chamran University of Ahvaz. Fish were acclimatized to laboratory conditions (with a 12 h light/ 12 h dark photoperiod) for two weeks in 2000-L tanks and fed with a commercial food twice a day during the acclimation period. The formulation and components of a commercial extruded basal diet are shown in Table 1.
After the acclimation period, fish were randomly distributed in 12 tanks with similar conditions of water volume, quantitative, and qualitative factors in the form of 4 treatments (30 fish in each replication). During the experimental period, the physicochemical factors of water were as follows, temperature (°C): 22.5 ± 0.7, dissolved oxygen (mg/L): 7 ± 0.3, salinity (ppt): 1.2, pH: 7.8 ± 0.3, total hardness (ppm): 250 ± 27.
Table 1
Ingredient of the experimental diets (g kg− 1 dry diet).
Ingredientsa | G1 | G2 | G3 | Control |
---|
NaDFh | 0. 5 | 1 | 1.5 | 0 |
Crude protein | 42.3 | 42.3 | 42.3 | 42.3 |
Crude lipid | 14.2 | 14.2 | 14.2 | 14.2 |
Ash | 8.80 | 8.80 | 8.80 | 8.80 |
Fiber | 3 | 3 | 3 | 3 |
hFormi® NaDF, Addcon Nordic AS, Porsgrunn, Norway.
2.2. Diet preparation and rearing period
For preparing the experimental diets, the basal diet (FFS2 and GFS1, Alltech Coppens Co, Germany) were formulated and supplemented with the Sodium diformate (Addcon Nordic AS, Porsgrunn, Norway) according to the methods reported in previous studies (Hoseinifar et al., 2017; Ng and Koh, 2017). Briefly, acidifiers were weighed with the desired concentrations (0.05, 0.1 and 0.15 g/Kg) and then dissolved in physiological serum and added to food with gelatin. The resultant doughy mixture was pelleted using a meat grinder. Then the pellets were air dried at room temperature for 1 h, packaged and stored in a refrigerator at 4°C until used. During the study, fish were fed 3% of body weight day− 1, three times daily at 08:00, 14:00 and 20:00 for 60 days. Feed pH was measured according to the method proposed by Baruah et al. (2005). Briefly, five grams of feed was poured into a china plant and mixed in 50 ml of deionized water for 1 minute using a magnetic stirrer. After feed homogenization, the pH of the solution was measured. Experimental diets were include control (without supplementing NaDF), NaDF 0.05 (0. 5 g/kg sodium diformate), NaDF 0.1 (1 g/kg sodium diformate), NaDF 0.15 (1.5 g/kg sodium diformate).
2.3. Sampling
Three fish from each tank were randomly collected and anaesthetized (0.5 ml l− 1 of 2-phenoxyethanol) and taken for blood samples and digestive enzyme analyses. Blood samples (3 fish per replicate, a total of 15 fish per treatment) were withdrawn from the caudal vein by a 2.5 ml sterile syringe and used to obtain serum. Then, the blood samples were kept at ambient temperature to clot and afterward centrifuged (3000 g for 10 min at a temperature of 4°C) for serum separation (Molayemraftar et al., 2022). Afterward, all the samples were stored at -80°C until analysis.
2.4. Growth measurement
At the beginning (0 days), middle (30 days), and end of the trial (60 days), total fish biomass in every tank was anaesthetized (0.5 ml/L of 2-phenoxyethanol) and their weight (g) and length (cm) were measured individually. The data obtained from each group were used to calculate the feed utilization and growth parameters. Daily weight gain (DWG), weight gain (WG), specific growth ratio (SGR), condition factor (CF), feed conversion ratio (FCR), and protein efficiency ratio (PER) was calculated for each group as follow (Chelemal Dezfoulnejad and Molayemraftar, 2021; Mohammadian et al., 2021):
WG (%) = [(WF – WI) / WI] × 100
DWG = (WF – WI) / days
SGR (% body weight / days) = [(Ln WF − Ln WI) / t] × 100
CF = (FW × 100) / standard length3 (cm)
FCR = feed intake (g) / weight gain (g)
PER = protein intake (g)∕weight gain (g)
Where WI is initial body weights; WF is final body weights (g); and t is the trial duration in days.
