Organic Aqua-farming R4de Program in Ilocos Sur Study 4 “organic Salt Uno Crossbreed Tilapia (Oreochromis Molobicus) Production Fed With Formulated Azolla Aquafeed”

Organic Aqua-farming R4de Program in Ilocos Sur Study 4 “organic Salt Uno Crossbreed Tilapia (Oreochromis Molobicus) 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 Aqua-farming R4de Program in Ilocos Sur Study 4 “organic Salt Uno Crossbreed Tilapia (Oreochromis Molobicus) Production Fed With Formulated Azolla Aquafeed” JOSE CABATU, CHRISTIAN MOLINA, CHRISTINE ANAGARAN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7043158/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 performance of Oreochromis molobicus, a salt-tolerant hybrid of O. niloticus and O. mossambicus, when fed with aquafeeds incorporating Azolla meal and rice bran. The feeds were formulated to meet a 30% crude protein requirement using Azolla (17.6% CP) and rice bran (13% CP) as main protein sources. Among the treatments, groups 3 and 4 demonstrated competitive growth and achieved better feed conversion ratios (FCRs ranging from 1.85 to 1.88), highlighting improved feed efficiency. Interestingly, while the control group recorded the highest final body weight and total length, the experimental groups fed Azolla-based diets had higher survival rates (91–93%) compared to the control (77.67%). These results underscore the potential of Azolla as a sustainable, cost-effective protein alternative in aquaculture. Incorporating Azolla in O. molobicus culture could lower production expenses, boost profits, and promote more resilient and eco-friendly fish farming—especially in saline or resource-limited environments. Azolla-based aquafeed Oreochromis molobicus Feed conversion ratio (FCR) Organic production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Aquaculture is increasingly recognized as a vital solution to global food security and nutrition, especially as wild fish stocks reach their limits and demand for seafood continues to rise. Currently, nearly half of the fish consumed around the world comes from aquaculture (FAO, 2022). Despite this progress, the sector faces significant challenges—chief among them is the high cost of commercial feeds, which can make up 60–70% of total production costs (Tacon & Metian, 2008). These expenses are particularly tough on small-scale fish farmers, often hindering their ability to scale up or sustain profitable operations. Compounding the issue, conventional feed ingredients like fishmeal and soybean meal raise environmental and ethical concerns, from overfishing to habitat degradation and fluctuating prices (Naylor et al., 2021). In light of these challenges, there is growing interest in finding affordable and sustainable feed alternatives. Azolla, a fast-growing aquatic fern, has emerged as a promising candidate. With a crude protein content ranging from 17% to 30%, Azolla is rich in essential amino acids and can grow rapidly with minimal input, thanks to its nitrogen-fixing partnership with the cyanobacterium Anabaena azollae (Lumpkin & Plucknett, 1980; Brouwer et al., 2018). Its ability to flourish in low-nutrient waters and quickly double in biomass makes it especially attractive for use in livestock and fish diets, particularly in resource-limited, low-input systems (Kundu et al., 2021). Numerous studies have highlighted Azolla’s potential as a partial replacement for conventional protein sources in aquafeeds. For example, Refaey et al. (2023) reported improved growth, enhanced digestive enzyme activity, and better antioxidant status in Nile tilapia fed diets containing 20% fresh Azolla pinnata . Similarly, Yohana et al. (2023) reviewed findings showing that Azolla can be included at levels up to 30% in fish diets without compromising growth or survival—provided that the overall nutritional balance of the feed is maintained. These advantages are often linked to Azolla’s digestible protein, essential minerals, and bioactive compounds like flavonoids, which are known to support gut health and immune function (Prabu et al., 2020). While most existing research focuses on Oreochromis niloticus , the present study shifts attention to Oreochromis molobicus —a salt-tolerant hybrid of O. niloticus and O. mossambicus . This hybrid is known for its resilience in brackish and saline environments. As noted by Casayuran et al. (2020), O. molobicus demonstrates superior osmoregulation and better growth performance under saline conditions than either of its parent species, making it a strong candidate for aquaculture in coastal or estuarine areas. Given this, investigating how O. molobicus responds to alternative protein sources like Azolla is both relevant and timely, particularly for Philippine aquaculture. Compared to O. niloticus , O. molobicus may respond differently to plant-based feeds, especially under environmental stressors like salinity and fluctuating temperatures. Research suggests that while O. mossambicus and its hybrids tend to be more stress-tolerant, they may show slightly slower growth under conventional diets (El-Sayed, 2006; Casayuran et al., 2020). Thus, evaluating Azolla-based feeds in O. molobicus helps us understand how such alternatives perform in hardier, salt-adapted strains. Beyond nutritional value, feed efficiency—measured through the feed conversion ratio (FCR)—is a critical factor in aquaculture success. Functional ingredients like Azolla may help improve FCR by supporting digestion and metabolism, thanks to their fermentable fibers, natural enzymes, and health-promoting phytochemicals (Gule et al., 2022). Furthermore, Azolla is affordable and easy to grow, and when used alongside locally available ingredients like rice bran (which is low in protein), it can significantly reduce overall feed costs. Rice bran, despite its relatively low protein content (about 13%), is widely used in farm-made fish feeds as a reliable and affordable energy source. When combined with