Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus)

Copyright © The Author(s). All Rights Reserved © 2025 GLOBAL PUBLICATION HOUSE |INT. Journal of Agriculture and Research| Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus) By Author(s): ________________________________________ Yuli Andriani 1) * , & Aisyah1) 1) Department of Fisheries, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Indonesia ________________________________________ Abstract: This study aims to evaluate the effect of adding various types of leafy greens in artificial feed formulations on the growth rate of red tilapia (Oreochromis niloticus) fingerlings. The research was conducted from January to March 2023 at the Aquatic Resource Management Laboratory, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jatinangor. The research method used was an experimental design with a Completely Randomized Design (CRD), consisting of five treatments with three replications each. The treatments included: Treatment A (control/no leafy green addition), Treatment B (10% Carica papaya leaf meal), Treatment C (10% Alocasia macrorrhiza leaf meal), Treatment D (10% Albizia falcataria leaf meal), and Treatment E (a combination of 3.33% the three various leaves). The results showed that the addition of leafy greens in the feed did not result in significant differences in growth rate and feed efficiency. However, the treatment with 10% Alocasia macrorrhiza leaf meal produced the highest daily growth rate at 1.22% and a feed efficiency of 26.21%. Based on these results, it can be concluded that adding up to 10% leafy greens in feed formulations can be considered an alternative source of plant-based protein for red tilapia fingerlings. Keywords: Alocasia macrorrhiza, Albizia falcataria, Flour, papaya leaves, red tilapia, How to cite: Andriani, Y., & Aisyah, A. (2025). Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus). GPH-International Journal of Agriculture and Research, 8(10), 27-43. https://doi.org/10.5281/zenodo.17587549 * e-ISSN 3050-9602 p-ISSN 2795-4340 ARTICLE ID: #02156 VOLUME 08 ISSUE 10 OCT - 2025 10.5281/ZENODO.17587549 Page 27 of 43 Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus) © 2025 Published by GLOBAL PUBLICATION HOUSE | International Journal of Agriculture & Research Introducation The aquaculture sector in Indonesia is targeted to reach a production volume of 5 million tons, representing a 56% increase in 2012 compared to 3.2 million tons achieved in 2011 (Ministry of Marine Affairs and Fisheries, 2012). One of the aquaculture species with significant potential for increased production is the red tilapia (Oreochromis niloticus), which is an important freshwater commodity in Indonesia. Farmers prefer to cultivate red tilapia because it is relatively easy to culture and market. Furthermore, market demand for red tilapia remains high across various size categories from fry to table-size fish due to its affordable price and wide consumer acceptance. Red tilapia possesses several advantageous traits, including rapid growth, tolerance to high stocking densities, a favourable feed conversion ratio, and resistance to diseases and suboptimal environmental conditions (Carman & Sucipto, 2009). In intensive aquaculture systems, feed management is one of the key determinants of production success. Feed composition both in quality and quantity strongly influences growth and production efficiency. However, the increasing dependency on imported raw materials such as fish meal, squid meal, crustacean meal, meat bone meal (MBM), poultry meat meal (PMM), soybean meal, wheat flour, and various vitamins and minerals (Directorate of Production, Directorate General of Aquaculture, 2009) has led to rising feed costs, which consequently affect profitability in aquaculture operations. To overcome these limitations, the development of locally available, affordable, and nutritionally balanced feed ingredients is essential. Soybean meal is a commonly used plantbased protein source due to its high protein content, yet the exploration of other local plant resources with comparable nutritional and functional properties is urgently needed. Several plant leaves such as Carica papaya, Albizia falcataria, and Alocasia macrorrhiza have shown potential as alternative protein sources for fish feed because of their nutritional adequacy, availability, and low cost. Carica papaya L. is widely cultivated across Indonesia and valued for its multifunctional uses. Its leaves contain 17.0% crude protein, 7.2% crude fiber, and 11.1% ash (Sutama, 2008), while Central Java, East Java, and West Java serve as major papaya producing regions (Kalie, 2010). Alocasia macrorrhiza is also used by farmers as supplementary