Effect of mulberry (Morus alba) leaf extract on growth and survival of labeo catla

Introduction

Aquaculture has become one of the fastest-growing food-producing sectors and plays a significant role in ensuring global food security, improving human nutrition and supporting economic development (Li et al., 2019; Perschbacher, 2015) [16] . It represents a major sector of the global food production industry, with an average annual growth rate of 2.3% and a total fish production of approximately 96.4 million tonnes recorded in 2020 (FAO, 2020). In recent decades, aquaculture has contributed more than half of the total fish consumed worldwide (Naderi et al., 2017) [14] . India is the second-largest producer of fish and aquaculture products globally, with inland fisheries contributing a major proportion of total fish production. Fish provide an excellent source of high-quality protein, essential fatty acids, vitamins and minerals that are important for human health and development. Among freshwater fish species, Labeo catla (Catla), belonging to the family Cyprinidae, is one of the most commercially important Indian major carps due to its rapid growth, high consumer preference and significant market value. It is commonly cultured in polyculture systems along with Labeo rohita and Cirrhinus mrigala, where it contributes substantially to increased fish production and farm profitability. Its high market demand and consumer acceptance make it an important species in freshwater aquaculture in India (Mohanty et al., 2013) [13] . In aquaculture, disease outbreaks and environmental stress remain major constraints affecting fish health, growth performance, survival and overall aquaculture productivity. Antibiotics and synthetic chemicals have been widely applied in aquaculture for the prevention and control of bacterial infections. The excessive use of antibiotics and synthetic chemicals for health management has raised concerns regarding antimicrobial resistance, chemical residues, environmental pollution and potential risks to human health. Therefore, there is an increasing need to develop safe, sustainable and eco-friendly alternatives that can improve fish health and performance. In recent years, the use of medicinal plants and their bioactive compounds has gained considerable attention as natural feed additives in aquaculture. Herbal supplements have been reported to enhance growth performance, survival and promote the overall health of cultured fish. Among these medicinal plants, Morus alba (white mulberry) has received significant attention because of its rich nutritional composition and diverse biological activities. The leaves of Morus alba, a plant native to Asian regions including India, China and Japan, have been extensively employed in traditional medicine, animal nutrition and sericultural practices. Mulberry is widely cultivated in tropical and subtropical areas due to its high nutritional value (Al-Kirshi et al., 2013) [1] . The leaves of M. alba are rich in crude protein, essential fatty acids, vitamins, minerals and various bioactive compounds such as flavonoids, alkaloids, polyphenols, polysaccharides, saponins, tannins, phytosterols and triterpenes (Nomura et al., 1983 [2,3,5,11,15] . These compounds exhibit antioxidant, antibacterial, antiinflammatory, immunomodulatory, hypolipidemic and antidiabetic activities, which may positively influence growth performance, digestive enzyme activity, antioxidant status and gut microbial balance in fish (Feng et [6,8,9,17] . However, the direct application of mulberry leaves may be limited due to the presence of certain anti-nutritional factors. Therefore, extraction methods such as ethanol extraction are employed to reduce undesirable compounds while preserving and concentrating valuable bioactive constituents. Considering the nutritional and pharmacological potential of M. alba, its application as a natural dietary supplement may provide an effective strategy to enhance growth performance and survival of cultured fish while reducing dependence on synthetic additives. Therefore, the present study was designed to evaluate the effects of mulberry leaf extract supplementation on the growth performance and survival of Labeo catla. This approach may contribute to the development of sustainable and environmentally friendly practices in freshwater aquaculture.

Materials and Methods

Location of experiment

The experiment was conducted at the Hands-on Training Centre, Department of Aquaculture, College of Fisheries Science, Kamdhenu University, Veraval, for a period of 60 days from October to December 2025. A total of 20 plastic aquarium tanks (50 L capacity; 2 × 1 × 1 ft) were arranged in a Completely Randomized Design (CRD) consisting of five treatments with four replicates each. The tanks were cleaned and disinfected with potassium permanganate (4 mg L⁻¹) before filling with clean water. Labeo catla were procured from a local hatchery, transported in oxygen-filled polythene bags and acclimatized for seven days in a 500 L FRP tank under proper aeration and feeding. After acclimatization, 200 fish were randomly stocked in the experimental tanks at a density of 10 fish per tank, and adequate aeration was maintained throughout the experimental period.