2.5. Digestive enzyme activity
The activity of chymotrypsin, trypsin, α-amylase, lipase, protease, and ALP was assayed in triplicates for each tank (using pooled samples from each tank) after 60 days feeding with different levels of Sodium diformate. Intestinal samples were completely homogenized with PBS buffer (1:5 w/v) on ice water bath. Then, the mixture was centrifuged at 12000 rpm for 20 min at 4°C and the supernatant was separated and used for the analysis of digestive enzyme activity (Regoli et al. 2012). Bovine serum albumin was used as a standard for the Bradford method to measure the total proteins in the crude enzyme extracts (Bradford, 1976). The levels of chymotrypsin and trypsin activity were kinetically measured using N-Benzoyl-L-tyrosine ethyl ester (BTEE) and Nα-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as substrates, respectively (Hummel, 1959; Bergmeyer, 1974). The level of alpha-amylase activity was measured using soluble starch as the substrate hydrolyzable to maltose when it was reacted with 3,5-Dinitrosalicylic acid solution (Bernfeld, 1951). The activity of lipase enzyme in crude enzyme extract was determined by hydrolysis of p-nitrophenolemyristate using spectrophotometer. The release of fatty acids as a result of enzymatic hydrolysis of triglycerides to glycerol in the stabilized emulsion of olive oil, Fluka TM (Borlongan, 1990), was used to assess lipase activity. Protease enzyme activity was measured using 2% azocasein substrate solution in 50 mM Tris / Hcl buffer at pH = 7.5 and using a spectrophotometer (Garcia-carreno et al., 1993). The activity of intestinal ALP was quantified using p-nitrophenyl phosphate (PNPP) as substrate by a commercially colorimetric kit (Pars Azmoon Co., Tehran, Iran).
2.6. Serum Immunity parameters
Total protein, albumin and globulin levels as well as lysozyme activity and alternative complement (ACH50) and bactericidal activities were detected in the sera samples.
Micrococcus lysodeikticus was used to determine serum lysozyme activity according to the turbidometric assay (Sharifuzzaman & Austin 2009). Briefly, sodium phosphate buffer (0.02 M, pH = 5.8, Sigma–Aldrich) was used. The phosphate buffer-free serum sample was applied as a negative control. The absorbance was recorded at 450 nm and expressed in the unit of lysozyme per ml serum when causing a reduction of 0.001 per min at 22°C.
Total protein (TP) content was determined according to Biuret method. The basis of this method is a formation of a Cu2+-protein complex in alkaline reagent and then measuring optical density at 540 nm by a spectrophotometer. Serum albumin (Alb) was also measured at 540 nm using bromcresol green complex (Pars Azmun, Iran). Finally, total globulin was calculated by subtracting of Alb from TP.
Agarose plates containing rabbit red blood cells were applied for detecting the activity of ACH50 (give refenece here). Several holes (diameter = 3 mm) were punched on a plate and then filled with 15 µl of serum. After 24 h of incubation at room temperature, the zone of lysis was measured and expressed as an arbitrary unit per ml of serum (Barta 1993; mohammadian et al 2021).
Serum bactericidal activity was determined by incubating (90 min at 25°c) the mixture of the diluted sera and L. garvieae as previously described by Gisbert et al. (2015). The bactericidal activity of serum was expressed as a percentage of the ratio of CFU in the experimental group to those in the control group.
2.7. Intestinal histomorphology
Intestinal histomorphologyAt the days 30 and 60 from the start of the experiment, the intestine of fish (n = 3) were dissected immediately out following euthanizing. The samples were then divided into three different sections, including proximal, middle and posterior parts and separately fixed in 10% neutral phosphate buffered formalin (pH = 7.2) and processed using the standard protocol for histopathological examination. After embedding the sample with paraffin wax, three separate cross sections with the thickness of ~ 5 µm were prepared using a microtome (Microtec CUT4050) and then have been stained with hematoxylin and eosin (H&E) for further histopathological investigations. The villi height, villi width and the thickness of epithelium, lamina propria and muscularis layers were determined under Nikon light microscope (Eclipse E600) by using of AxioVision 8.4 microscope software from Carl Zeiss (Oberkochen, Germany).
2.9. Statistical analysis
The statistical analysis of this study was conducted using SPSS-20 software (SPSS Inc, USA). The normality and variance homogeneity of the data was checked using the Kolmogorov–Smirnov and Levine test. The data were analyzed using the one-way analysis of variance (ANOVA), followed by Tukey’s test. The significance level was accepted at P < 0.05. All data are expressed as mean ± standard error (SE) for each experimental group.