feed in gourami (Osphronemus goramy) culture and contains 19.50% crude protein, 7.78% fat, and 17.33% crude fiber (Laboratory of Ruminant Nutrition and Feed Chemistry, 2013). Meanwhile, Albizia falcataria leaves containing 20.65% crude protein, 28.34% crude fiber, and 5.13% crude fat (Tanjung, 2000) are abundant by-products of the timber industry and therefore represent an economically valuable raw material for feed production. These plants contain diverse bioactive compounds such as alkaloids, polyphenols, flavonoids, saponins, tannins, and mimosine, which contribute to antioxidant, antibacterial, and immunostimulant activities. The role of natural antioxidants in fish physiology, Page No. 28 Andriani, Y., & Aisyah, A. (2025). Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus). GPH-International Journal of Agriculture and Research, 8(10), 27-43. https://doi.org/10.5281/zenodo.17587549 Volume 8 Issue No 10 (2025) Access: https://gphjournal.org/index.php/ar particularly in maintaining cellular integrity and pigmentation, has been widely recognized (Andriani et al., 2022). Such bioactive molecules can enhance color quality, immune response, and stress tolerance in fish (Andriani et al., 2020). Papaya leaves also contain proteolytic enzymes such as papain and chymoprotein that catalyze the hydrolysis of complex proteins into simpler, more digestible forms (Munajat & Budiana, 2003; Suhartono, 1992). On the other hand, antinutritional factors such as tannins and mimosine present in Albizia falcataria leaves can be reduced through pre-treatments like lime water (Ca(OH)₂) soaking and sun-drying (Saifullah, 2005; Santoso, 2001), improving their nutritional suitability. In addition, fermentation technology has become a promising approach to improving the nutritional and physical characteristics of plant-based and organic materials. Fermentation enhances digestibility, reduces antinutritional compounds, and modifies the texture and palatability of feed ingredients (Andriani et al., 2020; Sulistiyono et al., 2020). Fermented organic matter derived from domestic food waste has been demonstrated to improve fish growth performance and feed conversion efficiency (Andriani et al., 2023), indicating that controlled fermentation can transform underutilized biomass into high-value aquafeed resources. Furthermore, flavonoid compounds found in Alocasia macrorrhiza leaves exhibit antibacterial properties by forming complexes with extracellular proteins and disrupting bacterial membrane integrity (Haitami, 2011). Their mode of action involves protein denaturation and irreversible cell damage, which enhances the host’s disease resistance. Therefore, integrating various leaves combined with appropriate fermentation processes may produce functional fish feeds that promote growth, improve feed utilization, and strengthen immune responses against bacterial infections. Materials and Methods Based on the aforementioned background, this study was conducted to evaluate the effect of various types of plant leaves incorporated into formulated feed on the growth performance of red tilapia (Oreochromis niloticus) fingerlings. The experiment was carried out at the Laboratory of Aquatic Resource Management, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, from January to March 2023. The experimental fish used in this study were red tilapia fingerlings with an initial average body weight of 5 g per fish. The fish were obtained from the Citomi Community Hatchery Unit (Unit Pembenihan Rakyat, UPR Citomi) located in Tanggulun Barat Village, Kalijati District, Subang Regency, West Java, Indonesia. Page No. 29 Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus) © 2025 Published by GLOBAL PUBLICATION HOUSE | International Journal of Agriculture & Research Table 2. Formulation of experimental diets Feed ingredients Treatments / Crude Protein (%) A (27%) B (27%) C (27%) D (27%) E (27%) Fish meal 23,94 22,46 23,50 23,38 23,14 Soybean meal 23,94 22,46 23,50 23,38 23,14 Rice bran 21,05 17,53 16,50 16,61 16,85 Pollard (wheat bran) 21,05 17,53 16,50 16,61 16,85 Carica papaya leaf meal - 10 - - 3,33 Alocasia macrorrhiza leaf meal - - 10 - 3,33 Albizia falcataria leaf meal - - - 10 3,33 Tapioca flour 7 7 7 7 7 Premix (vitamin– mineral) 2 2 2 2 2 Fish oil 1 1 1 1 1 Total 100 100 100 100 100 The equipment used in this study included twenty aquaria (30 × 30 × 25 cm) that served as rearing tanks for red tilapia (Oreochromis niloticus) fingerlings. A digital balance (AND EK-120G, accuracy ±0.01 g) was used to weigh both the fish and feed ingredients. The aeration system consisted of a blower, aeration hoses, valves, air stones, and PVC pipes to provide continuous oxygen supply to each