Collection of feed ingredients to prepare diet

The experimental diets were formulated using various feed ingredients, including fish meal, groundnut oil cake, wheat flour, vegetable oil, fish oil, tapioca flour and vitaminmineral premix. All feed ingredients were procured from local markets, while fresh mulberry leaves were collected from a nearby area. The collected leaves were sun-dried for 3-4 days to reduce moisture content before processing. Mulberry leaf extract was prepared using ethanol as a solvent through a conventional solvent extraction technique. The proximate composition of all dietary ingredients was determined according to established standard analytical methods. All ingredients were finely ground using a grinder and sieved to obtain a uniform particle size. The processed powdered ingredients were then packed in high-density polyethylene (HDPE) bags and stored at room temperature until further use in diet preparation.

Preparation of mulberry leaf extract

Fresh leaves of Morus alba were collected and air-dried under natural conditions. The dried leaves were subsequently pulverized into a fine powder using an electric blender. Approximately 180 g of the powdered material was immersed in 70% ethanol in a conical flask and maintained at room temperature for seven days with intermittent shaking to facilitate efficient extraction. Following the extraction period, the supernatant was separated carefully, and the ethanol was removed using a rotary evaporator. The obtained crude extract was then freeze-dried, transferred into sterile containers and preserved at -20°C until further experimental use (De Oliveira et al., 2015) [5] .

Experimental diet preparation

Five experimental diets were formulated with a uniform crude protein level of 30%. The control diet (T0) was prepared without Morus alba leaf extract, while T1, T2, T3, and T4 diets were supplemented with 0.25%, 0.5%, 0.75% and 1% mulberry leaf extract, respectively. The feed ingredients were weighed according to the formulation, finely ground, sieved and thoroughly mixed. Water was added to obtain a homogeneous dough, which was steamcooked by autoclaving at 121°C and 15 lbs pressure for 15 min. After cooling, the required amounts of vitamin-mineral premix and mulberry leaf extract were incorporated and the mixture was pelletized using a mechanical pelletizer. The prepared pellets were sun-dried until the moisture content was reduced below 10% and stored in labelled airtight plastic containers until further use.

Measurement of growth and survival

At the completion of the 60-day experiment, the final body weight and survival rate of fish were recorded. Growth performance parameters including mean weight gain, specific growth rate (SGR) and feed conversion ratio (FCR) were calculated to evaluate the effect of the experimental diets. 1) Body weight gain (g) = (Final body weight -Initial body weight) 2) FCR (Feed conversion ratio) = Amount of feed given/Body Weight gain 3) SGR (Specific growth rate) = ln [(Final body weight)ln (Initial body weight)] × 100 / days of the experiment 4) Survival rate% = (No. of fish survived / No. of fish stocked) × 100

Analysis of water quality parameters

During the experiment period, water quality parameters such as temperature, pH, dissolved oxygen (DO) and total hardness were checked every week. The water temperature was recorded with a thermometer and pH was measured using a digital pH meter and pH indicator solution. The dissolved oxygen and hardness in the water was determined by titration method.

Statistical analysis

Statistical analysis was performed using one-way Analysis of Variance (ANOVA) in SPSS software. The obtained results were presented as mean ± standard error (SE) and significant differences among treatment means were determined using Duncan's Multiple Range Test (DMRT) at a significance level of p<0.05.

Results and Discussion

Effects of mulberry leaf extract (MLE) on the growth and survival of Labeo catla

The initial mean weights of catla in the five experimental treatments were 0.48 ± 0.010 g (T0), 0.47 ± 0.008 g (T1), 0.47 ± 0.017 g (T2), 0.50 ± 0.007 g (T3) and 0.48 ± 0.013 g (T4). After 60 days of the experimental period, the final mean weights increased to 1.53 ± 0.024 g in T0, 1.66 ± 0.015 g in T1, 1.85 ± 0.024 g in T2, 2.23 ± 0.021 g in T3 and 2.09 ± 0.012 g in T4, with the highest final weight observed in T3 and the lowest in T0.

The mean weight gain of Labeo calta was evaluated for all five treatments using the mean weight data. At the end of the 60-day experimental period, the highest mean weight gain was recorded in treatment T3 (1.73 ± 0.021 g), followed by T4 (1.59 ± 0.012 g), T2 (1.35 ± 0.024 g), T1

(1.16 ± 0.015 g) and the lowest value was observed in the control treatment T0 (1.03 ± 0.024 g). Statistical analysis indicated a significant difference (p<0.05) in mean weight gain among all the experimental treatments. The maximum mean weight gain was observed in treatment T3 (0.75% mulberry leaf extract), which was significantly higher than the other treatments, suggesting its better performance in promoting the growth of L. catla.

