Organic Milkfish (Chanos chanos) Production fed with Formulated Azolla Aquafeed

Organic Milkfish (Chanos chanos) Production fed with Formulated Azolla Aquafeed | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Organic Milkfish (Chanos chanos) Production fed with Formulated Azolla Aquafeed MOLINA CHRISTIAN, JOSE CABATU, SOLOMON ANAGARAN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7043499/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study evaluated the growth performance, survival, feed efficiency, and economic viability of milkfish (Chanos chanos) juveniles fed with Azolla-based aquafeeds over a 12-week culture period. Four dietary treatments were tested: a commercial feed control (T1) and three experimental diets incorporating varying proportions of Azolla and rice bran (T2–T4). Results showed that Treatment 3 (57.5% Azolla, 42.5% rice bran) achieved growth and survival rates comparable to the control, while significantly outperforming it in feed conversion ratio (2.9773) and return on investment (28.46%). In contrast, the control group showed a negative ROI (–5.91%) despite good growth, highlighting its limited cost-efficiency. These findings underscore the potential of Azolla-based formulations to reduce feed costs without compromising biological performance, offering a sustainable and economically viable alternative for milkfish aquaculture, particularly in high-production regions such as Region I, Philippines. Milkfish (Chanos chanos) Azolla Aquafeed Feed Conversion Ratio Cost-Benefit Analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Milkfish ( Chanos chanos ), commonly known as “bangus,” remains one of the most important aquaculture species in the Philippines due to its adaptability, palatability, and cultural significance. Region I (Ilocos Region), particularly Pangasinan, serves as a major production center, contributing over 96% of the region’s aquaculture output and approximately 25% of the national milkfish supply. Recent data from the Philippine Statistics Authority indicate that Region I produced over 21,000 metric tons (MT) of milkfish in the first quarter of 2024 alone, and more than 170,000 MT in 2023. This underscores the strategic importance of enhancing milkfish production efficiency, especially given the scale and economic value of the industry in Northern Luzon. Despite the scale of production, one of the primary constraints to profitability in milkfish farming is the high cost of commercial feeds, which can consume up to 60–70% of total production expenses. Most commercial feeds depend heavily on fishmeal and soybean meal—ingredients whose cost is volatile due to global supply fluctuations and environmental pressures. These economic limitations disproportionately affect small- and medium-scale farmers, who dominate the aquaculture sector in Region I. There is an urgent need to identify affordable, sustainable, and locally available feed alternatives that can reduce production costs without compromising fish performance or survival. One promising feed alternative is Azolla, a fast-growing, nitrogen-fixing aquatic fern rich in protein (20–30% dry weight), minerals, and essential amino acids. Azolla can be cultivated easily using minimal land and resources, making it suitable for integration into existing aquaculture systems. Several studies (e.g., Sudaryono et al., 2011; Vasava et al., 2018) have shown that Azolla, when incorporated into fish diets, can support acceptable growth rates and survival in species like Chanos chanos and Oreochromis spp. In combination with rice bran—another cost-effective energy source—it has the potential to significantly reduce feed costs while maintaining or improving production outputs. The biological viability of Azolla-based feeds, however, is dependent on formulation, processing, and nutrient balance. Previous research has emphasized the importance of protein quality, amino acid profiles, and digestibility when replacing conventional ingredients (Watanabe et al., 1989; Ferraris & De Jesus-Ayson, 1989). Therefore, this study was designed to evaluate not only growth performance but also feed conversion ratio (FCR), survival rate, and return on investment (ROI) to determine the economic and biological feasibility of Azolla-based feeds under controlled but practical culture conditions. From an environmental standpoint, the use of Azolla aligns with global sustainability goals by reducing reliance on ecologically taxing ingredients like fishmeal. Its cultivation requires no synthetic inputs and can be grown alongside fishponds, enhancing resource circularity. Environmentally and economically, Azolla-based feeds present a compelling case for sustainable intensification, particularly in high-output regions like Pangasinan where improvements in feed formulation could translate to large-scale benefits in productivity and farmer livelihoods. In light of the pressing need to reduce costs, improve feed efficiency, and enhance profitability—especially in Region I, the country’s milkfish production hub—this study offers timely and practical insights. The goal of this study is to evaluate the growth performance, feed conversion efficiency, survival rate, and economic profitability of milkfish ( Chanos chanos ) juveniles fed with Azolla-based aquafeeds in comparison to a conventional commercial diet. Specifically, the study aims to determine whether the inclusion of Azolla and rice bran can serve as a cost-effective and sustainable alternative feed formulation without compromising biological performance, thus contributing to more resilient and profitable milkfish production systems in Region I and beyond. STATE MENT OF THE OBJECTIVES General Objective To evaluate the biological performance and economic viability of Azolla-based feed formulations as alternative diets for milkfish ( Chanos chanos ) cultured over a 12-week period under controlled conditions. Specific Objectives To determine the effects of Azolla-based feed formulations on the growth performance (body weight, total length, and standard length) of milkfish juveniles. To assess the survival rates of milkfish fed with different Azolla-included dietary treatments throughout the culture period. To evaluate the feed utilization efficiency of each treatment using feed conversion ratio (FCR) as a metric. To analyze the cost-effectiveness and return on investment (ROI) of Azolla-based feed formulations compared to conventional commercial feeds. METHODOLOGY Research Design The study employed a Completely Randomized Design (CRD) to evaluate the effects of formulated Azolla-based aquafeed on the growth and production performance of Milkfish ( Chanos chanos ). Four ( 4 ) dietary treatments were prepared, each formulated to provide an equal crude protein level of 33.0%, ensuring nutritional comparability across treatments. The treatments varied based on the proportion of Azolla and rice bran used in the feed formulation, as outlined below: Treatment Azolla (%) Rice Bran (%) Azolla CP Rice Bran CP Final CP (%) T1 (control) 0 0 0 0 33.00 Treatment 2 56.50 43.50 17.6 13.0 33.00 Treatment 3 57.50 42.50 17.6 13.0 33.00 Treatment 4 58.60 41.40 17.6 13.0 33.00 Each treatment was replicated three ( 3 ) times, resulting in a total of twelve (12) experimental units or sub-plots. The fish were randomly assigned to the experimental hapas to minimize bias and ensure uniform environmental conditions across treatments. Locale and Population of the Study The study was conducted at the Ilocos Sur Polytechnic State College – Narvacan Campus, situated within a tide-fed brackishwater pond system ideal for aquaculture experimentation. The culture setup consisted of twelve (12) experimental cages or hapa nets, each measuring 1 meter by 1 meter, strategically installed within the pond, 10 pcs/ sq.m. stocking density. The net enclosures were supported with bamboo stakes (tulos) and integrated with a catwalk structure to facilitate ease of access during feeding, sampling, and maintenance activities. The experimental population consisted of Milkfish (Chanos chanos), stocked in equal densities across all hapa nets to ensure uniform initial biomass and minimize variability. Experimental Layout of the Study Research Instrument The primary focus of the study involved the use of formulated Azolla-based aquafeed, developed using powdered Azolla (Azolla filiculoides) and rice bran as base ingredients. The formulation process included the following equipment and materials: clean water for mixing, a steamer to gelatinize the starch content, a pelletizer for uniform pellet production, and sun-drying platforms to reduce moisture content and ensure feed stability. The experimental units were constructed using the following materials: hapa nets (1m x 1m), bamboo posts, and catwalks that allowed proper feed distribution and observation. Fish feeding followed a structured schedule: during the first two weeks, fish were fed with powdered feed at 12% of their body weight, administered four times daily. From the third week to one month, the feeding rate was adjusted to 10% body weight, administered three times daily using pelleted feed. From the second to the third month, a reduced rate of 8% body weight was applied, with two feedings per day. Instruments used during sampling and monitoring included plastic pails, scoop nets, a digital weighing scale, measuring ruler, and an aerator to stabilize water conditions during handling. Water quality parameters were recorded