Aquaculture produces high-quality animal protein, improves nutrition and generates money and employment worldwide. Concerned scientists and policymakers are both optimistic and disturbed by the tremendous increase in the production over the previous two decades. Aquaculture intensification, like any other sector, has had a severe influence on the environment, leading to the development of infectious illnesses and other environmental concerns. Economic losses are incurred as a result of disease outbreaks and environmental damage. The use of probiotics, may thus provide plenty of opportunity for fish farming enterprises to expand. Probiotics are currently used in both water and feed in the aquaculture. Several incidences of Aeromonas hydrophila infections have been reported in India’s Indian major carps in recent years (Shome et al., 2005). Increased uses of antimicrobial drugs, pesticides and disinfectants have all been used to combat this, leading in the rise of resistant bacterium strains. As a result, probiotics are becoming increasingly important in the aquaculture business, as the need for environmentally benign, long-term aquaculture grows. The term “probiotics” is derived from the Greek word meaning “life.” The term “probiotic” was given by Parker (1974). According to the World Health Organization and the Food and Agriculture Organization, probiotics are the live microorganisms that provide a health benefit to the host when provided in appropriate amounts. Probiotics (live bacteria) that colonise the gut and/or animal-derived microbial supplements, have recently been acknowledged to be advantageous to fish health (Dimitroglou et al., 2011). Due to rising demand for environment friendly aquaculture, application of probiotics in aquaculture is increasingly acknowledged. Lactic acid bacteria (LAB) has been widely employed as a food supplement to protect fish from numerous infectious illnesses in recent years (Geng et al., 2012). Lactobacillus has demonstrated to be effective in fish culture (Barbosa et al., 2011). Because it is resistant to environmental conditions and has a long life cycle, Lactobacillus is the most often utilised probiotic in aquaculture and the beneficial activities of these bacterial species in the aquaculture are well known (Wang et al., 2008; Kumar et al., 2019: Amit et al., 2021; Amit et al., 2022; Totewad and Gyananath, 2021; Gao et al., 2016; Sinha and Pandey, 2013).However, there is a clear need to increase our understanding of gut microbiology, as well as the proper dose and safety parameters. Due to its flavour and nutritional quality, Cirrhinus mrigala is one of the most significant candidate fish species in India, with the highest market demand and acceptability as food by people. Therefore, the goal of this study was to see how probiotic bacteria (Lactobacillus plantarum) affected the survival, growth and body composition of mrigal, Cirrhinus mrigala.

Experimental design
 
The experiment was conducted at the Fish Farm of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, in outdoor FRP ponds (1.5 x 1.0 x 0.75 m). The trial included five treatments (Table 1), each with triplicates. Rice bran, mustard meal, vitamin and mineral mixture and salt were used to make the basic diet (49, 49, 1.5 and 0.5 per cent respectively). At varied levels (@ 0 (D0), 106 CFU/g (D1), 107 CFU/g (D2), 108 CFU/g (D3) and 109 CFU/g (D4), probiotic bacterial strain L. plantarum FLB1 was added into basal meals (D0). Fish were stocked @ 15 fingerlings each FRP pool and supplementary diets were supplied ad libitum daily for 120 days (May to August).