aquarium. Water quality was monitored using a Water Quality Checker (Hanna Instruments, USA) to measure temperature, dissolved oxygen (DO), and pH. Water temperature was controlled using submersible water heaters (Rena, 75 W), with a total of 24 units installed across treatments. Ammonia concentrations were measured using an API® Ammonia Test Kit (Mars Fishcare, USA) to ensure that water quality remained within optimal conditions for fish growth. This experiment employed a completely randomized design (CRD) consisting of five treatments with three replicates each. The treatments represented different types and proportions of plant leaf meal used as a substitute for plant-based protein sources in the formulated diets, as follows: • A: Diet without any leaf meal (control) • B: Diet supplemented with 10% Carica papaya leaf meal • C: Diet supplemented with 10% Alocasia macrorrhiza leaf meal • D: Diet supplemented with 10% Albizia falcataria • E: Diet containing a combination of various leaf meals (3.33% each) Page No. 30 Andriani, Y., & Aisyah, A. (2025). Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus). GPH-International Journal of Agriculture and Research, 8(10), 27-43. https://doi.org/10.5281/zenodo.17587549 Volume 8 Issue No 10 (2025) Access: https://gphjournal.org/index.php/ar The linear model for this experimental design was expressed as follows (Gaspersz, 1991): Xij = μ + τi + εij Keterangan : Xij = observation of the response variable from the i-th treatment and j-th replication, μ = overall mean, τ = effect of the i-th treatment, and εij = random error associated with the i-th treatment and j-th replication. Research Procedure Preparation of Leaf Meals The preparation of the plant leaf meals began with the collection of fresh leaves, which were thoroughly washed with running water to remove impurities. The leaves were then soaked in a lime solution (Ca(OH)₂) at a concentration of 2% of the leaf weight for 20 minutes to eliminate antinutritional compounds. After soaking, the leaves were rinsed again with clean water and soaked in freshwater for 24 hours. The leaves were subsequently sundried until completely dry, separated from the stems, and ground into fine powder using a mechanical grinder. The resulting leaf meal was sieved to ensure uniform particle size and remove coarse residues. Feed Preparation Formulated feed was prepared by finely grinding all feed ingredients and sieving them through a standard mesh. Each ingredient was weighed according to the diet formulation calculated using the square method to achieve the desired protein level. The ingredients were thoroughly mixed, starting from those with the smallest proportion to those with the largest, to ensure homogeneity. The mixed feed was then moistened with hot water and stirred until a dough-like consistency was achieved. The dough was extruded using a pelleting machine, and the pellets were sun-dried to constant moisture content before storage and use in the feeding trial. Experimental Setup and Fish Maintenance Prior to the experiment, all aquaria and supporting equipment were cleaned thoroughly to prevent contamination and disease transmission. The aquaria were rinsed with clean water, air-dried, and arranged according to the experimental layout. Each aquarium was randomly labeled to represent different treatments. Freshwater sourced from a natural spring was used as the culture medium and aerated continuously for 24 hours before use to ensure adequate dissolved oxygen levels. The water temperature was maintained between 25°C and 28°C throughout the experiment. After acclimation, ten red tilapia (Oreochromis niloticus) fingerlings were stocked into each aquarium, corresponding to a stocking density of 1 fish per 2 liters of water. Fish were observed daily to monitor behavior and health condition during the rearing period. Page No. 31 Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus) © 2025 Published by GLOBAL PUBLICATION HOUSE | International Journal of Agriculture & Research Experimental Procedure A total of ten red tilapia (Oreochromis niloticus) fingerlings were stocked into each aquarium that had been prepared for the experiment. Fish were fed with the experimental diets at a feeding rate of 5% of total biomass per day, divided into three feeding times: 08:00, 12:00, and 16:00. Uneaten feed and fecal residues were removed daily by siphoning to maintain water quality throughout the rearing period. Water Quality Monitoring Water quality parameters observed during the study included temperature, pH, dissolved oxygen (DO), and ammonia (NH₃) concentration. Measurements were taken periodically to represent the environmental conditions within the rearing aquaria. Water temperature was recorded daily, while measurements of pH, dissolved oxygen, ammonia, and fish biomass were conducted every 10 days during the 40-day rearing period. These parameters were used to evaluate the environmental stability and suitability of the culture media during the experimental period. Observation Parameters The parameters measured during the experiment were used to evaluate the growth performance and water quality of red tilapia (Oreochromis niloticus) fingerlings. 