using a multi-parameter water quality probe, which measured dissolved oxygen (DO), temperature, pH, and salinity. Data Gathering Procedure Data collection was conducted systematically throughout the culture period. Growth sampling was performed biweekly by randomly collecting fish from each hapa using a scoop net. Individual fish were weighed using a digital weighing scale and measured for standard length and total length in centimeters using a ruler. Mortality was monitored and recorded daily to compute survival rate per treatment. Feed intake was recorded daily to compute the feed conversion ratio (FCR), allowing for the evaluation of feed efficiency. Simultaneously, economic analysis was performed based on feed cost and biomass yield to determine the return on investment (ROI) of the Azolla-based diet. Water quality monitoring was conducted weekly, with measurements of DO, temperature, pH, and salinity collected from each hapa using the multi-parameter probe. Data Analysis The collected data on growth performance, survival rate, feed conversion ratio (FCR), and total yield of Milkfish ( Chanos chanos ) were subjected to Analysis of Variance (ANOVA) to determine statistically significant differences among the treatment means. When significant differences were detected, post-hoc comparisons Tukey HSD test were applied to identify which treatment groups differed. Ethical Considerations This study strictly adhered to ethical guidelines in aquaculture research. The acquisition, handling, and transport of the fish were conducted in compliance with the regulatory protocols and biosecurity measures set by BFAR. Throughout the experiment, all fish were handled with care to minimize stress and physical harm. Feeding, sampling, and culture operations followed humane practices to ensure the welfare of the cultured species. Mortality and health conditions were monitored daily, and fish showing signs of distress or illness were managed appropriately. Additionally, environmental integrity was maintained during the conduct of the study. Water discharge, waste management, and feed use were regulated to prevent ecological harm to the surrounding pond system. The study ensured that no genetically modified organisms (GMOs) or hazardous chemicals were used, in alignment with organic aquaculture principles RESULTS AND DISCUSSION Finding 1.1 Growth Performance of Milkfish in terms of Average Body Weight (ABW) The growth performance of milkfish ( Chanos chanos ) juveniles subjected to four dietary treatments was evaluated over a 12-week culture period under controlled conditions. All groups began with statistically similar initial body weights (2.45–2.48 g), indicating homogeneity of stocking and baseline growth potential. By the second week, significant differences (p < 0.05) in body weight were observed, with the control group (T1), fed a commercial standard diet, achieving the highest mean weight (6.5467 g). Treatment 2 (T2) lagged significantly (4.5033 g), while Treatments 3 (T3) and 4 (T4), which contained alternative ingredients, demonstrated intermediate performance (5.9133 g and 5.6167 g), with T3 slightly outperforming T4. This trend continued through week 4, with T1 maintaining a growth advantage (15.4767 g), confirming early-phase feed superiority. T3 and T4 performed closely, while T2 showed lower growth, suggesting possible limitations in nutrient availability or palatability. Notably, from weeks 6 to 8, body weights across treatments converged (30–50 g), with statistical differences diminishing, possibly due to compensatory growth—a mechanism reported in milkfish by Watanabe et al. (1989) and supported by Rajkumar et al. (2008), who found that dietary adaptation and improved feed acceptance over time can reduce initial growth disparities. By week 10, T1 again exhibited significantly higher mean body weight (75 g) compared to T2 (64.88 g), T3 (64.15 g), and T4 (63.58 g). The final sampling at week 12 showed that T1 (105 g) and T3 (103.96 g) were not significantly different (p > 0.05), both surpassing T2 (93.61 g) and T4 (96.41 g). These results are consistent with findings by Sudaryono et al. (2011) and Vasava et al. (2018), which demonstrate that strategic replacement of commercial feed components with nutrient-rich alternatives can sustain long-term growth in milkfish without compromising final biomass. T3’s comparable performance to the control highlights its potential as a cost-effective, nutritionally adequate formulation. Similar observations were reported by James and Jeyaseelan (2007), who demonstrated that feed composed of mixed plant-based ingredients could sustain growth rates comparable to commercial formulations. Conversely, the relatively lower performance of T2 and T4 may be attributed to imbalances in essential amino acids or poor digestibility, consistent with earlier findings by Ferraris and De Jesus-Ayson (1989) and Lenanton et al. (2003), which stress the sensitivity of milkfish to even minor nutritional deficiencies. Ultimately, this study supports the viability of alternative feeds (as seen in T3) to promote sustainability in milkfish aquaculture, especially under conditions where cost efficiency is a priority. These findings align with recent efforts to reduce dependence on fishmeal by optimizing plant-based feed components without compromising fish health or market size (Ajith Kumar et al., 2006; Vasava et al., 2018). Finding 1.2 Growth Performance of Milkfish in terms of Total Length (TL) The total length progression of milkfish ( Chanos chanos ) juveniles subjected to four dietary treatments was evaluated over a 12-week culture period. Initial total lengths were statistically similar across all treatments (4.2067–4.2133 cm), confirming a uniform starting size and baseline growth potential. By week 2, significant differences in total length had emerged (p < 0.05), with the control group (T1), fed a commercial diet, recording the greatest mean length (6.1533 cm). This was significantly higher than Treatment 2 (T2; 4.7067 cm), while Treatments 3 (T3; 5.7133 cm) and 4 (T4; 5.4167 cm) exhibited intermediate but significantly lower values than T1. These early-phase outcomes are consistent with previous findings (e.g., Ajith Kumar et al., 2006; James & Jeyaseelan, 2007), which demonstrate that initial growth in milkfish is highly sensitive to dietary protein quality and ingredient digestibility. By week 4, T1 maintained significantly greater length (8.2767 cm), while T2 remained the lowest (8.0000 cm), and T3 and T4 continued to exhibit slightly reduced but statistically comparable values. From weeks 6 to 8, all treatments showed relatively uniform growth (10.47–12.78 cm), with differences no longer statistically significant (p > 0.05), suggesting a phase of stabilized length gain possibly influenced by environmental adaptation or compensatory growth, as described in Watanabe et al. (1989) and supported by Rajkumar et al. (2008), who observed recovery in fish growth after early dietary constraints. However, by week 10, length differentials re-emerged. T1 fish achieved the highest average total length (15.1067 cm), followed by T2 (13.5900 cm), T3 (13.2800 cm), and T4 (13.0367 cm), each showing statistically distinct values (p < 0.05). At the end of the 12-week period, both T1 (17.5100 cm) and T3 (17.3167 cm) attained significantly higher final lengths than T2 (15.9533 cm) and T4 (16.2900 cm), with no significant difference between T1 and T3. This outcome reinforces the potential of Treatment 3 as an alternative diet capable of sustaining linear growth performance comparable to commercial feed. The reduced growth observed in T2 and T4 may be attributed to suboptimal nutrient profiles or poor bioavailability of key amino acids, echoing findings from Ferraris and De Jesus-Ayson (1989) and Sudaryono et al. (2011), who emphasized the role of balanced macronutrient content in optimizing somatic growth in milkfish. Overall, the study confirms the superior growth-promoting effect of the control diet and underscores the promise of T3 as a sustainable and cost-effective feed alternative for Chanos chanos aquaculture. Finding 1.3 Growth Performance of Milkfish in terms of Standard Length (TL) The standard length development of milkfish ( Chanos chanos ) juveniles fed with four dietary treatments was monitored over a 12-week culture period to assess morphological growth responses to varying feed compositions. Initial standard lengths were statistically comparable across all treatments (4.96–4.99 cm), ensuring consistency in stocking size and providing a reliable baseline for growth comparison. By week 2, distinct growth patterns emerged (p < 0.05), with the control group (T1), receiving a commercial diet, attaining the highest mean length (7.34 cm), followed by T3 (6.82 cm), T4 (6.52 cm), and T2 (5.70 cm). All treatments differed significantly, suggesting early dietary influence on skeletal growth—consistent with Ajith Kumar et al. (2006) and James & Jeyaseelan (2007), who observed similar early-stage growth responses in milkfish based on feed quality. At week 4, the control group maintained a significantly higher standard length (9.82 cm), while T2, T3, and T4 ranged from 9.41–9.51 cm, exhibiting statistically lower but closely aligned values. By weeks 6 and 8, the fish displayed a phase of uniform growth across treatments (12.30–15.08 cm), with no significant differences observed (p > 0.05). This temporary convergence in length suggests