 
Experimental diets
 
The basal diet consisted of de-oiled rice bran (49 per cent), mustard meal (49 per cent), vitamin-mineral mixture (1.5 per cent) and salt (0.5 per cent). With the use of an electric lab pelletizer, the basal diet and experimental designed diets for the experiment were made into sinking pellets. L. plantarum FLB1 was isolated from the gut of rohu using de Man Rogosa and Sharpe (MRS) agar for the manufacture of experimental diets and the amplified 16S rRNA gene fragment was subjected to Sanger sequencing for genus confirmation in addition to biochemical testing. To determine probiotic qualities, the isolate was examined in vitro for hemolytic activity, acid and bile tolerance for growth kinetics, auto-aggregation, cell-surface hydrophobicity, phenol tolerance, cell adhesion and safety parameters (Zhang et al., 2014). L. plantarum FLB1 was isolated and the aliquots of L. plantarum FLB1 were stored at -80^oC in 30% glycerol stock (Kumar et al., 2019; Zhang et al., 2014 at the College of Fisheries, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana. For formulations of probiotic diet in the present study, an aliquot of L. plantarum FLB1 was removed from -80^oC storage and streaked on MRS agar plate. After incubation at 37^oC for 48 h, individual colonies were picked up for subculture in MRS broth at 37^oC. The cells were extracted by centrifugation  (2000 g) after incubation to obtain a microbial pellet. The pellet was washed three times in phosphate-buffered saline (pH 7.2), counted and mixed with control diet @106(D1), 107(D2), 108(D3) and 109(D4) CFU/g, respectively. The bacterial solution was gently added to feed pellets with a hand sprayer for uniform dispersion in the laminar airflow chamber under sterilized conditions to get correct final concentrations in the diet. The resulting diet was kept in sealed plastic zip lock bags at 4^oC until it was utilized. Diets were created fresh once a week to provide a high probiotic level in the supplemented diet. The proximate compositions of the test diets (Table 2), as well as nutritional composition of fish flesh were measured using the Association of Official Analytical Chemists’ standard techniques (AOAC, 2012).


 
Water parameters
 
Temperature, dissolved oxygen, pH, total alkalinity, nitrate, nitrite and ammonia were all measured every two weeks using APHA’s standard techniques (APHA, 2012).
 
Growth parameters
 
The following formulae (Halver, 1957) were used to compute total length gain (TLG), net weight gain (NWG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER) and condition factor (K) of fish for each treatment.
 

 TLG= Final total length (TL) (cm) - Initial total length (TL) (cm)

NWG = Final body weight (g) - Initial body weight (g)

 

 

 
 
Statistical analysis
 
The data was statistically analyzed using statistical software (SPSS 20.0, SPSS Inc. and Richmond, CA, USA). Means and standard errors of the mean were used to present the data. The effect of nutritional supplementation with probiotic bacteria (Lactobacillus plantarum) on survival, growth and body composition of fish (P<0.5) was determined using one-way ANOVA, followed by Duncan’s multiple comparison to determine significant differences among the treatments.

Water quality
 
Mean water temperature, pH, dissolved oxygen, total alkalinity, ammonia, nitrite and nitrate in different treatments ranged from 30.82 to 30.87^oC, 7.92 to 7.99, 7.79 to 7.91 mg/l, 219.96 to 228.81 mg/l, 0.054 to 0.061 mg/l, 0.048 to 0.053 mg/l and 0.25 to 0.31 mg/l respectively. The water quality parameters showed no significant differences (P<0.05) and remained well within the optimum rage for carps (Table 3).


 
Effect of diet supplementation with L. plantarum on growth and survival
 
D4 had the greatest values (29.00, 214.37, 0.95 and 1.12) of NWG (g), per cent NWG, SGR and condition factor (K) respectively (Table 4) while D0 had the lowest values (17.50, 126.94, 0.68 and 1.07) respectively at the end of the study period (120 days). At the end of the trial, the survival rate (per centage) in all probiotic fed treatments ranged from 91.11% to 97.78%, indicating that probiotic supplemented diet had no negative effects. Survival (per cent) improved in probiotic-incorporated diets (D1-D4), with a significant (P<0.05) improvement in D4, when compared to control (D0), PER and FCR also improved considerably (1.96 and 1.84, respectively) in D4 (Table 5).

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Effect of diet supplementation with L. plantarum on nutritional composition of fish
 
Fish flesh quality (wet weight basis) in terms of crude proteins, lipid, ash, total carbohydrate and moisture content was estimated at the end of the experiment and the results are provided in Table 6. In all treatments (D0 to D4), the crude protein content in the fish meat ranged from 13.90 to 14.63%. The crude protein content of treatments D0, D1, D2, D3 and D4 was 13.90, 13.97, 14.11, 14.35 and 14.63 per cent, respectively. The fish had a crude protein content of 13.66 per cent at the time of stocking. The variations in protein content between the treatments were significant (P<0.05) (D4>D3>D2>D1=D0>Initial). Probiotic in the diet significantly (P<0.05) improved meat quality in terms of crude protein content when compared to the control (D0) and time of stocking. In all treatments (D0 to D4), the total lipid content in the fish flesh varied from 2.22 to 3.10 g 100/g. Treatments D0, D1, D2, D3 and D4 had lipid content of 2.22, 2.51, 2.74, 2.91 and 3.10% respectively. Fish had a lipid content of 1.92 when they were stocked. There were considerable differences in lipid content amongst the treatments (D4>D3>D2>D1>D0>Initial). As a result, probiotic improved nutritional quality in terms of lipid content considerably (P<0.05). When compared to the control group, all probiotic fed groups demonstrated a substantial (P<0.05) increase in C. mrigala meat crude protein and lipid content as well as a significant (P<0.05) decrease in total ash, moisture and total carbohydrate content compared to the control (D0) in the present study. Overall, inclusion of probiotics improved nutritional quality of the fish.
       
