Effects of fructooligosaccharide on growth performance, survival rate, feed utilization and disease resistance against Aeromonas hydrophila in Labeo rohita

including the development of antibiotic-resistant bacteria, immune suppression and environmental contamination (Cabello, 2006; Reverter et al., 2014) [5,23] . Consequently, there is growing interest in eco-friendly alternatives such as functional feed additives, including prebiotics, to promote health and disease resistance in cultured species (Hoseinifar et al., 2015) [13] . Prebiotics are nondigestible dietary ingredients that beneficially affect the host by selectively stimulating the growth or activity of beneficial gut microorganisms (Ringø et al., 2010) [24] . Their inclusion in aquafeeds has shown positive effects on growth performance, nutrient utilization, immune response, gut microbiota composition and disease resistance (Guerreiro et al., 2018; Rohani et al., 2021) [12,16] . Common prebiotics used in aquaculture include β-glucans, inulin, mannanoligosaccharides (MOS), galactooligosaccharides (GOS) and fructooligosaccharides (FOS) (Wee et al., 2024). Among these, FOS naturally found in plants such as onions, garlic, asparagus and bananas-has emerged as a promising prebiotic that enhances fish growth, immunity and resistance to pathogens while reducing reliance on antibiotics (Fuller & Gibson, 1998; Sridevi et al., 2014) [10,29] . FOS are shortchain β-D-fructans synthesized enzymatically via transfructosylation of sucrose using β-fructofuranosidase from Aspergillus niger (Roberfroid, 2007) [25] . Given its potential benefits, the present study was conducted to evaluate the effects of fructooligosaccharide supplementation on the growth performance, feed utilization and disease resistance against Aeromonas hydrophila in Labeo rohita.

Materials and Methods

Experimental Setup

The experiment entitled "Effects of Fructooligosaccharide on Growth Performance, Survival Rate, Feed Utilization and Disease Resistance against Aeromonas hydrophila in Labeo rohita" was conducted at the Department of Aquaculture, College of Fisheries Science, Kamdhenu University, Veraval. The study was carried out for 60 days, from 19 September 2024 to 17 November 2024, followed by a 7-day disease challenge test against Aeromonas hydrophila. The experiment was designed under a Completely Randomized Design (CRD) with five treatment groups, each replicated four times.

Experimental Tanks

A total of 20 rectangular plastic tanks (2 × 1 × 1 ft, capacity 55 L) were used for the study. All tanks were thoroughly cleaned and disinfected with 4 ppm potassium permanganate (KMnO₄) solution before use and then filled with 35 L of filtered, disinfected freshwater. Continuous aeration was provided throughout the experimental period.

Experimental Fish

Healthy Labeo rohita fry were procured from a commercial Indian Major Carp hatchery and transported to the experimental facility in oxygenated polythene bags. Fish were acclimated for six days in a 500 L tank under continuous aeration and regular feeding. After acclimation, fish with an average initial body weight of 0.50±0.023 g were randomly distributed among 20 tanks, with 20 fish per tank (total 400 fish). Tapioca flour was finely ground using a grinder, while highquality fish meal and fish oil were obtained from a commercial fish processing unit in Veraval.

Treatment Details

Proximate Analysis of Ingredients

The proximate composition of the feed ingredients was analyzed following AOAC, 2019 procedures. Crude protein was estimated by the micro-Kjeldahl method, moisture content by oven drying at 100 °C and ash content using a muffle furnace at 550 °C until white ash was obtained.

Feed Formulation

Experimental feeds were formulated to contain 30% crude protein using Pearson's square method. Five diets were prepared as per treatment details (Table 3.3).

Challenge Test

After the feeding trial, ten fish from each treatment were injected intramuscularly with 0.1 ml of A. hydrophila suspension (1 × 10⁶ CFU/ml) using a 26-gauge needle. The fish were observed for 7 days for mortality and clinical symptoms. A positive control group received the same bacterial suspension, while the negative control group was injected with 0.1 ml of sterile saline solution.

Water Quality Management

Water quality parameters such as temperature, pH, dissolved oxygen (DO) and alkalinity were monitored weekly. Temperature was measured using a thermometer, pH with a digital pH meter and DO by Winkler's method. Alkalinity was determined by the titration method and expressed in mg/L. Continuous aeration was maintained, and tanks were siphoned daily to remove waste and replenish with fresh filtered water.