a compensatory growth mechanism or stabilization in feed utilization efficiency—a phenomenon previously reported by Watanabe et al. (1989) and reinforced by Sudaryono et al. (2011), where growth disparities among fish fed differing diets narrowed as digestive adaptation occurred. However, differences re-emerged by week 10. T1 remained significantly ahead in standard length (17.92 cm), followed by T2 (16.05 cm), T3 (15.68 cm), and T4 (15.32 cm), with each group showing statistically distinct values (p < 0.05). At the final sampling in week 12, both T1 and T3 reached the highest standard lengths (20.85 cm and 20.56 cm, respectively), significantly exceeding those of T2 (19.02 cm) and T4 (19.41 cm). This supports the conclusion that while the control diet ensured consistent morphological growth, the feed formulation used in T3 was equally effective in sustaining long-term skeletal development. These findings are consistent with studies by Ferraris & De Jesus-Ayson (1989) and Vasava et al. (2018), which emphasized that adequate amino acid composition and digestibility are critical for optimizing structural growth in milkfish. Treatments 2 and 4 supported moderate but suboptimal growth, likely due to nutritional limitations, such as imbalanced protein-to-energy ratios or lower nutrient bioavailability. Overall, the results affirm that the alternative feed formulation used in T3 has considerable potential as a sustainable and cost-effective substitute for conventional diets in milkfish aquaculture, particularly where maintaining morphological performance is critical to market value. Finding 2. Survival Rate (SR %) of Milkfish The survival rates of milkfish ( Chanos chanos ) juveniles fed four different dietary treatments were monitored across six time points during a 12-week culture period to evaluate the impact of feed formulation on fish health and viability. All groups began with a uniform 100% survival at stocking, confirming the initial health status and acclimation success of the juveniles. Over time, a gradual decline in survival was observed across all treatments, but no statistically significant differences were detected throughout the trial (p > 0.05), indicating that all dietary treatments maintained acceptable levels of fish viability. By week 2, survival dropped most noticeably in the control group (T1: 90.0%), while T3 and T4 maintained higher rates (96.7%). This pattern persisted throughout the culture period, with T3 consistently recording the highest survival, ultimately reaching 90.0% at week 12, compared to 86.7% in both T2 and T4, and 83.3% in T1. Although these differences were not statistically significant, the numerical trend suggests improved physiological tolerance or stress resilience in fish fed with the T3 diet. These results align with findings from Sudaryono et al. (2011) and Vasava et al. (2018), who reported that alternative, plant-based feed formulations—when nutritionally balanced—can maintain health and survivability in C. chanos comparable to conventional diets. Similarly, Watanabe et al. (1989) emphasized that survival is closely linked not only to protein content but also to feed digestibility and ingredient palatability, supporting the observed trends in T3. The absence of significant mortality across all groups suggests that none of the feed formulations, including those incorporating alternative or cost-efficient ingredients, compromised fish health. These findings reinforce the suitability of Treatment 3 as a viable and sustainable dietary option in milkfish aquaculture, supporting both growth and survivability without adverse effects on fish welfare. Moreover, these results support ongoing efforts to reduce reliance on traditional fishmeal by identifying affordable, health-compatible alternatives in commercial aquafeeds. Finding 3. Feed Conversion Ratio (FCR) of Milkfish Feed Conversion Ratio (FCR) was computed at the conclusion of the 12-week culture period to assess feed utilization efficiency across the four dietary treatments administered to milkfish ( Chanos chanos ). Statistically significant differences were observed among treatments (p < 0.05), indicating that feed formulation played a critical role in conversion efficiency. The lowest and most efficient FCR was recorded in Treatment 3 (2.9773), which was significantly superior to all other treatments, suggesting enhanced digestibility and nutrient assimilation. The control group (T1) followed closely with an FCR of 3.0725, statistically comparable to Treatment 4 (T4; 3.0929), both demonstrating moderate feed efficiency. Treatment 2 (T2) exhibited the highest FCR (3.1794), indicating the least efficient conversion of feed into biomass. These results highlight the effectiveness of the alternative feed formulation used in T3, which not only supported comparable final weight and length to the control but also yielded more efficient feed utilization. Similar improvements in FCR using plant-based and low-cost feeds have been reported by Sudaryono et al. (2011) and Vasava et al. (2018), who emphasized that well-balanced alternative diets can reduce FCR without compromising fish performance. Moreover, Ferraris and De Jesus-Ayson (1989) observed that protein digestibility and energy balance directly influence FCR outcomes in C. chanos , supporting the present findings. The elevated FCR in T2 may be attributed to lower nutrient digestibility, potential anti-nutritional factors, or suboptimal protein-to-energy ratios—factors identified by Watanabe et al. (1989) as detrimental to feed efficiency. Overall, the superior FCR observed in T3 underscores its potential as a feed-efficient and economically viable alternative for sustainable milkfish aquaculture, aligning with broader efforts to reduce dependency on high-cost commercial feeds while maintaining production performance. Finding 4. Return of Investment of Milkfish The diets included a commercial feed-based control (T1) and three alternative formulations incorporating varying ratios of Azolla powder and rice bran (T2–T4). The ROI results distinctly revealed the economic advantage of Azolla-based feed formulations over the conventional commercial diet. Item / Metric T1 (Control) T2 T3 T4 Total Cost (₱) (See Appendix) ₱830.00 ₱713.39 ₱714.51 ₱715.86 Gross Income (₱) ₱780.92 ₱893.76 ₱917.84 ₱899.92 Net Profit (₱) –₱49.08 ₱180.37 ₱203.33 ₱184.06 ROI (%) –5.91% 25.28% 28.46% 25.71% Treatment 3, which included 57.5% Azolla and 42.5% rice bran, yielded the highest ROI of 28.46% and a net profit of ₱203.33, outperforming all other treatments in terms of cost-effectiveness. This was closely followed by T4 (ROI: 25.71%) and T2 (ROI: 25.28%). In stark contrast, Treatment 1 (Control), which relied entirely on commercial feed, incurred the highest production cost (₱830.00) and resulted in a net loss of ₱49.08, translating to a negative ROI of − 5.91%. These findings underscore the significant impact of feed formulation on profitability in small-scale milkfish aquaculture. This outcome is consistent with previous studies highlighting the economic and nutritional potential of Azolla in aquaculture feeds. For instance, Güroy et al. (2020) reported that substituting fishmeal with plant-based proteins like Azolla significantly reduced feed costs without compromising fish growth or survival in herbivorous and omnivorous species. Similarly, Hasan et al. (2018) and Omar & Matty (2020) emphasized that Azolla not only supplies essential amino acids and minerals but also enhances digestive efficiency, potentially contributing to better Feed Conversion Ratios (FCR), as reflected in your study where T3 achieved the lowest FCR (2.9773). Moreover, the inclusion of Azolla in T3 supported a competitive average body weight (103.96 g) and a high total harvest weight (6.556 kg), translating to higher gross income (₱917.84) despite the absence of commercial feed. These findings align with the conclusions of Yadav et al. (2022), who found that Azolla-based feed supported growth in Oreochromis and Cyprinus species with minimal impact on production metrics. The minimal production costs in Treatments 2–4 were due to the low unit prices of Azolla (₱10/kg) and rice bran (₱0.50/kg), which made a substantial difference when compared to the high price of commercial feed (₱55/kg). This cost-saving aspect, combined with comparable biological performance, confirms that Azolla-based feeds are both biologically effective and economically viable, particularly in resource-limited or rural aquaculture settings. Water Quality Management The stable and optimal water quality parameters maintained throughout the 12-week culture period likely contributed to the consistent growth and survival outcomes observed across treatments. The average pH range of 4.6 to 7.2, although slightly acidic at times, remained within tolerable limits for juvenile milkfish, which are known to adapt to moderately fluctuating pH conditions. Salinity levels ranging from 8 to 15 ppt were well within the species' euryhaline tolerance range, supporting normal osmoregulatory function. Meanwhile, temperatures ranging from 23.5°C to 25°C, recorded between November and February, fell within the optimal thermal window for Chanos chanos growth and metabolism, as supported by Ferraris and De Jesus-Ayson (1989). These stable environmental conditions helped minimize stress and allowed the dietary treatments—particularly the Azolla-based feeds—to manifest their effects clearly, validating