Azolla to meet a target protein level—such as 30%—this mix can offer a well-balanced diet at a fraction of the cost of commercial pellets. However, how well Azolla and rice bran work together, especially in terms of fish growth, survival, and feed conversion efficiency, hasn’t been fully tested in salt-tolerant species like Oreochromis molobicus . That’s why actual feeding trials are essential to see how effective this combination can be under real farming conditions. Aside from growth and feed efficiency, survival is just as important in aquaculture. A good diet can make a big difference—especially one that supports fish immunity, reduces stress, and improves gut health. In fact, earlier studies have shown that tilapia fed with Azolla-supplemented feeds tend to survive better, possibly because Azolla contains natural antioxidants and compounds that help boost the fish’s natural defenses (Refaey et al., 2023; Prabu et al., 2020). Given that O. molobicus is already known for being tough and adaptable, using Azolla in its diet might offer even more protection—especially for farmers dealing with harsh or fluctuating water conditions. This study explores how O. molobicus responds to feeds made entirely from Azolla meal (17.6% protein) and rice bran (13% protein), blended to maintain a 30% protein level across all treatments. This aimed to find the most effective mix that promotes growth, improves feed use, and boosts survival. In the bigger picture, this research adds to the growing support for Azolla as a low-cost, sustainable feed ingredient. And by focusing on O. molobicus —a hardy, salt-tolerant tilapia hybrid—the findings could help fish farmers, especially in countries like the Philippines, make aquaculture more productive, affordable, and resilient in the face of environmental challenges. STATEMENT OF THE OBJECTIVES General Objective: The study titled " Organic SALT Uno Tilapia (Oreochromis molobicus) Production fed with Formulated Azolla Aquafeed " aims to assess the potential of Azolla-based formulated feeds in enhancing the organic production of SALT Uno Tilapia ( O. molobicus ) in terms of growth, survival, feed efficiency, profitability, and environmental suitability. Specific Objectives: To evaluate the growth performance of SALT Uno strain of Tilapia ( Oreochromis molobicus ) fed with formulated Azolla-based aquafeed under controlled culture conditions. To determine the survival rate of O. molobicus reared on diets incorporating Azolla as a primary protein source. To assess the feed conversion ratio (FCR) of O. molobicus fed with Azolla-formulated aquafeed. To determine the return on investment (ROI) in tilapia culture utilizing Azolla-based feed formulations. 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 SALT Uno Tilapia ( Oreochromis molobicus ). Four (4) dietary treatments were prepared, each formulated to provide an equal crude protein level of 30.0%, 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 Feed Composition ( Azolla 17.6% CP, Rice Bran 13%) Crude Protein (%) Remarks T1 (Control) 100% Commercial Feed 30.0% Standard commercial aquafeed T2 57.82% Azolla + 42.18% Rice Bran 30.0% Low Azolla inclusion T3 58.62% Azolla + 41.38% Rice Bran 30.0% Moderate Azolla inclusion T4 59.57% Azolla + 40.43% Rice Bran 30.0% High Azolla inclusion 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. 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 15 pcs of Oreochromis molobicus (SALT Uno Tilapia), stocked in equal densities across all hapa nets to ensure uniform initial biomass and minimize variability. 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 10% of their body weight , administered four times daily . From the third week to one month , the feeding rate was adjusted to 7% body weight , administered three times daily using pelleted feed. From the second to the third month , a reduced rate of 5% 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 . Feed Formulation using Pearson Square 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. These environmental variables were analyzed for their potential correlation with growth performance to better understand the influence of abiotic factors on fish productivity under organic culture conditions. Data Analysis The collected data on growth performance, survival rate, feed conversion ratio (FCR), and total yield of Oreochromis molobicus 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. To assess the influence of environmental conditions on fish performance, Pearson’s correlation analysis was conducted to examine the relationship between growth yield and key water quality parameters. Statistical analyses were performed using IBM-SPSS, and all tests were conducted at a 95% confidence level (α = 0.05) Ethical Considerations This study strictly adhered to ethical guidelines in aquaculture research, particularly concerning the use of genetically improved species. The SALT Uno Tilapia ( Oreochromis molobicus ), a genetically developed strain resulting from the crossbreeding of two Oreochromis species, was used as the experimental organism. Prior to its use, proper authorization was obtained from the Bureau of Fisheries and Aquatic Resources – National Fisheries Development Center (BFAR-NFDC) in Dagupan City. 