Statistical Analysis

All data were analyzed statistically using one-way ANOVA under a Completely Randomized Design (CRD) with SPSS software. Differences among means were compared using Duncan's Multiple Range Test (DMRT) at a 5% significance level (p<0.05).

Results and Discussion

Effects of Dietary Fructooligosaccharide Supplementation on the growth of Labeo rohita

The growth performance of Labeo rohita was evaluated by measuring mean weight, weight gain, percentage weight gain, specific growth rate (SGR) and total length across different dietary treatments of fructooligosaccharide (FOS) supplementation. The experiment was conducted for 60 days, during which samples were collected every 15 days to assess growth performance.

The initial average weight of L. rohita was 0.50±0.005 g across all treatment groups. After 60 days of feeding, the final mean weights were recorded as 2.66±0.046 g, 2.86±0.043 g, 2.88±0.049 g, 3.06±0.048 g and 3.14±0.048 g in the control group (T0), T1, T2, T3 and T4, respectively. The results revealed a significant difference (p<0.05) among the treatments, with T4 showing the highest final mean weight. Although T4 recorded a higher weight than T3, the difference was not statistically significant. In contrast, the control group exhibited the lowest mean weight. The consistent increase in mean weight across treatments indicates that dietary supplementation of FOS positively influenced growth in L. rohita. These findings align with Poolsawat et al. (2020) [22] , who reported a significant increase in final body weight of Oreochromis niloticus when fed a 2% FOS-supplemented diet. Similarly, El Latif et al. (2015) [9] observed improved growth performance in tilapia fed diets containing 2% FOS compared to the control. However, Chitmanat et al. (2017) [6] found no significant difference in Anabas testudineus, suggesting that variations in species, fish size and culture conditions can influence the growth response to prebiotic supplementation.

The percentage weight gain of L. rohita fry was calculated based on the recorded mean weights. The mean percentage weight gains for treatments T0, T1, T2, T3 and T4 were 423.03±9.84, 462.60±9.01, 489.77±2.44, 512.50±1.89 and 528.11±2.46, respectively. The results revealed that all FOS-supplemented diets significantly improved weight gain compared to the control (p<0.05), with T4 showing the highest percentage gain. Although T4 recorded higher values than T3, the difference was not statistically significant. These findings corroborate the results of Akrami et al. (2013) [3] , who reported higher weight gain percentages in juvenile stellate sturgeon (Acipenser stellatus) fed FOSsupplemented diets compared to controls. Likewise, Hoseinifar et al. (2014) [14] and Soleimani et al. (2012) [28] observed improved growth in common carp (Cyprinus carpio) and Rutilus rutilus, respectively, with 2% FOS inclusion. The present results therefore confirm that moderate FOS supplementation effectively enhances growth in L. rohita.

Throughout the experimental period, L. rohita exhibited consistent improvement in mean weight gain across all FOS-supplemented treatments compared to the control. At the end of 60 days, mean weight gains were recorded as 2.15±0.031 g (T0), 2.35±0.027 g (T1), 2.39±0.040 g (T2), 2.56±0.022 g (T3) and 2.64±0.039 g (T4). Among all treatments, T4 showed the highest mean weight gain, followed closely by T3, while the control group showed the lowest. Statistical analysis indicated that all treatments differed significantly from the control (p<0.05). The trend clearly demonstrates that increasing FOS inclusion up to 2% improved the growth performance of L. rohita. Similar growth-promoting effects of FOS have been reported by Poolsawat et al. (2020) [22] in tilapia and Soleimani et al. (2012) [28] in Caspian roach, where diets supplemented with 2% FOS yielded the highest growth rate. Conversely, Grisdale Helland et al. (2008) [11] found no significant effect of FOS supplementation on Salmo salar, possibly due to differences in species and gut microbiota composition.