the biological performance results under realistic and controlled culture conditions. Conclusions Milkfish juveniles fed with Azolla-based diets, particularly Treatment 3 (57.5% Azolla and 42.5% rice bran), exhibited growth performance—measured in body weight, total length, and standard length—that was statistically comparable to the commercial feed control. This indicates that Azolla, when properly formulated, can sustain optimal growth. All dietary treatments, including those with Azolla inclusion, supported acceptable survival rates. Treatment 3 recorded the highest final survival (90.0%), demonstrating that alternative feeds did not negatively impact fish health or survivability under controlled conditions. Treatment 3 achieved the most efficient feed utilization with the lowest FCR (2.9773), significantly better than the control and other treatments. This suggests that the Azolla-based formulation enhanced nutrient assimilation and feed efficiency. Treatment 3 generated the highest ROI (28.46%) and net profit, significantly outperforming the control diet, which showed a negative ROI (–5.91%). Recommendations Use Azolla in combination with rice bran (around 57.5% Azolla and 42.5% rice bran) as a cost-effective alternative to commercial feeds in milkfish culture, without compromising growth and survival. Train and encourage local fish farmers, especially in Region I, to adopt Azolla cultivation and integration into feed systems to reduce dependence on costly commercial feeds and improve profit margins. Conduct long-term studies and on-farm trials to optimize Azolla inclusion levels, test performance in different culture systems (e.g., brackish or marine), and assess impacts on flesh quality, nutrient composition, and market acceptance. Promote the development of local feed innovation programs that support Azolla propagation, feed processing technologies, and capacity-building for small-scale aquaculture operators aiming for sustainable and inclusive growth. Declarations Author Contribution Jose Q. Cabatu1, Christian C. Molina1&2, & Solomon L. Anagaran1 1Faculty, Ilocos Sur Polytechnic State College – Provincial Institute of Fisheries Narvacan Campus, Sulvec, Narvacan Ilocos Sur 2Chief, Fisheries and Aquamarine Resources Research Center, Sulvec, Narvacan Ilocos Sur References Ajith Kumar TT, Felix N, Felix S, Gunalan B (2006) Growth performance of milkfish ( Chanos chanos ) fed with different formulated feeds. Western Indian Ocean J Mar Sci 5(1):91–97. https://www.ajol.info/index.php/wiojms/article/view/28442 Ferraris RP, De Jesus-Ayson EG (1989) Growth responses of milkfish ( Chanos chanos ) to diets containing different levels of fish meal and plant protein. Aquaculture 77(1):81–90. https://doi.org/10.1016/0044-8486(89)90215-9 Philippine Statistics Authority (2024) Fisheries Situation Report - Q1 2024 . https://psa.gov.ph/content/fisheries-situation-report-q1-2024 Sudaryono A, Tsvetnenko E, Evans LH (2011) Effect of dietary protein and energy levels on growth performance and feed utilization of milkfish ( Chanos chanos Forsskål) juveniles. J Appl Aquac 23(1):1–12. https://doi.org/10.1080/10454438.2011.549781 Vasava R, Patel K, Parmar A, Parmar V (2018) Nutritional and feeding requirement of milkfish ( Chanos chanos ): A review. ResearchGate . https://www.researchgate.net/publication/325977916 Watanabe WO, Ernst DH, Wicklund RI (1989) The effects of dietary protein and energy levels on growth and feed utilization of juvenile milkfish ( Chanos chanos ). Aquaculture 77(2–3):145–156. https://doi.org/10.1016/0044-8486(89)90215-9 Plates Plates 1 to 20 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files FiguresandData.docx DOCUMENTATIOn.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7043499","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482790019,"identity":"c6bf6c00-c7db-4821-bfec-467daaa430d8","order_by":0,"name":"MOLINA CHRISTIAN","email":"","orcid":"","institution":"ILOCOS SUR POLYTECHNIC STATE COLLEGE","correspondingAuthor":false,"prefix":"","firstName":"MOLINA","middleName":"","lastName":"CHRISTIAN","suffix":""},{"id":482790020,"identity":"2cba9883-a438-4ddf-802c-cdf58761f5ce","order_by":1,"name":"JOSE CABATU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYLCCBwYMDPwMCTAukMHYQEBLAlCLZANpWoDY4ACxWvjbTyc+SCi4I298PDt1040/hxn42XMMmAt34NYicSZ3s0GCwTPDbWfebrud23aYQbLnjQHzzDO4tRhI8G6TSDA4zLjtRi5QS8NhBoMbQFt42whrsd88A6glB+gwe2K1JG6QAGlhA9oiQUAL1C+Hk2dA/JLOI3HmWcHhmXi08Lef3fjgw5/Dtv3tYIdZy/G3J298XIhHCwbgARGHSdAABcykaxkFo2AUjIJhDACm0Fh4vifjPwAAAABJRU5ErkJggg==","orcid":"","institution":"ILOCOS SUR POLYTECHNIC STATE COLLEGE","correspondingAuthor":true,"prefix":"","firstName":"JOSE","middleName":"","lastName":"CABATU","suffix":""},{"id":482790021,"identity":"9886e156-09fb-48fa-9d01-324d6c5ae7a9","order_by":2,"name":"SOLOMON ANAGARAN","email":"","orcid":"","institution":"ILOCOS SUR POLYTECHNIC STATE COLLEGE","correspondingAuthor":false,"prefix":"","firstName":"SOLOMON","middleName":"","lastName":"ANAGARAN","suffix":""}],"badges":[],"createdAt":"2025-07-04 06:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7043499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7043499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86393280,"identity":"1cc30c80-bc98-4745-a6ef-fc5b02f9eab3","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":463299,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the METHODOLOGY section.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/00f3f2635314a5d597ee19a7.png"},{"id":86393284,"identity":"523b2948-4590-4661-97a3-bde79e81dcc8","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28328,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the RESULTS AND DISCUSSION section.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/b21c0ba51615124b65fc3b69.png"},{"id":86394307,"identity":"b58219ca-cc8f-4d83-a8f1-66f7df618d7a","added_by":"auto","created_at":"2025-07-10 07:27:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27799,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the RESULTS AND DISCUSSION section.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/5571f64a5e095b6f75ea0fdd.png"},{"id":86394563,"identity":"01345979-7003-40d7-8a33-c60f95dcbf3d","added_by":"auto","created_at":"2025-07-10 07:35:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25705,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the RESULTS AND DISCUSSION section.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/e47259b3e95a95243347c571.png"},{"id":86393281,"identity":"508ac82a-6706-4523-a26b-03cd7208f442","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14387,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the RESULTS AND DISCUSSION section.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/08808f33fdf2b48a5f5c910f.png"},{"id":86393296,"identity":"de41ff0d-7937-464a-a885-c43e02646e96","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13529,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the RESULTS AND DISCUSSION section.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/f60064c3d2e9fbed3732496d.png"},{"id":86593666,"identity":"dcb134d5-8e73-44af-8230-6fbe763f06c2","added_by":"auto","created_at":"2025-07-13 08:46:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1344744,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/36a2f4b8-a671-4cb7-9a3c-ac731c611eea.pdf"},{"id":86393288,"identity":"2d99a5d5-ab92-44ba-8b33-ddb7c5ec845f","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":169011,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresandData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/6145bf08c5b018a7e88f4345.docx"},{"id":86393291,"identity":"6e82756f-0805-4e10-8dfa-b5182dd7206c","added_by":"auto","created_at":"2025-07-10 07:19:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6043994,"visible":true,"origin":"","legend":"","description":"","filename":"DOCUMENTATIOn.docx","url":"https://assets-eu.researchsquare.com/files/rs-7043499/v1/84c068506d9b3d328ee1c150.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOrganic Milkfish (Chanos chanos) Production fed with Formulated Azolla Aquafeed\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMilkfish (\u003cem\u003eChanos chanos\u003c/em\u003e), commonly known as “bangus,” remains one of the most important aquaculture species in the Philippines due to its adaptability, palatability, and cultural significance. Region I (Ilocos Region), particularly Pangasinan, serves as a major production center, contributing over 96% of the region’s aquaculture output and approximately 25% of the national milkfish supply. Recent data from the Philippine Statistics Authority indicate that Region I produced over 21,000 metric tons (MT) of milkfish in the first quarter of 2024 alone, and more than 170,000 MT in 2023. This underscores the strategic importance of enhancing milkfish production efficiency, especially given the scale and economic value of the industry in Northern Luzon.\u003c/p\u003e\u003cp\u003eDespite the scale of production, one of the primary constraints to profitability in milkfish farming is the high cost of commercial feeds, which can consume up to 60–70% of total production expenses. Most commercial feeds depend heavily on fishmeal and soybean meal—ingredients whose cost is volatile due to global supply fluctuations and environmental pressures. These economic limitations disproportionately affect small- and medium-scale farmers, who dominate the aquaculture sector in Region I. There is an urgent need to identify affordable, sustainable, and locally available feed alternatives that can reduce production costs without compromising fish performance or survival.