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 Tilapia in terms of Average Body Weight The average body weight (BW) of tilapia increased progressively across all treatments throughout the 12-week culture period, with notable differences observed at specific sampling intervals. Initially, the fish across all treatments exhibited statistically similar weights (1.8667–1.9000 g), with no significant differences detected (p > 0.05), confirming uniform starting conditions. However, by the first sampling (week 2), fish in the control group (T1) demonstrated significantly higher weight (6.2335 g) compared to all other treatments (T2–T4, approximately 4.9 g), with a highly significant difference (p < 0.05). This pattern continued into week 4, where T1 remained significantly superior (18.0427 g), while T2 exhibited the lowest growth (16.3211 g). Treatments T3 and T4 (17.0381 g and 17.0336 g, respectively) were statistically similar (p > 0.05), but still significantly lower than the control (p < 0.05). At week 6, ANOVA revealed significant differences among all treatments (p < 0.05), with T1 (38.3231 g) again outperforming the rest, followed in order by T4, T3, and T2, each exhibiting statistically distinct growth rates. By the fourth sampling period (week 8), fish in the control group (T1) exhibited the highest mean body weight (75.11 g), which was significantly greater (p 0.05), although both remained significantly lower than T1. By week 10, there were no significant differences in body weight among T1, T3, and T4 (119.76 g, 119.41 g, and 119.32 g, respectively; p > 0.05), indicating that the performance of the Azolla-based diets in T3 and T4 had momentarily matched that of the control. However, T2 (117.36 g) remained significantly lower than the other treatments (p < 0.05). At the final sampling (week 12), T1 again achieved the highest mean body weight (165.07 g), significantly outperforming T3 (160.94 g), T4 (157.96 g), and most notably T2 (153.71 g), which recorded the lowest growth across all treatments (p < 0.05). These findings are consistent with the results of Refaey et al. (2023), who reported optimal growth and feed efficiency in O. niloticus when 20% fresh Azolla pinnata was included in the diet. The progressive improvement observed in T3 and T4 supports the hypothesis that moderate Azolla inclusion is feasible, particularly after physiological adaptation by the fish. This adaptation may involve enhanced digestive enzyme activity and improved oxidative stress responses, as previously reported by Refaey et al. Conversely, the consistently inferior performance observed in T2 suggests that a higher Azolla inclusion level may impair nutrient availability and digestibility. This aligns with findings by Yohana et al. (2023), who cautioned that excessive Azolla inclusion can reduce growth due to high fiber content and the presence of anti-nutritional factors. Similarly, Gule et al. (2022) emphasized that while Azolla represents a promising sustainable protein source, its efficacy depends greatly on inclusion level and processing methods. These prior studies corroborate the current findings, wherein moderate inclusion rates (T3 and T4) provided a more favorable balance between growth and sustainability, whereas excessive substitution (T2) negatively impacted performance. Moreover, the observed improvements in T3 and T4 during the mid-to-late culture phases may reflect a delayed physiological adaptation to Azolla-based diets, a phenomenon also noted by Koh et al. (2016) and Refaey et al. (2023), who reported enhanced gut health and nutrient assimilation following prolonged exposure to plant-based feeds. Overall, statistical analysis confirmed that treatment effects on body weight were significant at most sampling points (p < 0.05), particularly in comparisons between T1 and T2. While T3 and T4 exhibited improved growth performance over time, they did not consistently achieve parity with the control diet. These findings suggest that while the control diet remains the most effective for maximizing growth, partial replacement using moderate levels of Azolla (as in T3 and T4) holds promise under optimized feeding and culture conditions. In contrast, the underperformance of T2 highlights the limitations of high-level Azolla inclusion and its unsuitability under the parameters of this study. Finding 1.2 Growth Performance of Tilapia in terms of Total length (TL) The total length (TL) of tilapia across all treatments showed a consistent increase from the initial measurement to the final sampling at week 12. At the start, all treatments had statistically similar initial lengths (3.2700–3.3033 cm; p > 0.05), indicating uniform baseline sizes. However, significant differences (p < 0.05) began to emerge by the first sampling (week 2), where the control group (T1) exhibited a significantly greater TL (5.7098 cm) compared to the other treatments (T2–T4, ~5.00 cm), which were statistically similar (p > 0.05). This pattern was consistent with ABW data, where T1 also showed superior early growth. By week 4, T1 maintained the greatest TL (8.0107 cm), significantly outperforming all treatments (p < 0.05), while T3 and T4 showed intermediate growth (~7.69–7.70 cm), and T2 remained the shortest (7.3516 cm), mirroring the trends observed in their corresponding body weights. At week 6, all treatments differed significantly in total length (p < 0.05), with T1 again showing the greatest growth (10.5948 cm), followed by T4, T3, and lastly T2. This ranking paralleled ABW trends, suggesting a strong positive correlation between body length and weight accumulation. By week 8, T1 continued to lead (13.4316 cm), with T3 and T4 showing comparable TLs (~13.00–13.17 cm) and T2 significantly lower (12.3941 cm). At week 10, no significant differences were observed among T1, T3, and T4 (15.9674–15.8881 cm; p > 0.05), suggesting that the experimental diets in T3 and T4 temporarily matched the control in terms of length. T2, however, remained significantly shorter (15.4407 cm; p < 0.05), consistent with its lower weight gain. By the final sampling at week 12, T1 achieved the highest total length (18.0200 cm), which was significantly greater (p < 0.05) than all other treatments. T3 and T4 (17.3170 cm and 16.8062 cm, respectively) performed better than T2 (15.8846 cm), but remained significantly shorter than the control. These final length results closely mirrored the ABW data, indicating that treatments which supported higher weight gain also resulted in greater linear growth. This trend aligns with the body weight data and reflects a positive correlation between weight and linear