The initial average length of L. rohita was 3.58±0.006 cm across all treatments. After 60 days, the mean lengths recorded were 5.80±0.022 cm (T0), 6.27±0.030 cm (T1), 6.33±0.026 cm (T2), 6.50±0.046 cm (T3) and 6.56±0.039 cm (T4). Treatment T4 showed a significantly higher length compared to the control group (p<0.05). These findings are consistent with Sepulveda Quiroz et al. (2021), who observed increased total length in Atractosteus tropicus fed diets containing 5-10 g/kg FOS and Lima Paz et al. (2019) [8] , who reported similar results in Colossoma macropomum. The improved length in FOS-supplemented groups suggests that prebiotics enhance nutrient absorption and feed utilization efficiency.

The specific growth rate (SGR) was significantly influenced by dietary FOS supplementation. The mean SGR values for treatments T0, T1, T2, T3 and T4 were 2.76±0.032, 2.87±0.027, 2.95±0.007, 3.02±0.005 and 3.06±0.006 %, respectively. Treatment T4 recorded the highest SGR, followed by T3, while the control group showed the lowest. Statistical analysis revealed significant differences (p<0.05) between all treatments and the control. The enhanced SGR in the FOS-supplemented groups indicates better growth efficiency and metabolic performance. These observations agree with Ahmed et al. (2019) [1] , who found the best SGR in Cyprinus carpio fed 10 g/kg FOS and Akrami et al. (2013) [3] , who noted higher SGR at 1% inclusion in A. stellatus. Such variations in optimal FOS levels among species may be due to differences in digestive physiology and gut microbial communities. [9] also reported that Nile tilapia fed with 2% FOS exhibited a significantly better FCR and FCE than the control group, supporting the present findings. Similarly, Ahmed et al. (2019) [1] found that common carp fed 1% FOS had improved feed efficiency and growth performance. The observed enhancement in protein efficiency may be due to improved intestinal morphology and increased nutrient digestibility caused by the fermentation of FOS in the gut, which produces short-chain fatty acids beneficial for metabolism. Zhou et al. (2010) [34] reported that Red drum fish fed diets with FOS exhibited a higher PER than those fed other prebiotics. However, other studies such as Ai et al.

(2011) [2] on Larimichthys crocea and Grisdale Helland et al.

(2008) [11] on Salmo salar found no significant improvements in feed utilization at high inclusion levels, suggesting that the effects of FOS can vary with species, inclusion levels and culture conditions. Overall, the present study concludes that dietary inclusion of 2% fructooligosaccharide optimizes feed utilization efficiency in Labeo rohita. The improved FCR, FCE and PER values in FOS-supplemented groups demonstrate that FOS serves as an effective prebiotic feed additive, enhancing nutrient absorption, digestion and protein utilization, which ultimately promotes better growth and performance in Labeo rohita.

Influence of Fructooligosaccharide-Incorporated Diet on Disease Resistance in Labeo Rohita Against

Aeromonas hydrophila

At the end of the 60-day experimental period, a disease challenge test was conducted to evaluate the resistance of Labeo catla against Aeromonas hydrophila. Ten fish from each treatment group were randomly selected and injected intramuscularly with A. hydrophila at a concentration of 1 × 10⁶ cells/ml, ensuring minimal stress during handling.

Mortality was observed for seven days post-challenge, and survival percentages were recorded.

Dietary supplementation with fructooligosaccharide (FOS) significantly improved the survival rate of L. catla compared to the control group. After 7 days, the control group (T0) exhibited a survival rate of 50%, whereas fish fed with FOS-supplemented diets showed higher survival rates. Treatment T4 (2% FOS) achieved the highest survival rate of 80%, followed by T2 and T3 (both 70%), and T1 (60%). This suggests that incorporating 2% FOS into the diet enhances disease resistance in L. catla against A. hydrophila, demonstrating better protection compared to the control. These findings align with the results of Poolsawat et al.

(2020) [22] , who observed that supplementation with FOS improved immune responses and disease resistance in tilapia. Similarly, Zhang et al. (2014) [33] , El Latif et al.

(2015) [9] and Lima paz et al. (2019) [8] reported that FOS supplementation enhanced the survival of blunt snout bream, Nile tilapia and tambaqui, respectively, following exposure to A. hydrophila. Moreover, Soleimani et al.

(2011) [28] demonstrated that 2% FOS supplementation yielded better survival and growth in Caspian roach than other tested levels. These studies collectively support the role of FOS as a beneficial prebiotic additive for improving disease resistance and overall health in fish.