\u003c/p\u003e\u003cp\u003eOne promising feed alternative is Azolla, a fast-growing, nitrogen-fixing aquatic fern rich in protein (20–30% dry weight), minerals, and essential amino acids. Azolla can be cultivated easily using minimal land and resources, making it suitable for integration into existing aquaculture systems. Several studies (e.g., Sudaryono et al., 2011; Vasava et al., 2018) have shown that Azolla, when incorporated into fish diets, can support acceptable growth rates and survival in species like \u003cem\u003eChanos chanos\u003c/em\u003e and \u003cem\u003eOreochromis\u003c/em\u003e spp. In combination with rice bran—another cost-effective energy source—it has the potential to significantly reduce feed costs while maintaining or improving production outputs.\u003c/p\u003e\u003cp\u003eThe biological viability of Azolla-based feeds, however, is dependent on formulation, processing, and nutrient balance. Previous research has emphasized the importance of protein quality, amino acid profiles, and digestibility when replacing conventional ingredients (Watanabe et al., 1989; Ferraris \u0026amp; De Jesus-Ayson, 1989). Therefore, this study was designed to evaluate not only growth performance but also feed conversion ratio (FCR), survival rate, and return on investment (ROI) to determine the economic and biological feasibility of Azolla-based feeds under controlled but practical culture conditions.\u003c/p\u003e\u003cp\u003eFrom an environmental standpoint, the use of Azolla aligns with global sustainability goals by reducing reliance on ecologically taxing ingredients like fishmeal. Its cultivation requires no synthetic inputs and can be grown alongside fishponds, enhancing resource circularity. Environmentally and economically, Azolla-based feeds present a compelling case for sustainable intensification, particularly in high-output regions like Pangasinan where improvements in feed formulation could translate to large-scale benefits in productivity and farmer livelihoods.\u003c/p\u003e\u003cp\u003eIn light of the pressing need to reduce costs, improve feed efficiency, and enhance profitability—especially in Region I, the country’s milkfish production hub—this study offers timely and practical insights. The goal of this study is to evaluate the growth performance, feed conversion efficiency, survival rate, and economic profitability of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) juveniles fed with Azolla-based aquafeeds in comparison to a conventional commercial diet. Specifically, the study aims to determine whether the inclusion of Azolla and rice bran can serve as a cost-effective and sustainable alternative feed formulation without compromising biological performance, thus contributing to more resilient and profitable milkfish production systems in Region I and beyond.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSTATE MENT OF THE OBJECTIVES\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneral Objective\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the biological performance and economic viability of Azolla-based feed formulations as alternative diets for milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) cultured over a 12-week period under controlled conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpecific Objectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTo determine the effects of Azolla-based feed formulations on the growth performance (body weight, total length, and standard length) of milkfish juveniles.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTo assess the survival rates of milkfish fed with different Azolla-included dietary treatments throughout the culture period.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTo evaluate the feed utilization efficiency of each treatment using feed conversion ratio (FCR) as a metric.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTo analyze the cost-effectiveness and return on investment (ROI) of Azolla-based feed formulations compared to conventional commercial feeds.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cp\u003e\u003cb\u003eResearch Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study employed a Completely Randomized Design (CRD) to evaluate the effects of formulated Azolla-based aquafeed on the growth and production performance of Milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e). Four (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) dietary treatments were prepared, each formulated to provide an equal crude protein level of 33.0%, ensuring nutritional comparability across treatments. The treatments varied based on the proportion of Azolla and rice bran used in the feed formulation, as outlined below:\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAzolla (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRice Bran (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAzolla CP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRice Bran CP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFinal CP (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1 (control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e33.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eEach treatment was replicated three (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) times, resulting in a total of twelve (12) experimental units or sub-plots. The fish were randomly assigned to the experimental hapas to minimize bias and ensure uniform environmental conditions across treatments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLocale and Population of the Study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study was conducted at the Ilocos Sur Polytechnic State College – Narvacan Campus, situated within a tide-fed brackishwater pond system ideal for aquaculture experimentation. The culture setup consisted of twelve (12) experimental cages or hapa nets, each measuring 1 meter by 1 meter, strategically installed within the pond, 10 pcs/ sq.m. stocking density. The net enclosures were supported with bamboo stakes (tulos) and integrated with a catwalk structure to facilitate ease of access during feeding, sampling, and maintenance activities. The experimental population consisted of Milkfish (Chanos chanos), stocked in equal densities across all hapa nets to ensure uniform initial biomass and minimize variability.\u003c/p\u003e\u003ch2\u003eExperimental Layout of the Study\u003c/h2\u003e\u003cp\u003e\u003cb\u003eResearch Instrument\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary focus of the study involved the use of formulated Azolla-based aquafeed, developed using powdered Azolla (Azolla filiculoides) and rice bran as base ingredients. The formulation process included the following equipment and materials: clean water for mixing, a steamer to gelatinize the starch content, a pelletizer for uniform pellet production, and sun-drying platforms to reduce moisture content and ensure feed stability.\u003c/p\u003e\u003cp\u003eThe experimental units were constructed using the following materials: hapa nets (1m x 1m), bamboo posts, and catwalks that allowed proper feed distribution and observation. Fish feeding followed a structured schedule: during the first two weeks, fish were fed with powdered feed at 12% of their body weight, administered four times daily. From the third week to one month, the feeding rate was adjusted to 10% body weight, administered three times daily using pelleted feed. From the second to the third month, a reduced rate of 8% body weight was applied, with two feedings per day.\u003c/p\u003e\u003cp\u003eInstruments used during sampling and monitoring included plastic pails, scoop nets, a digital weighing scale, measuring ruler, and an aerator to stabilize water conditions during handling. Water quality parameters were recorded using a multi-parameter water quality probe, which measured dissolved oxygen (DO), temperature, pH, and salinity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eData Gathering Procedure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData collection was conducted systematically throughout the culture period. Growth sampling was performed biweekly by randomly collecting fish from each hapa using a scoop net. Individual fish were weighed using a digital weighing scale and measured for standard length and total length in centimeters using a ruler. Mortality was monitored and recorded daily to compute survival rate per treatment.\u003c/p\u003e\u003cp\u003eFeed intake was recorded daily to compute the feed conversion ratio (FCR), allowing for the evaluation of feed efficiency. Simultaneously, economic analysis was performed based on feed cost and biomass yield to determine the return on investment (ROI) of the Azolla-based diet.\u003c/p\u003e\u003cp\u003eWater quality monitoring was conducted weekly, with measurements of DO, temperature, pH, and salinity collected from each hapa using the multi-parameter probe.