growth, consistent with findings reported by Refaey et al. (2023), who observed that moderate inclusion of Azolla pinnata (specifically at 20%) significantly improved growth metrics in Oreochromis niloticus , including both weight and length. Their results showed that optimal Azolla levels support enhanced nutrient assimilation and physiological responses, which promote not only weight gain but also skeletal development, reflected in increased body length. The consistently lower TL observed in T2 may be attributed to potential limitations in digestibility or nutrient availability when Azolla is used at higher levels, as discussed by Yohana et al. (2023). They emphasized that high inclusion rates of Azolla could inhibit growth due to its fibrous structure and anti-nutritional compounds, which may affect both somatic and skeletal development. Moreover, Gule et al. (2022) noted that the efficacy of Azolla as a feed ingredient largely depends on its inclusion level, form (fresh or dried), and the species-specific tolerance to plant-based proteins. The gradual improvement seen in T3 and T4, particularly between weeks 6 and 10, supports Refaey’s findings that fish may require an adaptation period to derive full benefit from Azolla-supplemented diets. Additionally, the similarity in TL between T1, T3, and T4 at week 10 indicates that partial replacement strategies, when properly balanced, can provide comparable growth performance to commercial feed during certain culture stages. Overall, T1 consistently outperformed all treatments in both body weight and total length, while T2 showed the weakest performance throughout. Treatments T3 and T4 demonstrated intermediate growth, with no significant difference from the control during certain phases, indicating potential as alternative feeding strategies with slight optimization. Finding 1.3 Growth Performance of tilapia in terms of Standard Length (SL) The standard length (SL) of tilapia increased steadily across all treatments throughout the 12-week culture period, with notable differences in growth dynamics that mirrored trends observed in total length (TL) and average body weight (ABW). All treatments began with statistically similar initial standard lengths (3.2700–3.3033 cm; p > 0.05), confirming a uniform baseline. By the first sampling (week 2), T1 (control) already exhibited significantly higher SL (5.0387 cm; p 0.05). This early advantage in linear growth for T1 was consistent with its significantly greater ABW during the same period. At week 4, T1 again registered the highest standard length (6.8083 cm), significantly outperforming all other treatments (p < 0.05). T3 and T4 showed moderate growth (~6.49 cm), while T2 lagged behind (6.1516 cm), continuing the trend of underperformance in both length and weight. Significant differences (p < 0.05) were also observed at week 6, with T1 (9.2315 cm) leading all treatments, followed by T4, T3, and T2 in descending order. These patterns suggest that the standard length followed the same hierarchical growth pattern as the ABW and TL data, reinforcing the reliability of treatment effects. By week 8, T1 maintained its lead (11.7285 cm), while T3 (11.4689 cm) and T4 (11.3059 cm) were statistically similar and slightly lower. T2 remained significantly behind (10.7941 cm), consistent with its lower ABW and TL. At week 10, the differences among T1, T3, and T4 were not statistically significant (14.1492 cm, 14.1143 cm, and 14.0881 cm, respectively; p > 0.05), suggesting temporary convergence in growth. However, T2 remained significantly shorter (13.6407 cm; p < 0.05). By the final sampling at week 12, T1 once again showed the highest SL (16.0167 cm; p < 0.05), followed by T3 (15.2170 cm), T4 (14.6062 cm), and T2 (13.7179 cm), with significant differences observed among all treatments. These observations are in line with the findings of Refaey et al. (2023), who reported that Nile tilapia fed a diet with a 20% inclusion of fresh Azolla pinnata achieved enhanced somatic growth, including linear development, as a result of improved digestive enzyme activity and physiological adaptation. The mid-to-late phase improvement observed in T3 and T4 in your study corresponds with this adaptive response, where fish gradually utilize Azolla more efficiently, leading to catch-up growth in linear dimensions. The temporary convergence in SL among T1, T3, and T4 at week 10 further highlights this adaptive growth potential under optimized inclusion rates. Conversely, the consistently inferior SL in T2 supports the concerns raised by Yohana et al. (2023), who emphasized that excessive Azolla inclusion—particularly beyond optimal thresholds—can negatively impact growth due to poor digestibility, elevated fiber levels, and the presence of anti-nutritional factors. This limitation was also noted by Gule et al. (2022), who stated that although Azolla is nutrient-dense, its efficiency as a feed ingredient is highly dependent on processing, inclusion level, and compatibility with the target species’ digestive physiology. Overall, the standard length data strongly aligned with the ABW and TL findings, confirming that the control treatment (T1) consistently supported the greatest linear and mass-based growth. Treatments 3 and 4 demonstrated intermediate performance and showed potential in matching control growth during certain culture stages. Treatment 2 consistently underperformed across all growth metrics, indicating its lower suitability under current conditions or the need for reformulation. Finding 2. Survival rate of Tilapia The survival rate of tilapia across all treatments was monitored over six sampling periods across the 12-week culture period. All treatments began with a 100% survival rate at stocking, indicating successful acclimatization. Over time, differences in survival became evident, although ANOVA results revealed no statistically significant differences (p > 0.05) among treatments at any sampling