Physico-Chemical Parameters

Throughout the 60-day experimental period, the water quality parameters of all experimental tanks were carefully monitored on a weekly basis to ensure an optimal environment for the growth and survival of Labeo rohita. The parameters measured included water temperature, dissolved oxygen (DO), pH and alkalinity.

Conclusion

The results of the experiment revealed that incorporating fructooligosaccharide (FOS) into the diet of Rohu (Labeo rohita) enhanced growth performance, survival rate and resistance to Aeromonas hydrophila infection compared to the control group. Among all treatments, the diet containing 2% FOS showed the best outcomes, with the highest weight gain, specific growth rate (SGR), feed conversion ratio (FCR), feed efficiency ratio, protein efficiency ratio and disease resistance, indicating its superior potential to support fish health and development.

In summary, this study confirms that dietary supplementation of Labeo rohita with fructooligosaccharide has a beneficial effect on overall performance. Fish fed with 2% FOS demonstrated significant improvements in growth parameters and survival. Additionally, FOS inclusion enhanced immune response, resulting in increased resistance to disease challenges. Therefore, the use of 2% FOS in Rohu diets can be considered an effective and sustainable approach to promote better growth, survival and health in aquaculture systems while reducing dependence on antibiotics and chemical additives.

References

1. Ahmed VM, Abdulrahman NM, Amin SAH, Hassan BR, Mohammed SJ. Effect different level of fructooligosaccharide (fos) on growth parameters and feed utilization in Common carp (Cyprinus carpio L.). Journal of Animal and Poultry Production. 2019;10(9):285-287.
2. Ai Q, Xu H, Mai K, Xu W, Wang J, Zhang W. Effects of dietary supplementation of Bacillus subtilis and International Journal of Advanced Biochemistry Research https://www.biochemjournal.com fructooligosaccharide on growth performance, survival, non-specific immune response and disease resistance of juvenile large yellow croaker, Larimichthys crocea. Aquaculture. 2011;317(1-4):155-161.
3. Akrami R, Iri Y, Rostami HK, Mansour MR. Effect of dietary supplementation of fructooligosaccharide (FOS) on growth performance, survival, lactobacillus bacterial population and hemato-immunological parameters of Stellate sturgeon (Acipenser stellatus) juvenile. Fish & Shellfish Immunology. 2013;35(4):1235-1239.
4. AOAC. Association of Official Analytical Chemist. Washington, DC, USA: AOAC; 2019.
5. Cabello FC. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environmental Microbiology. 2006;8(7):1137-1144.
6. Chitmanat C, Thongsri S, Phimpimon T. The influences of dietary supplementation with fructooligosaccharide on growth and immune responses of climbing perch (Anabas testudineus). International Journal of Agriculture & Biology. 2017;19(4):787-791.
7. Chondar SL. Biology of Finfish and Shell fish. West Bengal: SCSC Publishers (India); 1999. p. 199-211.
8. De Lima Paz A, da Silva JM, da Silva KMM, Val AL. Protective effects of the fructooligosaccharide on the growth performance, hematology, immunology indicators and survival of Tambaqui (Colossoma macropomum, Characiformes: Serrasalmidae) infected by Aeromonas hydrophila. Aquaculture Reports. 2019;15:100222.
9. El-latif A, Ashraf M, El-Gawad A, Emam M. Effect of dietary fructooligosaccharide supplementation on feed utilization and growth performance of Nile tilapia (Oreochromis niloticus) fingerlings. Egyptian Journal for Aquaculture. 2015;5(3):1-16.
10. Fuller R, Gibson GR. Probiotics and prebiotics: microflora management for improved gut health. Clinical Microbiology and Infection. 1998;4(9):477480.