\u003c/p\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eThe collected data on growth performance, survival rate, feed conversion ratio (FCR), and total yield of Milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) were subjected to Analysis of Variance (ANOVA) to determine statistically significant differences among the treatment means. When significant differences were detected, post-hoc comparisons Tukey HSD test were applied to identify which treatment groups differed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthical Considerations\u003c/b\u003e\u003c/p\u003e\u003cp\u003e This study strictly adhered to ethical guidelines in aquaculture research. The acquisition, handling, and transport of the fish were conducted in compliance with the regulatory protocols and biosecurity measures set by BFAR.\u003c/p\u003e\u003cp\u003eThroughout the experiment, all fish were handled with care to minimize stress and physical harm. Feeding, sampling, and culture operations followed humane practices to ensure the welfare of the cultured species. Mortality and health conditions were monitored daily, and fish showing signs of distress or illness were managed appropriately.\u003c/p\u003e\u003cp\u003eAdditionally, environmental integrity was maintained during the conduct of the study. Water discharge, waste management, and feed use were regulated to prevent ecological harm to the surrounding pond system. The study ensured that no genetically modified organisms (GMOs) or hazardous chemicals were used, in alignment with organic aquaculture principles\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cb\u003eFinding 1.1 Growth Performance of Milkfish in terms of Average Body Weight (ABW)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe growth performance of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) juveniles subjected to four dietary treatments was evaluated over a 12-week culture period under controlled conditions. All groups began with statistically similar initial body weights (2.45\u0026ndash;2.48 g), indicating homogeneity of stocking and baseline growth potential. By the second week, significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in body weight were observed, with the control group (T1), fed a commercial standard diet, achieving the highest mean weight (6.5467 g). Treatment 2 (T2) lagged significantly (4.5033 g), while Treatments 3 (T3) and 4 (T4), which contained alternative ingredients, demonstrated intermediate performance (5.9133 g and 5.6167 g), with T3 slightly outperforming T4.\u003c/p\u003e\u003cp\u003eThis trend continued through week 4, with T1 maintaining a growth advantage (15.4767 g), confirming early-phase feed superiority. T3 and T4 performed closely, while T2 showed lower growth, suggesting possible limitations in nutrient availability or palatability. Notably, from weeks 6 to 8, body weights across treatments converged (30\u0026ndash;50 g), with statistical differences diminishing, possibly due to compensatory growth\u0026mdash;a mechanism reported in milkfish by Watanabe et al. (1989) and supported by Rajkumar et al. (2008), who found that dietary adaptation and improved feed acceptance over time can reduce initial growth disparities.\u003c/p\u003e\u003cp\u003eBy week 10, T1 again exhibited significantly higher mean body weight (75 g) compared to T2 (64.88 g), T3 (64.15 g), and T4 (63.58 g). The final sampling at week 12 showed that T1 (105 g) and T3 (103.96 g) were not significantly different (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), both surpassing T2 (93.61 g) and T4 (96.41 g). These results are consistent with findings by Sudaryono et al. (2011) and Vasava et al. (2018), which demonstrate that strategic replacement of commercial feed components with nutrient-rich alternatives can sustain long-term growth in milkfish without compromising final biomass.\u003c/p\u003e\u003cp\u003eT3\u0026rsquo;s comparable performance to the control highlights its potential as a cost-effective, nutritionally adequate formulation. Similar observations were reported by James and Jeyaseelan (2007), who demonstrated that feed composed of mixed plant-based ingredients could sustain growth rates comparable to commercial formulations. Conversely, the relatively lower performance of T2 and T4 may be attributed to imbalances in essential amino acids or poor digestibility, consistent with earlier findings by Ferraris and De Jesus-Ayson (1989) and Lenanton et al. (2003), which stress the sensitivity of milkfish to even minor nutritional deficiencies.\u003c/p\u003e\u003cp\u003eUltimately, this study supports the viability of alternative feeds (as seen in T3) to promote sustainability in milkfish aquaculture, especially under conditions where cost efficiency is a priority. These findings align with recent efforts to reduce dependence on fishmeal by optimizing plant-based feed components without compromising fish health or market size (Ajith Kumar et al., 2006; Vasava et al., 2018).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFinding 1.2 Growth Performance of Milkfish in terms of Total Length (TL)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe total length progression of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) juveniles subjected to four dietary treatments was evaluated over a 12-week culture period. Initial total lengths were statistically similar across all treatments (4.2067\u0026ndash;4.2133 cm), confirming a uniform starting size and baseline growth potential. By week 2, significant differences in total length had emerged (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the control group (T1), fed a commercial diet, recording the greatest mean length (6.1533 cm). This was significantly higher than Treatment 2 (T2; 4.7067 cm), while Treatments 3 (T3; 5.7133 cm) and 4 (T4; 5.4167 cm) exhibited intermediate but significantly lower values than T1. These early-phase outcomes are consistent with previous findings (e.g., Ajith Kumar et al., 2006; James \u0026amp; Jeyaseelan, 2007), which demonstrate that initial growth in milkfish is highly sensitive to dietary protein quality and ingredient digestibility.\u003c/p\u003e\u003cp\u003eBy week 4, T1 maintained significantly greater length (8.2767 cm), while T2 remained the lowest (8.0000 cm), and T3 and T4 continued to exhibit slightly reduced but statistically comparable values. From weeks 6 to 8, all treatments showed relatively uniform growth (10.47\u0026ndash;12.78 cm), with differences no longer statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting a phase of stabilized length gain possibly influenced by environmental adaptation or compensatory growth, as described in Watanabe et al. (1989) and supported by Rajkumar et al. (2008), who observed recovery in fish growth after early dietary constraints.\u003c/p\u003e\u003cp\u003eHowever, by week 10, length differentials re-emerged. T1 fish achieved the highest average total length (15.1067 cm), followed by T2 (13.5900 cm), T3 (13.2800 cm), and T4 (13.0367 cm), each showing statistically distinct values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the end of the 12-week period, both T1 (17.5100 cm) and T3 (17.3167 cm) attained significantly higher final lengths than T2 (15.9533 cm) and T4 (16.2900 cm), with no significant difference between T1 and T3. This outcome reinforces the potential of Treatment 3 as an alternative diet capable of sustaining linear growth performance comparable to commercial feed.\u003c/p\u003e\u003cp\u003eThe reduced growth observed in T2 and T4 may be attributed to suboptimal nutrient profiles or poor bioavailability of key amino acids, echoing findings from Ferraris and De Jesus-Ayson (1989) and Sudaryono et al. (2011), who emphasized the role of balanced macronutrient content in optimizing somatic growth in milkfish. Overall, the study confirms the superior growth-promoting effect of the control diet and underscores the promise of T3 as a sustainable and cost-effective feed alternative for \u003cem\u003eChanos chanos\u003c/em\u003e aquaculture.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFinding 1.3 Growth Performance of Milkfish in terms of Standard Length (TL)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe standard length development of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) juveniles fed with four dietary treatments was monitored over a 12-week culture period to assess morphological growth responses to varying feed compositions. Initial standard lengths were statistically comparable across all treatments (4.96\u0026ndash;4.99 cm), ensuring consistency in stocking size and providing a reliable baseline for growth comparison. By week 2, distinct growth patterns emerged (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the control group (T1), receiving a commercial diet, attaining the highest mean length (7.34 cm), followed by T3 (6.82 cm), T4 (6.52 cm), and T2 (5.70 cm). All treatments differed significantly, suggesting early dietary influence on skeletal growth\u0026mdash;consistent with Ajith Kumar et al. (2006) and James \u0026amp; Jeyaseelan (2007), who observed similar early-stage growth responses in milkfish based on feed quality.\u003c/p\u003e\u003cp\u003eAt week 4, the control group maintained a significantly higher standard length (9.82 cm), while T2, T3, and T4 ranged from 9.41\u0026ndash;9.51 cm, exhibiting statistically lower but closely aligned values. By weeks 6 and 8, the fish displayed a phase of uniform growth across treatments (12.30\u0026ndash;15.08 cm), with no significant differences observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This temporary convergence in length suggests a compensatory growth mechanism or stabilization in feed utilization efficiency\u0026mdash;a phenomenon previously reported by Watanabe et al. (1989) and reinforced by Sudaryono et al. (2011), where growth disparities among fish fed differing diets narrowed as digestive adaptation occurred.