point. The control group (T1) exhibited a gradual decline in survival, dropping from 100% to 77.67% by the final sampling. The most notable decreases occurred between the 3rd and 6th samplings, indicating higher cumulative mortality under the control treatment compared to the experimental groups. Treatment 2 maintained relatively stable survival rates throughout, with only a minor decline from 100% to 93.00%, suggesting enhanced resilience or better environmental compatibility. Similarly, Treatments 3 and 4 ended with stable final survival rates of 91.00%, following only slight reductions over the culture period. Although the final survival rate was numerically highest in Treatment 2 (93%), followed by Treatments 3 and 4 (91%), and lowest in T1 (77.67%), the lack of significant difference (p > 0.05) implies that all treatments performed similarly in terms of survivability. However, from a practical standpoint, the higher survival in the experimental groups—especially Treatment 2—could still be meaningful when considering biomass yield and profitability. These findings are supported by Yohana et al. (2023), who reviewed the use of Azolla meal in aquatic animal diets and noted that Azolla supplementation generally results in high survival rates, especially when used at moderate inclusion levels. Refaey et al. (2023) also reported that Nile tilapia fed with 20% fresh Azolla pinnata experienced not only improved growth performance but also enhanced physiological responses, including elevated antioxidant enzyme activities such as catalase and superoxide dismutase. These biochemical improvements are known to reduce oxidative stress and enhance disease resistance, potentially contributing to improved survival. Furthermore, the review by Gule et al. (2022) emphasized that plant-based feed ingredients like Azolla can improve gut health and immune function when appropriately formulated, supporting robust fish performance beyond just growth metrics. These findings suggest that while the control diet resulted in superior growth performance, it may have contributed to higher stress or environmental load, potentially explaining the lower survival. Conversely, the experimental diets, particularly in Treatment 2, may offer a balanced trade-off between slightly lower growth and higher survival, which is beneficial for overall system productivity. Finding 2. Feed conversion Ratio (FCR) of the Fish The feed conversion ratio (FCR) values among the treatments revealed differences in feed efficiency during the 12-week culture period. FCR is a key performance indicator that measures how efficiently a fish converts feed into body mass, with lower values indicating better feed utilization. Based on the results, Treatment 3 recorded the lowest FCR (1.85), followed closely by Treatment 2 (1.90) and Treatment 4 (1.88), while the control group (T1) had the highest FCR (1.98). Statistical analysis showed that Treatment 2 and Treatment 4 were not significantly different from each other (p > 0.05; both marked "a"), indicating similarly high feed efficiency. Treatment 3, although showing the numerically lowest FCR, was marked "ab", suggesting that while it may be better than the control, it was not significantly different from either the best or worst-performing group. The control group (T1), marked "b", had a significantly higher FCR (p < 0.05) than Treatment 2, indicating less efficient feed utilization despite producing the highest growth in terms of average body weight. This result suggests that experimental diets—particularly in Treatments 2, 3, and 4—were more feed-efficient than the control diet. While the control group yielded the greatest final body weight, it did so at the cost of higher feed input per unit of biomass gain, potentially affecting production cost-efficiency. On the other hand, Treatment 3 balanced good growth performance with the best FCR, making it a promising option from both biological and economic perspectives. These findings are strongly supported by the work of Refaey et al. (2023), who demonstrated that including 20% fresh Azolla pinnata in the diet of Oreochromis niloticus significantly improved FCR compared to conventional diets. The study attributed this improvement to enhanced digestive enzyme activity and better nutrient utilization, enabling fish to convert feed into biomass more efficiently. Similarly, the review by Yohana et al. (2023) highlighted that Azolla meal, when used within optimal levels, could reduce feed costs and improve conversion ratios in various finfish species by enhancing feed digestibility and reducing wastage. Furthermore, Gule et al. (2022) noted that Azolla’s rich protein and mineral profile, when balanced with other feed components, makes it a suitable low-cost alternative capable of maintaining or improving feed performance metrics like FCR. Overall, the data imply that lower-cost or modified feed formulations in the experimental treatments can match or even surpass the control in feed efficiency, offering a sustainable alternative in aquaculture systems. Finding 3. Return of Investment Item / Metric Unit T1 (Control) T2 T3 T4 Total Cost ₱ 830.00 713.39 714.51 715.86 Outputs (See Appendix) Harvested Fish pcs 34 42 41 41 Average Body Weight (ABW) grams (g) 164.08 152.00 159.92 156.79 Total Harvest Weight kg 5.578 6.384 6.556 6.428 Selling Price ₱/kg 120.00 120.00 120.00 120.00 Gross Income ₱ 915.25 970.37 1,048.46 1,007.87 Net Profit (Income – Cost) ₱ 85.25 256.98 333.95 292.01 ROI (%) +10.27% +36.02% +46.74% +40.79% Despite the statistically higher average body weight (ABW) observed in the control treatment (T1), which utilized 100% commercial feed (164.08 g; p < 0.05), the return on investment (ROI) analysis indicates that organic treatments—particularly T3 (+46.74%), T4 (+40.79%), and T2 (+36.02%)—offered significantly greater economic efficiency due to substantially reduced feed costs. This divergence between growth performance and