1. Daily Growth Rate (DGR) Daily growth rate was calculated to determine the average daily increase in fish body weight during the experimental period. The formula used was as follows (Effendi, 1997): DGR (%/day) = x 100% where: Wo = average final body weight (g), Wt = average initial body weight (g), t = duration of the experiment (days). 2. Feed Efficiency (FE) Feed efficiency was calculated to determine the ability of fish to convert the feed consumed into body weight gain. The formula used was as follows (Zonneveld et al.1991): FE (%) x 100% Where: F = total feed given (g) Wo = average initial body weight (g) Wt = average final body weight (g) Page No. 32 Andriani, Y., & Aisyah, A. (2025). Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus). GPH-International Journal of Agriculture and Research, 8(10), 27-43. https://doi.org/10.5281/zenodo.17587549 Volume 8 Issue No 10 (2025) Access: https://gphjournal.org/index.php/ar The data obtained from this experiment were analyzed using analysis of variance (ANOVA) to determine the effect of each treatment. When significant differences were detected (p < 0.05), the results were further analyzed using Duncan’s Multiple Range Test (DMRT) at a 95% confidence level, in order to identify which treatments differed significantly from each other. The experimental feed used in this study was prepared in crumb form, containing approximately 27% crude protein. The feed ingredients consisted of fish meal, soybean meal, rice bran, pollard (wheat bran), oil, premix, and various types of leaf meal according to the respective treatment formulation. All plant leaves used in the preparation of experimental feeds were sourced from the same location, namely Karang Bahagia, Bekasi Regency, West Java, Indonesia, to ensure consistency in raw material characteristics and minimize variability among treatments. Results and Discussion Growth is defined as an increase in body length and weight over a certain period of time. It is a complex biological process influenced by various intrinsic and extrinsic factors. Growth occurs as a result of cellular division through mitosis, leading to an increase in both tissue mass and body size. According to Effendi (1997), the factors influencing fish growth can be categorized into internal and external factors. Internal factors include heredity, sex, and age, whereas external factors involve environmental conditions such as feed availability and quality, water temperature, parasitic infections, and disease occurrence. Among these, feed quality particularly its protein content derived from both animal and plant sources play a crucial role in supporting optimal growth and metabolic processes in fish. Figure 1. Average body weight of red tilapia (Oreochromis niloticus) during the experimental period. Based on the results presented in Figure 6, the inclusion of different types of plant leaf meal in the formulated diets resulted in variations in body weight gain of red tilapia Page No. 33 Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus) © 2025 Published by GLOBAL PUBLICATION HOUSE | International Journal of Agriculture & Research (Oreochromis niloticus). The highest average body weight at the end of the rearing period was observed in Treatment A (control, without leaf meal addition), reaching 7.40 g, followed by Treatment E, which received a combination of Carica papaya leaf meal (3.33%), Alocasia macrorrhiza leaf meal (3.33%), and Albizia falcataria leaf meal (3.33%), with an average weight of 7.36 g. Treatment D, containing 10% Albizia falcataria leaf meal, produced an average body weight of 7.19 g, while Treatment C, with 10% Alocasia macrorrhiza leaf meal, reached 7.02 g. The lowest average body weight was recorded in Treatment B, which contained 10% papaya leaf meal, at 6.31 g. These findings indicate that while the control diet (without leaf meal) produced the highest growth, the combination of the three leaf types in Treatment E showed a comparable growth trend, suggesting potential synergistic effects of mixed plant ingredients on fish performance. Table 1. Average daily growth rate of red tilapia (Oreochromis niloticus) fingerlings Treatment Mean daily growth rate (%, ±SD) A (TH) 1,17±0,1464 B (TDP 10%) 1,01±0,1706 