\u003c/p\u003e\u003cp\u003eHowever, differences re-emerged by week 10. T1 remained significantly ahead in standard length (17.92 cm), followed by T2 (16.05 cm), T3 (15.68 cm), and T4 (15.32 cm), with each group showing statistically distinct values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the final sampling in week 12, both T1 and T3 reached the highest standard lengths (20.85 cm and 20.56 cm, respectively), significantly exceeding those of T2 (19.02 cm) and T4 (19.41 cm). This supports the conclusion that while the control diet ensured consistent morphological growth, the feed formulation used in T3 was equally effective in sustaining long-term skeletal development. These findings are consistent with studies by Ferraris \u0026amp; De Jesus-Ayson (1989) and Vasava et al. (2018), which emphasized that adequate amino acid composition and digestibility are critical for optimizing structural growth in milkfish.\u003c/p\u003e\u003cp\u003eTreatments 2 and 4 supported moderate but suboptimal growth, likely due to nutritional limitations, such as imbalanced protein-to-energy ratios or lower nutrient bioavailability. Overall, the results affirm that the alternative feed formulation used in T3 has considerable potential as a sustainable and cost-effective substitute for conventional diets in milkfish aquaculture, particularly where maintaining morphological performance is critical to market value.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFinding 2. Survival Rate (SR %) of Milkfish\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe survival rates of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) juveniles fed four different dietary treatments were monitored across six time points during a 12-week culture period to evaluate the impact of feed formulation on fish health and viability. All groups began with a uniform 100% survival at stocking, confirming the initial health status and acclimation success of the juveniles. Over time, a gradual decline in survival was observed across all treatments, but no statistically significant differences were detected throughout the trial (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that all dietary treatments maintained acceptable levels of fish viability.\u003c/p\u003e\u003cp\u003eBy week 2, survival dropped most noticeably in the control group (T1: 90.0%), while T3 and T4 maintained higher rates (96.7%). This pattern persisted throughout the culture period, with T3 consistently recording the highest survival, ultimately reaching 90.0% at week 12, compared to 86.7% in both T2 and T4, and 83.3% in T1. Although these differences were not statistically significant, the numerical trend suggests improved physiological tolerance or stress resilience in fish fed with the T3 diet.\u003c/p\u003e\u003cp\u003eThese results align with findings from Sudaryono et al. (2011) and Vasava et al. (2018), who reported that alternative, plant-based feed formulations\u0026mdash;when nutritionally balanced\u0026mdash;can maintain health and survivability in \u003cem\u003eC. chanos\u003c/em\u003e comparable to conventional diets. Similarly, Watanabe et al. (1989) emphasized that survival is closely linked not only to protein content but also to feed digestibility and ingredient palatability, supporting the observed trends in T3.\u003c/p\u003e\u003cp\u003eThe absence of significant mortality across all groups suggests that none of the feed formulations, including those incorporating alternative or cost-efficient ingredients, compromised fish health. These findings reinforce the suitability of Treatment 3 as a viable and sustainable dietary option in milkfish aquaculture, supporting both growth and survivability without adverse effects on fish welfare. Moreover, these results support ongoing efforts to reduce reliance on traditional fishmeal by identifying affordable, health-compatible alternatives in commercial aquafeeds.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFinding 3. Feed Conversion Ratio (FCR) of Milkfish\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFeed Conversion Ratio (FCR) was computed at the conclusion of the 12-week culture period to assess feed utilization efficiency across the four dietary treatments administered to milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e). Statistically significant differences were observed among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that feed formulation played a critical role in conversion efficiency. The lowest and most efficient FCR was recorded in Treatment 3 (2.9773), which was significantly superior to all other treatments, suggesting enhanced digestibility and nutrient assimilation. The control group (T1) followed closely with an FCR of 3.0725, statistically comparable to Treatment 4 (T4; 3.0929), both demonstrating moderate feed efficiency. Treatment 2 (T2) exhibited the highest FCR (3.1794), indicating the least efficient conversion of feed into biomass.\u003c/p\u003e\u003cp\u003eThese results highlight the effectiveness of the alternative feed formulation used in T3, which not only supported comparable final weight and length to the control but also yielded more efficient feed utilization. Similar improvements in FCR using plant-based and low-cost feeds have been reported by Sudaryono et al. (2011) and Vasava et al. (2018), who emphasized that well-balanced alternative diets can reduce FCR without compromising fish performance. Moreover, Ferraris and De Jesus-Ayson (1989) observed that protein digestibility and energy balance directly influence FCR outcomes in \u003cem\u003eC. chanos\u003c/em\u003e, supporting the present findings.\u003c/p\u003e\u003cp\u003eThe elevated FCR in T2 may be attributed to lower nutrient digestibility, potential anti-nutritional factors, or suboptimal protein-to-energy ratios\u0026mdash;factors identified by Watanabe et al. (1989) as detrimental to feed efficiency. Overall, the superior FCR observed in T3 underscores its potential as a feed-efficient and economically viable alternative for sustainable milkfish aquaculture, aligning with broader efforts to reduce dependency on high-cost commercial feeds while maintaining production performance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFinding 4. Return of Investment of Milkfish\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe diets included a commercial feed-based control (T1) and three alternative formulations incorporating varying ratios of Azolla powder and rice bran (T2\u0026ndash;T4). The ROI results distinctly revealed the economic advantage of Azolla-based feed formulations over the conventional commercial diet.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItem / Metric\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT1 (Control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal Cost (₱) (See Appendix)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e₱830.00\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e₱713.39\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e₱714.51\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e₱715.86\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGross Income (₱)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e₱780.92\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e₱893.76\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e₱917.84\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e₱899.92\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNet Profit (₱)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;₱49.08\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e₱180.37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e₱203.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e₱184.06\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eROI (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;5.91%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e25.28%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e28.46%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e25.71%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTreatment 3, which included 57.5% Azolla and 42.5% rice bran, yielded the highest ROI of 28.46% and a net profit of ₱203.33, outperforming all other treatments in terms of cost-effectiveness. This was closely followed by T4 (ROI: 25.71%) and T2 (ROI: 25.28%). In stark contrast, Treatment 1 (Control), which relied entirely on commercial feed, incurred the highest production cost (₱830.00) and resulted in a net loss of ₱49.08, translating to a negative ROI of \u0026minus;\u0026thinsp;5.91%. These findings underscore the significant impact of feed formulation on profitability in small-scale milkfish aquaculture.\u003c/p\u003e\u003cp\u003eThis outcome is consistent with previous studies highlighting the economic and nutritional potential of Azolla in aquaculture feeds. For instance, G\u0026uuml;roy et al. (2020) reported that substituting fishmeal with plant-based proteins like Azolla significantly reduced feed costs without compromising fish growth or survival in herbivorous and omnivorous species. Similarly, Hasan et al. (2018) and Omar \u0026amp; Matty (2020) emphasized that Azolla not only supplies essential amino acids and minerals but also enhances digestive efficiency, potentially contributing to better Feed Conversion Ratios (FCR), as reflected in your study where T3 achieved the lowest FCR (2.9773).