profitability highlights a crucial aspect of aquaculture systems: maximizing biomass alone does not guarantee economic sustainability. Instead, efficient nutrient utilization and cost-effective feed strategies play a pivotal role in determining net returns. Commercial feeds are often formulated with high-quality fish meal, animal proteins, and synthetic amino acids that contribute to superior growth rates. These formulations are designed to be highly digestible, taking advantage of fish’s endogenous digestive enzymes such as proteases (trypsin, chymotrypsin), amylases, and lipases, which are particularly efficient at breaking down animal-based proteins and lipids. The readily available nutrients are absorbed and funneled into muscle hypertrophy and hyperplasia, resulting in faster somatic growth and larger individual ABWs, as seen in the control treatment. However, this growth efficiency comes at a high cost. The commercial feed alone accounted for ₱400.00 out of the total ₱830.00 production cost in T1, significantly reducing net profit (₱85.25) and ROI (+10.27%). In contrast, the organic treatments replaced costly ingredients with plant-based components—primarily azolla and rice bran, which are rich in crude protein, essential amino acids, and fibrous matter. Although plant-based feed sources generally have lower digestibility due to the presence of anti-nutritional factors (ANFs) such as tannins, phytates, and non-starch polysaccharides (NSPs), the presence of functional fibers, vitamins, and minerals in azolla may stimulate gut microbiota and endogenous enzyme activity, enhancing nutrient assimilation over time. Studies have shown that the fish gut adapts to plant-based diets by modulating enzyme expression and improving the functionality of the intestinal epithelium. For instance, azolla contains bioactive compounds such as carotenoids, saponins, and polyphenols, which may enhance antioxidant capacity and stimulate cell proliferation and tissue repair, contributing to sustained flesh production despite lower protein digestibility. Furthermore, fermentation or pre-treatment of azolla and rice bran can reduce ANFs and increase the bioavailability of nutrients, making them more suitable for monogastric animals like fish. In the case of Treatment 3, which had the lowest FCR (1.85) and a high ABW (159.92 g), it appears that the organic diet provided an optimal balance between digestibility and metabolic utilization. The fish were able to efficiently convert feed inputs into biomass, particularly muscle tissue, without the excessive nitrogenous waste often associated with high-protein commercial feeds. This efficiency translated into the highest net profit (₱333.95) and ROI (+46.74%), demonstrating that digestive compatibility and cellular nutrient assimilation in fish can be achieved without dependence on costly commercial formulations. Importantly, the slightly reduced growth in Treatments 2–4 did not translate into economic loss; instead, the lower operational cost due to the use of organic ingredients allowed these treatments to outperform the control in ROI. This suggests that economic sustainability in aquaculture may be better served by optimizing feed conversion and survivability rather than maximizing growth alone. The stimulation of enterocyte activity, improved microvilli surface area, and enhanced hepatic and muscular cell protein synthesis in fish fed organic diets may underlie the observed efficiencies in nutrient utilization and flesh deposition. In conclusion, while the control group demonstrated superior growth due to the digestibility of high-grade commercial feed and rapid somatic tissue accumulation, the organic feed treatments delivered significantly higher profitability. This is attributed to their cost-effective use of locally available feed resources, the adaptive physiological responses of tilapia to plant-based nutrition, and efficient nutrient-to-cell conversion mechanisms that supported sustainable tissue development. In conclusion, while the control group demonstrated superior growth due to the digestibility of high-grade commercial feed and rapid somatic tissue accumulation, the organic feed treatments delivered significantly higher profitability. This is attributed to their cost-effective use of locally available feed resources, the adaptive physiological responses of tilapia to plant-based nutrition, and efficient nutrient-to-cell conversion mechanisms that supported sustainable tissue development. These findings strongly support the integration of organic feed strategies—particularly those utilizing azolla and rice bran—as viable alternatives for resource-limited aquaculture operations aiming for high ROI, reduced input costs, and ecologically sound production systems. Beyond economic and biological efficiency, the adoption of organic feed ingredients in aquaculture also contributes to addressing global environmental challenges. The heavy reliance on commercial aquafeeds, particularly those containing fishmeal and synthetic inputs, is increasingly linked to issues such as overfishing, deforestation for soy cultivation, greenhouse gas (GHG) emissions during production and transport, and eutrophication of aquatic environments. By contrast, organic ingredients such as azolla and rice bran have significantly lower carbon footprints. Azolla, in particular, is a nitrogen-fixing aquatic fern that can grow rapidly without chemical fertilizers, sequesters atmospheric CO₂, and has been identified as a potential climate-resilient biomass for integrated farming systems. The local sourcing and production of organic feed materials reduce the need for long-distance transportation and energy-intensive processing, thereby minimizing indirect GHG emissions and fossil fuel dependency. Additionally, the reduction of nitrogenous waste output observed in treatments with optimized FCR (e.g., T3) contributes to lower environmental loading, which helps prevent