C (TDS 10%) 1,22±0,1127 D (TDSN 10%) 1,15±0,0709 E (TDP 3.33% + TDS 3.33% + TDSN 3.33% 1,17±0,1350 Note: TH = without leaf meal (control); TDP = Carica papaya leaf meal; TDS = Alocasia macrorrhiza leaf meal; TDSN = Albizia falcataria leaf meal. Based on the analysis of variance, the addition of various plant leaf meals in the formulated diets showed no significant effect on the daily growth rate of red tilapia (Oreochromis niloticus) fingerlings during the 40-day rearing period. The mean daily growth rate of red tilapia ranged between 1.01% and 1.22% (Table 4). As shown in Table 4, the highest daily growth rate was observed in Treatment Cwith a value of 1.22%. This result indicates that the diet formulated with Alocasia macrorrhiza leaf meal was well digested by the fish and effectively supported growth. These findings are consistent with the study by Komariah (2002), who reported that the inclusion of Alocasia macrorrhiza leaf meal at 10% in gourami feed produced the highest growth rate (4.02%) compared to fish fed diets without Alocasia macrorrhiza leaf meal (3.75%). The proximate composition analysis revealed that diets in Treatments C, D, and E contained the lowest crude fiber level (4.38%) among all treatments. The control diet (Treatment A) had a crude fiber content of 4.74%, while Treatment B exhibited the highest crude fiber level (5.33%), which likely contributed to its lowest growth rate (1.01%). Fish growth is generally associated with the protein and fiber content of the diet. In this study, crude fiber levels ranged from 4.38% to 5.33%. According to Haetami et al. (2006), the tolerance limit for crude fiber in tilapia fingerlings is approximately 4%. Excessive fiber levels can reduce nutrient digestibility. Wahju (1997) stated that high fiber Page No. 34 Andriani, Y., & Aisyah, A. (2025). Effect of Various Leaf Meal Supplementation as a Source of Plant-Based Protein on the Growth of Red Tilapia (Oreochromis niloticus). GPH-International Journal of Agriculture and Research, 8(10), 27-43. https://doi.org/10.5281/zenodo.17587549 Volume 8 Issue No 10 (2025) Access: https://gphjournal.org/index.php/ar content in fish feed increases fecal output, as undigested fiber binds digestible nutrients and carries them out of the body. Similarly, Mudjiman (2002) noted that digestibility is influenced by feed composition, particularly protein and crude fiber levels. Crude fiber consists of organic components resistant to strong acid and alkali hydrolysis, such as cellulose, hemicellulose, lignin, chitin, and algin. According to Halver (1989), fish have a limited ability to digest crude fiber because they lack gut microorganisms capable of producing the enzymes amylase and cellulase. Nonetheless, moderate fiber content is beneficial for intestinal function and metabolic waste excretion. However, when fiber levels are too high, digestibility decreases, as the fish’s capacity to degrade fiber depends on the cellulolytic activity of intestinal microflora (Bureau, 1999). The proximate composition analysis (Table 2) also indicated that the highest crude protein content was found in papaya leaf meal (21.88%), although it did not result in superior growth compared with other leaf meals. This is likely due to the higher crude fiber content (12.16%) in papaya leaf meal compared to Alocasia macrorrhiza (6.21%) and Albizia falcataria (7.10%) leaf meals, which could have reduced feed digestibility. Protein is one of the most essential nutrients for fish, serving as a structural component for tissue formation and repair, enzyme and hormone synthesis, antibody production, plasma protein formation, and as a secondary energy source. The protein content of the experimental diets (≈27%) was within the optimal range for tilapia growth. This finding aligns with Sahwan (2003), who reported that the protein requirement for tilapia fingerlings ranges between 25–30%. According to Affandi and Tang (2002), ingested feed undergoes digestion, absorption, transport, and metabolism. Undigested portions are excreted as feces, whereas absorbed nutrients are transported to target organs. Part of the absorbed nutrients is catabolized to produce energy, while the remainder contributes to cell formation, tissue repair, and other vital physiological processes. Furthermore, Handajani and Widodo (2010) explained that protein digestion in fish is mediated by digestive enzymes secreted by the pancreas, intestinal wall, and stomach. Proteinases such as pepsin play a key role in protein hydrolysis, where dietary proteins are denatured by hydrochloric acid (HCl) in the stomach and subsequently hydrolyzed into simpler peptides for absorption. 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