Introduction
Fish is among the best sources of animal protein but aquaculture industry have faced to many challenges. Global aquaculture productions are vulnerable, and the increasing prevalence of diseases has caused a partial and overall reduction in aquaculture production (Bondad-Reantaso et al., 2005). Factors such as overcrowding in ponds, periodic movement and manipulation, sudden temperature changes, water quality deterioration, and poor nutritional conditions, along with physiological changes in fish such as stress, have heightened susceptibility to infections (Quesada-García et al., 2013). In aquaculture, the application of natural immuno-stimulants to enhance fish health and boost their resistance to pathogens has shown encouraging results (Habibnia et al., 2024).
Methodology
This study examined the impact of incorporating probiotic Lactobacillus acidophilus (LA) and polyphenol ferulic acid (FA) into the diet of goldfish (Carassius auratus) on growth performance, as well as factors related to immunity and antioxidant activity. A total of 240 fish, initially weighing 3.34 ± 0.35 g, were divided into four experimental groups: a control treatment with no food supplement (T0), fed with 6 x 10⁸ CFU/g LA probiotic (T1), fed with 100 mg/kg FA (T2), and fed with a combination of LA and FA (T3) for eight weeks. At the end of feeding trial, the fish were fasted for 24 hours and all the fish in each tank were sampled and then anesthetized with benzocaine at a concentration of 120 mg per liter and weighed individually. Growth performance was then assessed. Thereafter, nine fish (per replication) were randomly selected. The selected fish were then subjected to anesthesia with Benzocaine at a concentration of 120 mg per liter to minimize handling stress. The fish underwent dissection, with the intestine and liver tissues being meticulously separated. Subsequently, these tissues were promptly subjected to freezing using liquid nitrogen and were subsequently preserved at a temperature of -80 °C. To measure lysozyme activity, the sample was added to a suspension of Micrococcus lysodeikticus prepared in 0.1 molar citrate phosphate buffer at a pH of 5.8. Optical density was read at a wavelength of 410 nanometers for 5 minutes, with readings taken every 30 seconds. Total immunoglobulin (Ig) levels were determined based on the method by Siwicki et al. (1994). In brief, total protein from the homogenate sample was measured using the microprotein method, followed by precipitation of immunoglobulin molecules with a 12% polyethylene glycol solution. The protein level was then re-measured, and the difference in protein content was considered as the Ig content. The levels of lipid peroxidation products in the fish homogenate were determined based on a previous study (Kei, 1978). Briefly, trichloroacetic acid (20%) (1.25 mL) was mixed with the fish homogenate (0.25 mL) and centrifuged at 2000 g for 10 minutes. Then, 1.25 mL of sulfuric acid (0.05 molar) and 1 mL of thiobarbituric acid (0.2%) were added to the collected precipitate and boiled for 30 minutes. After adding 2 mL of n-butanol, centrifugation (2000 g) was performed for another 10 minutes. The final absorbance was recorded at a wavelength of 532 nanometers. Catalase activity in the fish homogenate was assessed based on a previous study (Goth, 1991). The reaction buffer (1 mL) consisted of hydrogen peroxide (65 mM) and sodium-potassium phosphate buffer (60 mM) mixed with the fish homogenate (0.5 mL) for 1 minute at 37°C. The enzymatic reaction was terminated by adding 1 mL of ammonium molybdate (32.4 mM), and absorbance was recorded at 405 nanometers. Superoxide dismutase activity in the fish homogenate was determined based on a previous study (Nishiimi et al., 1997). The reaction buffer included 2.6 mL of phosphate buffer (0.017 mM), 0.1 mL of phenazine methosulfate, and 0.1 mL of nitro blue tetrazolium mixed with the fish homogenate (0.5 mL). After adding 0.1 mL of NADH (2.34 mM), absorbance was recorded at 560 nanometers for 3.5 minutes. Glutathione peroxidase activity in the fish homogenate was assessed according to a previous study (Pagalia & Valentine, 1967). The reaction buffer (0.88 mL) included GSH (1 mM), NADPH (150 mM), and sodium azide (100 mM) mixed with the fish sample (20 µL). Absorbance was recorded at a wavelength of 340 nanometers for 1 minute. The normality of the data and the homogeneity of variances were analyzed using the Kolmogorov-Smirnov test and Levene's test, respectively. A one-way analysis of variance (ANOVA) was conducted to assess significant differences among treatments. Tukey's post-hoc tests were employed to evaluate differences between treatments. These analyses were performed using SPSS 22 software (SPSS, Richmond, VA, USA) with a 95% confidence level.
Results
The results showed that the final weight, weight gain and specific growth rate were significantly influenced by LA and FA, with the T3 treatment resulting in the highest growth observed. Furthermore, levels of total immunoglobulin, IgM, and lysozyme increased in fish fed diets containing FA or LA. Additionally, malondialdehyde (MDA) activity was reduced by the experimental diets, with its lowest value significantly observed in the T3 treatment compared to the control group. Fish fed diets containing LA and/or FA also displayed significant increases in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activity compared to the control group. The findings suggest that these nutritional supplements at optimal doses have potential to enhance growth as well as innate immunity and antioxidant factors in goldfish. Furthermore, both supplements exhibited synergistic effects; therefore, it is recommended to use these compounds simultaneously for sustainable aquaculture practices.
Discussion and conclusion
Our results showed that supplementing with FA and/or LA can significantly activate antioxidant activity, effectively scavenging excess free radicals and regulating ROS balance in the body, thereby enhancing antioxidant potential. In this study, the beneficial effects of the probiotic Lactobacillus acidophilus and the polyphenol ferulic acid were examined in carp fish. These additives in the diet had potential effects on growth, immune factors, and antioxidant capacity in carp, and simultaneous use of these compounds is recommended for sustainable aquaculture.
Conflict of Interest
Authors have no conflict of interest.