Effect of azolla-based feed on growth and survivability of Indian Butter catfish (Ompok bimaculatus)

Introduction

Despite significant advances in global food production, approximately 370 million people, representing over 5% of the world's population, are projected to experience hunger by 2060, particularly in developing nations (Parry et al., 2014) [23] . The accessibility and affordability of protein sources for aquafeed production represent critical factors determining the sustainability and success of aquaculture operations (FAO, 2018). In recent decades, plantbased protein sources have gained recognition as sustainable alternatives to fishmeal in aquafeeds, owing to their environmental benefits, cost efficiency, and reliable availability (Gatlin et al., 2007; Ahmadifar et al., 2019; Elumalai et al., 2020) [2,14,18] . Among these alternatives, Azolla, a floating aquatic fern commonly found in stagnant or slow-moving freshwater ecosystems across Africa, Asia, and parts of Australia, has emerged as particularly promising due to its rapid biomass production, high nutritional value, and adaptability to diverse environmental conditions. Azolla pinnata establishes a symbiotic association with the nitrogen-fixing cyanobacterium Anabaena azollae, which inhabits the dorsal leaf cavities of the fern. This unique biological relationship enables Azolla to thrive in nitrogen-deficient environments, making it an excellent candidate for sustainable feed production. With a crude protein content ranging between 13% and 30%, complemented by a rich profile of essential amino acids, particularly lysine, Azolla demonstrates superior nutritional quality compared to many other aquatic macrophytes and conventional green feed crops (Brouwer et al., 2018) [8] . The practical application of Azolla in aquaculture has been validated through its successful incorporation into diets for species such as Thai silver barb (Barbonymus gonionotus) and Nile tilapia (Oreochromis niloticus), where it has demonstrated positive effects on growth performance and feed utilization efficiency (Das et al., 2018; Sivakumar and Solaimalai, 2003) [11,26] . Furthermore, unlike seasonal crops, such as soybeans, Azolla offers the distinct advantage of continuous cultivation and harvest throughout the year, providing a consistent and economically viable protein source for aquaculture systems (Anglade et al., 2015) [3] . The Indian butterfish (O. bimaculatus), an economically important freshwater species, presents considerable potential for aquaculture development due to its high market demand and excellent nutritional profile. However, the cultivation of this species faces significant challenges related to feed formulation and growth optimization. Fish growth represents a complex biological process influenced by multiple dietary and environmental factors, typically quantified through various performance indices including daily weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, feed acceptability, and survival rate (Lupatsch et al., 2003; Simensen et al., 2000) [ 20,25] . The implementation of Azolla-based feed formulations for O. bimaculatus holds promise for enhancing growth performance, given its favorable nutritional composition and cost-effectiveness. Maintaining optimal water quality parameters represents a fundamental requirement for successful aquaculture production, as these conditions directly influence fish health, feed utilization efficiency, and overall growth performance. Critical water quality parameters requiring careful management include temperature, pH, dissolved oxygen concentration, alkalinity, and nutrient levels (Bhatnagar and Devi, 2013) [6] . Hypoxia, characterized by dissolved oxygen concentrations below 1 to 2 mg/L, can induce severe physiological stress in fish populations, leading to growth impairment and potentially significant mortality events (Silviana et al., 2022) [24] . A comprehensive understanding of the complex interactions between feed composition, water quality parameters, and growth performance remains essential for developing sustainable culture practices for O. bimaculatus.

Materials and methods

Experimental fish and setup

The present study was carried out at the College of Fisheries Sciences, Chaudhary Charan Singh Haryana Agricultural University (CCS HAU), Hisar. The study was conducted to investigate the effects of Azolla-based feed on the growth performance and survivability of Indian butterfish (O. bimaculatus) during the advanced fingerling stage. The experiment was carried out in 12 disinfected glass aquarium tanks, each with a capacity of 150 liters, over 60 days from October 5, 2022, to December 4, 2022. The experimental design included four treatments (T1: control; T2: 20% Azolla; T3: 30% Azolla; T4: 100% Azolla), each with three replications. Each replication was stocked with 10 acclimatized fingerlings, arranged in a randomized block design (RBD). Fish survival was monitored daily, and growth data were recorded weekly.