11. Grisdale-Helland B, Helland SJ, Gatlin DM III. The effects of dietary supplementation with mannanoligosaccharide, fructooligosaccharide or galactooligosaccharide on the growth and feed utilization of Atlantic salmon (Salmo salar). Aquaculture. 2008;283(1-4):163-167.
12. Guerreiro I, Oliva-Teles A, Enes P. Prebiotics as functional ingredients: focus on Mediterranean fish aquaculture. Reviews in Aquaculture. 2018;10(4):800832.
13. Hoseinifar SH, Esteban MÁ, Cuesta A, Sun Y-Z. Prebiotics and fish immune response: a review of current knowledge and future perspectives. Reviews in Fisheries Science & Aquaculture. 2015;23(4):315-328.
14. Hoseinifar SH, Sharifian M, Vesaghi MJ, Khalili M, Esteban MA. The effects of dietary xylooligosaccharide on mucosal parameters, intestinal microbiota and morphology and growth performance of Caspian white fish (Rutilus frisii kutum) fry. Fish & Shellfish Immunology. 2014;39(2):231-236.
15. Huntingford F, Kadri S, Jobling M. Introduction: Aquaculture and Behaviour. In: Aquaculture and Behavior. 2012. p. 1-35.
16. Jahan N, Islam SM, Rohani MF, Hossain MT, Shahjahan M. Probiotic yeast enhances growth performance of rohu (Labeo rohita) through upgrading haematology and intestinal microbiota and morphology. Aquaculture. 2021;545:737243.
17. Kaur R, Ram RN. Temperature's influence on the embryonic development of Labeo rohita (Hamilton, 1822). Journal of Entomology and Zoology Studies. 2017;5(2):1172-1176.
18. Khan HA, Jhingran VG. Synopsis of biological data on rohu, Labeo rohita (Hamilton, 1822). FAO Fish Synopsis. 1975;(111):100.
19. Magnadottir B. Immunological control of fish diseases. Marine Biotechnology. 2010;12:361-379.
20. Sahu NP, Mohanta K. Present status and future scope of feed-based aquaculture in India. 2024.
21. Pohlenz C, Gatlin DM III. Interrelationships between fish nutrition and health. Aquaculture. 2014;431:111117.
22. Poolsawat L, Li X, Yang H, Yang P, Kabir Chowdhury MA, Yusuf A, Leng X. The potentials of fructooligosaccharide on growth, feed utilization, immune and antioxidant parameters, microbial community and disease resistance of tilapia (Oreochromis niloticus × O. aureus). Aquaculture Research. 2020;51(11):4430-4442.
23. Reverter M, Bontemps N, Lecchini D, Banaigs B, Sasal P. Use of plant extracts in fish aquaculture as an alternative to chemotherapy: current status and future perspectives. Aquaculture. 2014;433:50-61.
24. Ringø E, Olsen RE, Gifstad TO, Dalmo RA, Amlund H, Hemre GI, Bakke AM. Prebiotics in aquaculture: a review. Aquaculture Nutrition. 2010;16(2):117-136.
25. Roberfroid M. Prebiotics: the concept revisited. Journal of Nutrition. 2007;137(3):830S-837S.
26. Sahu S, Das BK, Pradhan J, Mohapatra BC, Mishra BK, Sarangi N. Effect of Magnifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings. Fish & Shellfish Immunology. 2007;23(1):109-118.
27. Sepulveda-Quiroz CA, Peña-Marín ES, Pérez-Morales A, Martínez-García R, Alvarez-Villagomez CS, Maytorena-Verdugo CI, et al. Fructooligosaccharide supplementation in diets for tropical gar (Atractosteus tropicus) juveniles: Effects on morphophysiology and intestinal barrier function. Aquaculture Research. 2021;52(1):37-50.
28. Soleimani N, Hoseinifar SH, Merrifield DL, Barati M, Abadi ZH. Dietary supplementation of fructooligosaccharide (FOS) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach (Rutilus rutilus) fry. Fish & Shellfish Immunology. 2012;32(2):316-321.
29. Sridevi V, Sumathi V, Prasad MG, Kumar MS. Fructooligosaccharides-type prebiotic: A review. Journal of Pharmacy Research. 2014;8(3):321-330.
30. Stickney RR, Gatlin DM III. Aquaculture: An Introductory Text. CABI; 2022.
31. Talwar PK, Jhingran AG. Inland fishes of India and adjacent countries. Vol. 2. CRC Press; 1991.
32. Wee W, Hamid NKA, Mat K, Khalif RIAR, Rusli ND, Rahman MM, et al. The effects of mixed prebiotics in aquaculture: A review. Aquaculture and Fisheries. 2024;9(1):28-34.