However, based on growth and immunological parameters, there is limited information on the integration of probiotics into carp cultures (Chi et al., 2014; Gupta et al., 2014; Kumar et al., 2019: Amit et al., 2021; Amit et al., 2022; Totewad and Gyananath, 2021; Gao et al., 2016; Sinha and Pandey, 2013), particularly Cirrhinus mrigala. The majority of study in this field, namely probiotic supplementation in fish, has been focused on species such as trout, tilapia and catfish, as well as shellfish (shrimp, prawns and oyster). Probiotics are typically described as multifunctional and can be used on a variety of species under a variety of growth conditions. Apart from these advantages, probiotics are more effective at converting organic matter to CO₂, hence high doses of probiotics in production ponds are advised (Wang and Wang, 2008). When fingerlings of Cyprinus carpio were fed L. plantarum @ 4.5 x 10⁶ CFU/mg, Valiallahi et al., (2018) observed an enhanced survival rate, specific growth rate and reduced FCR. Gupta et al., (2014) observed that feeding Bacillus coagulans, B. licheniformis and P. polymyxa 109/g incorporated food to Cyprinus carpio resulted in improved growth, a minimised feed conversion ratio and an enhanced protein efficiency ratio. Abumourad et al., (2013) found similar results in O. niloticus and Enferadi et al., (2018) found similar outcomes in O. mykiss when fed L. plantarum.
       
Furthermore, differences in the condition factor (K) of fish between treatments were significant when compared to the control, suggesting that probiotic incorporation had no negative impact on the condition of fish, which is used to measure the health of fish and indicates proportional weight gain with length gain (Nash et al., 2006).
       
When compared to control, all probiotic fed groups showed better protein and lipid in Cirrhinus mrigala meat, whereas D4 had lower ash, moisture and carbohydrate values in this study. Overall, the results demonstrated that supplementing the diet with probiotics improved the nutritional quality of the mrigal flesh with probiotic treatment could be related to increased fish growth, as seen in this study and in a previous investigation (Amit et al., 2021; Ghosh et al., 2003; Giri et al., 2013). Improved protein efficiency ratio may have also aided in the improvement of fish flesh quality (improved PER in D4). Probiotics also increased feed efficiency, growth and improved health parameters in fish, according to Valiallahi et al., (2018). Adding probiotics to fish’s diets improved the efficiency of converting feed protein to flesh Gatesoupe, (1999). Probiotics have been found to eliminate anti-nutritional components in feeds, resulting in improved nutritional quality and bioavailability as seen by higher growth and nutritional composition.

It can be concluded that L. plantarum boosted feed efficacy and protein deposition in fish flesh as well. Overall, adding probiotics to the diet improved water quality, fish’s growth and nutritional quality in a synergistic way. As a result, incorporating L. plantarum, in fish feed improved survival, growth, feed efficacy, K-value and nutritional quality of mrigal fingerlings significantly (P<0.05), with the best results in D4 (@109 CFU/g feed). Hence, it can be added to the mrigal’s diet during the growth period of the fish in a semi-intensive culture system. With the drive for environment friendly sustainable aquaculture, probiotics have become an integral element of the process. The information gathered in this study will aid in boosting fish production and productivity in a sustainable manner, as well as raising fish farmers’ revenue and contributing to the nation’s nutritional security.

The Dean, College of Fisheries and Dr. Vaneet Inder Kaur, Principal Scientist, Department of Aquaculture, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, are to be acknowledged for their assistance in performing this study.
 

All authors declared that there is no conflict of interest.