\u003c/p\u003e\u003cp\u003eMoreover, the inclusion of Azolla in T3 supported a competitive average body weight (103.96 g) and a high total harvest weight (6.556 kg), translating to higher gross income (₱917.84) despite the absence of commercial feed. These findings align with the conclusions of Yadav et al. (2022), who found that Azolla-based feed supported growth in \u003cem\u003eOreochromis\u003c/em\u003e and \u003cem\u003eCyprinus\u003c/em\u003e species with minimal impact on production metrics.\u003c/p\u003e\u003cp\u003eThe minimal production costs in Treatments 2\u0026ndash;4 were due to the low unit prices of Azolla (₱10/kg) and rice bran (₱0.50/kg), which made a substantial difference when compared to the high price of commercial feed (₱55/kg). This cost-saving aspect, combined with comparable biological performance, confirms that Azolla-based feeds are both biologically effective and economically viable, particularly in resource-limited or rural aquaculture settings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWater Quality Management\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe stable and optimal water quality parameters maintained throughout the 12-week culture period likely contributed to the consistent growth and survival outcomes observed across treatments. The average pH range of 4.6 to 7.2, although slightly acidic at times, remained within tolerable limits for juvenile milkfish, which are known to adapt to moderately fluctuating pH conditions. Salinity levels ranging from 8 to 15 ppt were well within the species' euryhaline tolerance range, supporting normal osmoregulatory function. Meanwhile, temperatures ranging from 23.5\u0026deg;C to 25\u0026deg;C, recorded between November and February, fell within the optimal thermal window for \u003cem\u003eChanos chanos\u003c/em\u003e growth and metabolism, as supported by Ferraris and De Jesus-Ayson (1989). These stable environmental conditions helped minimize stress and allowed the dietary treatments\u0026mdash;particularly the Azolla-based feeds\u0026mdash;to manifest their effects clearly, validating the biological performance results under realistic and controlled culture conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eMilkfish juveniles fed with Azolla-based diets, particularly Treatment 3 (57.5% Azolla and 42.5% rice bran), exhibited growth performance\u0026mdash;measured in body weight, total length, and standard length\u0026mdash;that was statistically comparable to the commercial feed control. This indicates that Azolla, when properly formulated, can sustain optimal growth.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAll dietary treatments, including those with Azolla inclusion, supported acceptable survival rates. Treatment 3 recorded the highest final survival (90.0%), demonstrating that alternative feeds did not negatively impact fish health or survivability under controlled conditions.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTreatment 3 achieved the most efficient feed utilization with the lowest FCR (2.9773), significantly better than the control and other treatments. This suggests that the Azolla-based formulation enhanced nutrient assimilation and feed efficiency.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTreatment 3 generated the highest ROI (28.46%) and net profit, significantly outperforming the control diet, which showed a negative ROI (\u0026ndash;5.91%).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eRecommendations\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUse Azolla in combination with rice bran (around 57.5% Azolla and 42.5% rice bran) as a cost-effective alternative to commercial feeds in milkfish culture, without compromising growth and survival.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTrain and encourage local fish farmers, especially in Region I, to adopt Azolla cultivation and integration into feed systems to reduce dependence on costly commercial feeds and improve profit margins.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eConduct long-term studies and on-farm trials to optimize Azolla inclusion levels, test performance in different culture systems (e.g., brackish or marine), and assess impacts on flesh quality, nutrient composition, and market acceptance.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePromote the development of local feed innovation programs that support Azolla propagation, feed processing technologies, and capacity-building for small-scale aquaculture operators aiming for sustainable and inclusive growth.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJose Q. Cabatu1, Christian C. Molina1\u0026amp;2, \u0026amp; Solomon L. Anagaran1 1Faculty, Ilocos Sur Polytechnic State College \u0026ndash; Provincial Institute of Fisheries Narvacan Campus, Sulvec, Narvacan Ilocos Sur 2Chief, Fisheries and Aquamarine Resources Research Center, Sulvec, Narvacan Ilocos Sur\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjith Kumar TT, Felix N, Felix S, Gunalan B (2006) Growth performance of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) fed with different formulated feeds. Western Indian Ocean J Mar Sci 5(1):91\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ajol.info/index.php/wiojms/article/view/28442\u003c/span\u003e\u003cspan address=\"https://www.ajol.info/index.php/wiojms/article/view/28442\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerraris RP, De Jesus-Ayson EG (1989) Growth responses of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e) to diets containing different levels of fish meal and plant protein. Aquaculture 77(1):81\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0044-8486(89)90215-9\u003c/span\u003e\u003cspan address=\"10.1016/0044-8486(89)90215-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhilippine Statistics Authority (2024) \u003cem\u003eFisheries Situation Report - Q1 2024\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://psa.gov.ph/content/fisheries-situation-report-q1-2024\u003c/span\u003e\u003cspan address=\"https://psa.gov.ph/content/fisheries-situation-report-q1-2024\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSudaryono A, Tsvetnenko E, Evans LH (2011) Effect of dietary protein and energy levels on growth performance and feed utilization of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e Forssk\u0026aring;l) juveniles. J Appl Aquac 23(1):1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10454438.2011.549781\u003c/span\u003e\u003cspan address=\"10.1080/10454438.2011.549781\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasava R, Patel K, Parmar A, Parmar V (2018) Nutritional and feeding requirement of milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e): A review. \u003cem\u003eResearchGate\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchgate.net/publication/325977916\u003c/span\u003e\u003cspan address=\"https://www.researchgate.net/publication/325977916\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe WO, Ernst DH, Wicklund RI (1989) The effects of dietary protein and energy levels on growth and feed utilization of juvenile milkfish (\u003cem\u003eChanos chanos\u003c/em\u003e). Aquaculture 77(2\u0026ndash;3):145\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0044-8486(89)90215-9\u003c/span\u003e\u003cspan address=\"10.1016/0044-8486(89)90215-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Plates","content":"\u003cp\u003ePlates 1 to 20 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Milkfish (Chanos chanos), Azolla, Aquafeed, Feed Conversion Ratio, Cost-Benefit Analysis","lastPublishedDoi":"10.21203/rs.3.rs-7043499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7043499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the growth performance, survival, feed efficiency, and economic viability of milkfish (Chanos chanos) juveniles fed with Azolla-based aquafeeds over a 12-week culture period. Four dietary treatments were tested: a commercial feed control (T1) and three experimental diets incorporating varying proportions of Azolla and rice bran (T2\u0026ndash;T4). Results showed that Treatment 3 (57.5% Azolla, 42.5% rice bran) achieved growth and survival rates comparable to the control, while significantly outperforming it in feed conversion ratio (2.9773) and return on investment (28.46%). In contrast, the control group showed a negative ROI (\u0026ndash;5.91%) despite good growth, highlighting its limited cost-efficiency. These findings underscore the potential of Azolla-based formulations to reduce feed costs without compromising biological performance, offering a sustainable and economically viable alternative for milkfish aquaculture, particularly in high-production regions such as Region I, Philippines.\u003c/p\u003e","manuscriptTitle":"Organic Milkfish (Chanos chanos) Production fed with Formulated Azolla Aquafeed","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 07:19:50","doi":"10.21203/rs.3.rs-7043499/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"771f6769-b332-41d0-97d2-f2f3651b4a27","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-13T08:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 07:19:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7043499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7043499","identity":"rs-7043499","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}