Preparation of experimental diet

Fresh Azolla was obtained from the Azolla culture unit of the College of Fisheries Science, CCS HAU, Hisar. The Azolla was thoroughly washed, air-dried under sunlight for 48 hours, ground into fine powder, and stored at 4°C to prevent carotenoid oxidation. The experimental diets were formulated by incorporating Azolla powder at proportions of 0%, 20%, 30%, and 100% into a commercial feed using the Pearson square method to balance protein levels. The feed mixture was combined with a small amount of vegetable oil and water to form a dough, which was extruded through a hand-operated pelletizer (1 mm pore size). The pellets were dried in a hot air oven at 55°C and stored in airtight glass containers in a refrigerator at 4°C.

Feeding Protocol

The experimental fish were fed thrice daily at a rate of 5% of their body weight. Initial measurements of body length (mm) and weight (g) were recorded before the experiment, and feed quantities were adjusted weekly based on the mean weight of the fish. Growth parameters were measured at weekly intervals, and final measurements were recorded after 60 days of rearing.

Physico-Chemical Analysis of Water

Water quality parameters were monitored fortnightly between 9:00 a.m. and 10:00 a.m. throughout the experimental period using standard methods (APHA, 1998). Temperature was measured using a mercury thermometer, while pH levels were determined with a digital pH meter (Aquasol, AM-P-PH). Salinity was assessed using a microprocessor salinity meter (LT-51), and dissolved oxygen (DO) was estimated through the modified Winkler method using an Aquasol DO kit (AEDO8). Ammonia concentration was quantified using a visual colorimetric method with an AQUASOL testing kit, and alkalinity was determined through titration with sulfuric acid, employing phenolphthalein and methyl orange indicators. These parameters were carefully recorded to ensure the optimal rearing environment for the experimental fish and to evaluate any potential variations across treatments. Alkalinity: Measured through titration with sulfuric acid and indicators (phenolphthalein and methyl orange).

Growth Parameters

Growth parameters like Average daily weight gain (ADG), Feed conversion ratio (FCR), Specific growth rate (SGR), the percentage increase in length and weight, and survival rate, total weight gains were recorded fortnightly and evaluated using the provided formulas -

Proximate Composition Analysis

The proximate composition of the experimental diets was analyzed to assess their nutritional quality, following the guidelines of AOAC (1995). Moisture content was determined by drying the feed samples in an oven at 105°C until a constant weight was achieved. Crude protein was estimated using the Kjeldahl method, while crude lipid content was analyzed by Soxhlet extraction. Crude fiber was measured through sequential digestion with dilute acid and alkali, and ash content was determined by combusting the samples in a muffle furnace at 600°C. These analyses ensured that the formulated diets met the nutritional requirements of the fish and provided insights into the role of Azolla in enhancing dietary quality.

Statistical Analysis

All data were analyzed using a randomized block design (RBD) and ANOVA to determine treatment effects, with a significance threshold of 𝑃<0.05. Statistical analyses were performed using OpStat software, while graphs and bar charts were generated using Microsoft Excel 2010.

Results and discussion

Growth performance and survival rates of O. bimaculatus were significantly influenced by dietary treatments and sampling periods (p < 0.05). Among experimental groups, the diet incorporating 20% Azolla (T₂) yielded the highest average daily weight gain (ADG: 0.31 g), followed by T₁ (0.26 g), T₃ (0.24 g), and T₄ (0.04 g). A parallel trend was observed in specific growth rate (SGR), with T₂ achieving the highest SGR (1.75%) and T₄ the lowest (0.39%). The feed conversion ratio (FCR), a critical indicator of nutrient utilization efficiency, varied significantly between control and treatment groups. Particularly, T₂ demonstrated superior FCR (1.61 ± 0.03) compared to T₁ (1.87 ± 0.03), T₃ (1.96 ± 0.02), and T₄ (6.61 ± 0.62), underscoring the efficacy of 20% Azolla inclusion. These results align with established optimal FCR ranges (1.5-2.0) for fish growth (Afia et al., 2019) [1] and corroborate findings in O. bimaculatus (1.58-1.85; Chavez et al., 2011) [9] and other teleosts (Cremer et al., 2003; Jayakumar et al., 2014) [10,19] . [22] . Statistical analysis confirmed significant interactions (p<0.05) between dietary treatments and sampling periods, reinforcing the benefits of 20% Azolla supplementation. These findings align with studies reporting enhanced growth in cyprinids with Azolla inclusion (Ayapan, 1992; Panigrahi et al., 2014; Datta, 2011) [12,22] , though optimal levels are species-specific (e.g., 10-25% for grass carp; Majhi, 2006; Tuladhar, 2003; Gangadhar et al., 2017) [17,21,27] . Water quality parameters remained within optimal thresholds throughout the trial, supporting physiological performance. Temperatures (22.73-24.06°C) and dissolved oxygen (5.7-6.07 mg/L) aligned with metabolic requirements for growth (Boyd, 1982; Bailey & Alanara, 2006) [5,7] . Stable pH (7.33-7.93) and alkalinity (115.83-142.39 mg/L), coupled with non-toxic ammonia (<0.035 mg/L), nitrite, nitrate, and salinity (0.24-0.28 ppt), ensured species-appropriate conditions (Dubey et al., 2022; Bhatnagar et al., 2013) [6,13] . Regular waste removal and 20% daily water exchange maintained these parameters, emphasizing the necessity of integrated husbandry practices in aquaculture systems (Majhi et al., 2006; Gangadhar et al., 2015) [17,21] .