Conclusion

A 60-day feeding experiment was carried out to assess the influence of dietary supplementation of Mulberry (Morus alba) leaf extract on the growth performance and survival of Labeo catla. Five experimental diets containing different levels of mulberry leaf extract (0-1%) were prepared with a uniform crude protein content of 30%. Among the tested diets, the treatment supplemented with 0.75% mulberry leaf extract exhibited the best growth performance, showing maximum mean weight gain and specific growth rate (SGR), along with the lowest feed conversion ratio (FCR) and highest survival rate. The findings suggest that the inclusion of 0.75% mulberry leaf extract in the diet can effectively improve the growth and survival of L. catla, indicating its potential as a natural feed additive for promoting sustainable aquaculture.

References

1. Al-Kirshi RA, Alimon A, Zulkifli I, Atefeh S, Zahari MW, Ivan M. Nutrient digestibility of mulberry leaves (Morus alba). Ital J Anim Sci. 2013;12(2):e36.
2. Chan W, Lye PY, Wong SK. Phytochemistry, pharmacology, and clinical trials of Morus alba. Chin J Nat Med. 2016;14:17-30.
3. Chen C, Mohamad RU, Saikim FH, Mahyudin A, Mohd NN. Morus alba L. plant: bioactive compounds and potential as a functional food ingredient. Foods. 2021;10(3):689.
4. Chen CC, Liu LK, Hsu JD, Huang HP, Yang MY, Wang CJ. Mulberry extract inhibits the development of atherosclerosis in cholesterol-fed rabbits. Food Chem. 2005;91(4):601-7.
5. De Oliveira AM, Mesquita MDS, Da Silva GC, De Oliveira Lima E, De Medeiros PL, Paiva PMG, et al. Evaluation of toxicity and antimicrobial activity of an ethanolic extract from leaves of Morus alba L. (Moraceae). Evid Based Complement Alternat Med. 2015;2015:513978.
6. El-Sayyad HI. Cholesterol overload impairing cerebellar function: the promise of natural products. Nutrition. 2015;31(5):621-30.
7. Food and Agriculture Organization of the United Nations. The state of world fisheries and aquaculture 2020: sustainability in action. Rome: FAO; 2020.
8. Feng QF, Li ZF, Hang XZ, Li H, Zhai XL, Xue Y, et al. Effects of dietary supplementation of mulberry leaf extract and 1-deoxynojirimycin on growth, digestion, immune ability and intestinal flora of Andrias davidianus. Acta Hydrobiol Sin. 2021;45:582-9.
9. Hao JY, Wan Y, Yao XH, Zhao WG, Hu RZ, Chen C, et al. Effect of different planting areas on the chemical compositions and hypoglycemic and antioxidant activities of mulberry leaf extracts in Southern China. PLoS One. 2018;13(6):e0198072.
10. He X, Fang J, Ruan Y, Wang X, Sun Y, Wu N, et al. Structures, bioactivities and future prospective of polysaccharides from Morus alba (white mulberry): a review. Food Chem. 2018;245:899-910.
11. Kusano G, Orihara S, Tsukamoto D, Shibano M, Coskun M, Guvenc A, et al. Five new nortropane alkaloids and six new amino acids from the fruit of Morus alba L. growing in Turkey. Chem Pharm Bull (Tokyo). 2002;50(2):185-92.
12. Li Z, Wang G, Yu E, Zhang K, Yu D, Gong W, et al. Artificial substrata increase pond farming density of grass carp (Ctenopharyngodon idella) by increasing the bacteria that participate in nitrogen and phosphorus cycles in pond water. PeerJ. 2019;7:e7906.
13. Mohanty BP, Banerjee S, Bhattacharjee S, Mitra T, Purohit GK, Sharma AP, et al. Muscle proteomics of the Indian major carp catla (Catla catla, Hamilton). J Proteomics Bioinform. 2013;6(11):252-63.
14. Naderi M, Keyvanshokooh S, Salati AP, Ghaedi A. Combined or individual effects of dietary vitamin E and selenium nanoparticles on humoral immune status and serum parameters of rainbow trout (Oncorhynchus mykiss) under high stocking density. Aquaculture. 2017;474:40-7.
15. Nomura T, Fukai T, Hano Y, Nemoto K, Terada S, Kuramochi T. Constituents of cultivated mulberry tree. Planta Med. 1983;47(3):151-6.
16. Perschbacher PW. The future of fish as food. J Fish Livest Prod. 2015;3:3.
17. Wang W, Zu Y, Fu Y, Efferth T. In vitro antioxidant and antimicrobial activity of extracts from Morus alba L. leaves, stems and fruits. Am J Chin Med. 2012;40(2):349-56.