Collectively, these findings demonstrate the nutritional viability of 20% Azolla in O. bimaculatus diets, optimizing growth and survival without compromising water quality.

Conclusion

This study conclusively demonstrates that a 20% Azolla inclusion (T₂) optimizes growth performance (ADG: 0.31 g; SGR: 1.75%), feed efficiency (FCR: 1.61 ± 0.03), and survival in O. bimaculatus fingerlings, significantly outperforming diets with higher Azolla concentrations. The severe decline in growth and survival observed at 100% inclusion (T₄), coupled with the significant treatment × time interaction (p < 0.05), underscores the critical need for moderated supplementation, aligning with optimal ranges (10-25%) established for cyprinids (Majhi, 2006; Panigrahi et al., 2014) [21,22] . Stable maintenance of water quality parameters, temperature (22.73-24.06°C), dissolved oxygen (5.7-6.07 mg/L), pH (7.33-7.93), and ammonia (<0.035 mg/L) within species-optimal thresholds (Boyd, 1982; Bhatnagar et al., 2013) [6,7] was fundamental to these outcomes, highlighting the essential synergy between nutritional strategy and environmental management. We therefore recommend incorporating 20% Azolla into commercial O. bimaculatus diets to maximize costefficiency and productivity, while strongly advising against inclusions exceeding 30% due to significant survival compromises. Future research should prioritize defining species-specific tolerance thresholds, evaluating long-term physiological and product quality impacts, and assessing ecological implications of scaled Azolla utilization, alongside standardized water management protocols to ensure replicability.

Acknowledgement

AcknowledgmentsThe first author extends thanks to the Vice Chancellor, Director of Research of the CCS Haryana Agriculture University, Hisar, and the Dean, College of Fisheries Science, CCS Haryana Agriculture University, Hisar, Haryana, for the infrastructure support in carrying out the research work.

References

1. Afia OE, David GS, Udo IU. Studies on the effects of feeding levels on growth response and nutrient utilization of Heteroclarias (hybrid catfish). J Appl Sci. 2019;19(7):725-730.
2. Ahmadifar E, Sheikhzadeh N, Roshanaei K, Dargahi N, Faggio C. Can dietary ginger (Zingiber officinale) alter biochemical and immunological parameters and gene expression related to growth, immunity, and antioxidant system in zebrafish (Danio rerio)? Aquaculture. 2019;507(1):341-348.
3. Anglade J, Billen G, Garnier J. Relationships for estimating N2 fixation in legumes: incidence for N balance of legume-based cropping systems in Europe. Ecosphere. 2015;6(3):1-24.
4. American Public Health Association. Standard methods for the examination of water and wastewater. 20th ed. Washington (DC): American Public Health Association; 1998. p.1-541.
5. Bailey J, Alanärä A. Digestible energy need (DEN) of selected farmed fish species. Aquaculture. 2006;251(24):438-455.
6. Bhatnagar A, Devi P. Water quality guidelines for the management of pond fish culture. Int J Environ Sci. 2013;3(6):1980-2009.
7. Boyd CE. Water quality management for pond fish culture. Amsterdam: Elsevier Scientific Publishing Co.; 1982.
8. Brouwer P, Schluepmann H, Nierop KG, Elderson J, Bijl PK, van der Meer I, et al. Growing Azolla to produce sustainable protein feed: the effect of differing species and CO2 concentrations on biomass productivity and chemical composition. J Sci Food Agric. 2018;98(12):4759-4768.
9. Chavez HM, Fang AL, Carandang AA. Effect of stocking density on growth performance, survival and production of silver pompano, Trachinotus blochii (Lacépède, 1801) in marine floating cages. Asian Fish Sci. 2011;24(3):321-330.
10. Cremer MC, Jian Z, Lan HP. Growth performance of golden fin pompano at two stocking densities in nearshore ocean cages at Hainan, China. Results of ASA/China. 2003;4(5):12.
11. Das M, Rahim FI, Hossain MA. Evaluation of fresh Azolla pinnata as a low-cost supplemental feed for Thai silver barb Barbonymus gonionotus. Fishes. 2018;3(1):15.
12. Datta SN. Culture of Azolla and its efficacy in diet of Labeo rohita. Aquaculture. 2011;310(3-4):376-379.
13. Dubey SK, Padiyar AP, Shenoy N, Gaikawd A, Mohanty B, Baliarsingh BK, et al. Climate-smart and nutrition-sensitive aquaculture in Odisha, India: a new horizon in sustainability, adaptation, and mitigation. In: Handbook on climate change and disasters. Edward Elgar Publishing; 2022. p.574-593.
14. Elumalai P, Kurian A, Lakshmi S, Faggio C, Esteban MA, Ringø E. Herbal immune modulators in aquaculture. Rev Fish Sci Aquac. 2020;29(1):33-57.
15. Food and Agriculture Organization of the United Nations. The state of world fisheries and aquaculture: opportunities and challenges. Rome: FAO; 2014.
16. Gangadhar B, Sridhar N, Saurabh S, Raghavendra CH, Hemaprasanth KP, Raghunath MR, et al. Effect of Azolla-incorporated diets on the growth and survival of Labeo fimbriatus during fry-to-fingerling rearing. Cogent Food Agric. 2015;1(1):1055539.
17. Gatlin DM, Barrows FT, Brown P, Dabrowski K, Gaylord TG, Hardy RW, et al. Expanding the utilization of sustainable plant products in aqua feeds: a review. Aquac Res. 2007;38(6):551-579.
18. Jayakumar R, Nazar AA, Tamilmani G, Sakthivel M, Kalidas C, Ramesh Kumar P, et al. Evaluation of growth and production performance of hatchery produced silver pompano Trachinotus blochii (Lacépède, 1801) fingerlings under brackishwater pond farming in India. Indian J Fish. 2014;61(3):58-62.
19. Lupatsch I, Kissil GW, Sklan D. Comparison of energy and protein efficiency among three fish species: gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus): energy expenditure for protein and lipid deposition. Aquaculture. 2003;225(1-4):175-189.
20. Majhi SK, Das A, Mandal BK. Growth performance and production of organically cultured grass carp Ctenopharyngodon idella (Val.) under mid-hill conditions of Meghalaya, North Eastern India. Turk J Fish Aquat Sci. 2006;6(2).
21. Panigrahi S, Choudhury D, Sahoo JK, Das SS, Rath RK. Effect of dietary supplementation of Azolla on growth and survivability of Labeo rohita fingerlings. Asian J Anim Sci. 2014;9(1):33-37.
22. Parry M. Effects of climate change on potential food production and risk of hunger. In: Plant genetic resources and climate change. Wallingford (UK): CABI; 2014. p.61-77.
23. Silviana NR, Pamungkas W, Grandiosa R. Utilizing black cumin (Nigella sativa) flour to increase the immunity system of tilapia (Oreochromis niloticus) against Aeromonas hydrophila bacterial attack. Jurnal Akuakultur Indonesia. 2022;21(2):161-177.
24. Simensen LM, Jonassen TM, Imsland AK, Stefansson SO. Photoperiod regulation of growth of juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture. 2000;190(1-2):119-128.
25. Sivakumar C, Solaimalai A. Weed management in ricefish-Azolla farming system: a review. Agric Rev. 2003;24:190-196.
26. Tuladhar B. Comparative study of fish yields with plant protein sources and fish meal. Our Nature. 2003;1(1):26-29.