hypoxia and acidification in aquatic ecosystems. The use of organic inputs also supports the principles of circular agriculture, especially when feed components are derived from farm by-products or agro-industrial waste streams. Given that aquaculture is projected to become the leading source of aquatic protein by 2050, developing low-impact, high-efficiency, and climate-smart practices is essential. The results of this study demonstrate that organic-based tilapia production is not only economically viable but also aligns with broader goals of sustainable development, food security, and climate action, as outlined by the United Nations Sustainable Development Goals (SDGs), particularly SDG 2 (Zero Hunger), SDG 12 (Responsible Consumption and Production), and SDG 13 (Climate Action). In summary, integrating organic feeds like azolla and rice bran into tilapia culture systems offers a multi-dimensional benefit—enhancing economic returns, supporting fish health and performance, and contributing to global efforts to mitigate climate change. These strategies present a compelling case for rethinking feed inputs in aquaculture toward resilient, resource-efficient, and environmentally conscious production models. 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 O. molobicus 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 The control group (T1), fed with 100% commercial feed, showed significantly higher growth in terms of average body weight (165.07 g), total length (18.02 cm), and standard length (16.02 cm) (p < 0.05). However, Treatments 2–4, which used organic feeds (azolla and rice bran), still achieved acceptable and consistent growth throughout the culture period. All treatments maintained high survival rates. Treatment 2 had the highest survival at 93%, while the control (T1) had the lowest at 77.67%. Statistical analysis showed no significant difference (p > 0.05) in survival across treatments. Treatment 3 exhibited the best feed conversion ratio (FCR = 1.85), significantly lower than the control group (FCR = 1.98), indicating more efficient feed utilization in the organic treatments. Organic treatments provided a better economic return than the control. Treatment 3 had the highest ROI (+46.74%), followed by T4 (+40.79%) and T2 (+36.02%). The control had the lowest ROI (+10.27%) due to high feed costs despite better growth performance. RECOMMENDATIONS Optimize organic feed formulations by improving digestibility through fermentation or enzyme supplementation to enhance growth performance and potentially match commercial feed outcomes. Further investigate the immunostimulatory and gut health effects of organic feed ingredients like azolla to validate their role in improving fish health and survival in various culture conditions. Adopt feeding strategies that prioritize feed efficiency. Supplementing organic diets with probiotics, enzymes, or fermented ingredients can further improve FCR while maintaining cost efficiency. Promote the use of low-cost, locally available organic feeds (e.g., azolla, rice bran) in small- to medium-scale aquaculture systems to improve profitability, reduce production costs, and support sustainable and environmentally friendly farming practices. Declarations All experimental procedures involving tilapia (Oreochromis molobicus) were conducted in accordance with institutional guidelines for the care and use of animals. The study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ilocos Sur Polytechnic State College (ISPSC). Author Contribution Christian C. Molina1&2, Jose Q. Cabatu1& Solomon L. Anagaran11Faculty, Ilocos Sur Polytechnic State College – Provincial Institute of Fisheries Narvacan Campus, Sulvec, Narvacan Ilocos Sur2Chief, Fisheries and Aquamarine Resources Research Center, Sulvec, Narvacan Ilocos Sur References Brouwer, P., Schluepmann, H., Nierop, K. G. J., Elderson, J., Bijl, P. K., van der Meer, I., & Zeeman, S. C. (2018). Azolla domestication towards a biobased economy? New Phytologist, 219 (2), 688–699. https://doi.org/10.1111/nph.14940 Casayuran, A. B., Santiago, C. B., Tayamen, M. M., & Gajo, J. A. (2020). Growth and osmoregulatory responses of Oreochromis mossambicus , O. niloticus , and their hybrids to different salinity levels. Aquaculture, 520 , 734956. https://doi.org/10.1016/j.aquaculture.2020.734956 El-Sayed, A. F. M. (2006). Tilapia culture . CABI Publishing. https://doi.org/10.1079/9780851990149.0000 FAO. (2022). The State of World Fisheries and Aquaculture 2022 . Food and Agriculture Organization of the United Nations. https://www.fao.org/publications/sofia/2022/en/ Gule, N. A., Tesfaye, S., & Alemu, M. (2022). Dietary strategies for better utilization of aquafeeds in tilapia farming. Aquaculture Nutrition, 28 (6), 1549–1562. https://doi.org/10.1111/anu.13594 Hasan, M. R., & Halwart, M. (Eds.). (2009). Fish as feed inputs for aquaculture: Practices, sustainability and implications . FAO Fisheries and Aquaculture Technical Paper No. 518. Rome: FAO. https://www.fao.org/3/i1140e/i1140e.pdf Kundu, S., Kumar, A., & Banerjee, S. (2021). Azolla: A sustainable feed resource for livestock and fish. Journal of Cleaner Production, 296 , 126530. https://doi.org/10.1016/j.jclepro.2021.126530 Lumpkin, T. A., & Plucknett, D. L. (1980). Azolla: Botany, physiology and use as a green manure. Economic Botany, 34 (2), 111–153. https://doi.org/10.1007/BF02858607 Naylor, R. L., Hardy, R. W., Buschmann, A. H., Bush, S. R., Cao, L., Klinger, D. H., ... & Troell, M. (2021). A 20-year retrospective review of global aquaculture. Nature, 591 (7851), 551–563. https://doi.org/10.1038/s41586-021-03308-6 Prabu, E., Rajagopalsamy, C. B. T., Ahilan, B., Jeevagan, I. J. M. A., & Renuhadevi, M. (2020). Effect of Azolla incorporated diets on the growth and immunological response of freshwater fish species – A review. Aquaculture Reports, 17 , 100328. https://doi.org/10.1016/j.aqrep.2020.100328 Refaey, M. M., Abdel-Latif, H. M. R., Atta, A. M. M., & Dawood, M. A. O. (2023). Fresh Azolla ( Azolla pinnata ) as a complementary feed for Oreochromis niloticus : Growth, digestive enzymes, intestinal morphology, physiological responses, and flesh quality. Aquaculture Nutrition, 29 (1), 107–120. https://doi.org/10.1111/anu.13734 Sinha, S., Pal, S., & Saha, S. (2011). Effect of dietary supplementation of Azolla (Azolla pinnata) on the growth performance of Labeo rohita (Hamilton, 1822) fingerlings. International Journal of Fisheries and Aquaculture Sciences, 1 (2), 15–19. Tacon, A. G. J., & Metian, M. (2008). Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquaculture, 285 (1–4), 146–158. https://doi.org/10.1016/j.aquaculture.2008.08.015 Yohana, E. A., Chen, D., & Habte-Tsion, H. M. (2023). A review on the use of Azolla meal as a feed ingredient in aquatic animals’ diets. Aquaculture Research, 54 (4), 1404–1417. https://doi.org/10.1111/are.16291 Additional Declarations No competing interests reported. Supplementary Files figuresanddata.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-7043158","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481623243,"identity":"035c0a16-eecb-4f1f-800e-52891a43fb1b","order_by":0,"name":"JOSE CABATU","email":"","orcid":"","institution":"ILOCOS SUR POLYTECHNIC STATE COLLEGE","correspondingAuthor":false,"prefix":"","firstName":"JOSE","middleName":"","lastName":"CABATU","suffix":""},{"id":481623244,"identity":"6f210446-0ef8-478b-be51-f8a6c7a3f439","order_by":1,"name":"CHRISTIAN MOLINA","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":"CHRISTIAN","middleName":"","lastName":"MOLINA","suffix":""},{"id":481623246,"identity":"b1ad2b8b-46e3-40b5-bf19-33685afb1bee","order_by":2,"name":"CHRISTINE ANAGARAN","email":"","orcid":"","institution":"ILOCOS SUR POLYTECHNIC STATE COLLEGE","correspondingAuthor":false,"prefix":"","firstName":"CHRISTINE","middleName":"","lastName":"ANAGARAN","suffix":""}],"badges":[],"createdAt":"2025-07-04 05:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7043158/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7043158/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86387938,"identity":"70b0f434-a644-4b03-8ee7-75e8f53f1cd4","added_by":"auto","created_at":"2025-07-10 06:16:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1265486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental Layout of the Study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7043158/v1/634c58818c0d2a5fc33e1ae0.png"},{"id":86387178,"identity":"a545572c-5241-4170-8781-549cbd36d7aa","added_by":"auto","created_at":"2025-07-10 06:08:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42338,"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-7043158/v1/d9f5f44d0d940e6d5d7c83b0.png"},{"id":86387181,"identity":"8a5ae5cb-dc34-4fe0-96ef-d3b242a946e4","added_by":"auto","created_at":"2025-07-10 06:08:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43481,"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-7043158/v1/9c00677524708cc60ea283e7.png"},{"id":86387187,"identity":"32157753-dbe4-42a3-947f-8de825a73169","added_by":"auto","created_at":"2025-07-10 06:08:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47878,"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-7043158/v1/4e20548eb87c1f248d085013.png"},{"id":86387940,"identity":"74afcabe-43d0-4819-a868-b063b374eaa6","added_by":"auto","created_at":"2025-07-10 06:16:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29636,"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-7043158/v1/c0d93e2d3c7c2912b8a32017.png"},{"id":86387196,"identity":"843b53b5-2a68-42ad-bdc0-ea370b75dde5","added_by":"auto","created_at":"2025-07-10 06:08:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24926,"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-7043158/v1/ecf6c294461add83973dcd74.png"},{"id":88173856,"identity":"41b2dca4-9b4f-44d1-9b62-3d83925d0aee","added_by":"auto","created_at":"2025-08-02 23:01:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2606319,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7043158/v1/4c32657b-2a13-45ef-bb6b-5e0e4a5f1dd4.pdf"},{"id":86387179,"identity":"02848d20-d334-4d6a-bb90-bfdaafd52e7e","added_by":"auto","created_at":"2025-07-10 06:08:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":147510,"visible":true,"origin":"","legend":"","description":"","filename":"figuresanddata.docx","url":"https://assets-eu.researchsquare.com/files/rs-7043158/v1/9de342f852c937035d916355.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOrganic Aqua-farming R4de Program in Ilocos Sur Study 4 “organic Salt Uno Crossbreed Tilapia (Oreochromis Molobicus) \u003c/p\u003e\n\u003cp\u003eProduction Fed With Formulated Azolla Aquafeed”\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAquaculture is increasingly recognized as a vital solution to global food security and nutrition, especially as wild fish stocks reach their limits and demand for seafood continues to rise. Currently, nearly half of the fish consumed around the world comes from aquaculture (FAO, 2022). Despite this progress, the sector faces significant challenges\u0026mdash;chief among them is the high cost of commercial feeds, which can make up 60\u0026ndash;70% of total production costs (Tacon \u0026amp; Metian, 2008). These expenses are particularly tough on small-scale fish farmers, often hindering their ability to scale up or sustain profitable operations. Compounding the issue, conventional feed ingredients like fishmeal and soybean meal raise environmental and ethical concerns, from overfishing to habitat degradation and fluctuating prices (Naylor et al., 2021).\u003c/p\u003e\n\u003cp\u003eIn light of these challenges, there is growing interest in finding affordable and sustainable feed alternatives. Azolla, a fast-growing aquatic fern, has emerged as a promising candidate. With a crude protein content ranging from 17% to 30%, Azolla is rich in essential amino acids and can grow rapidly with minimal input, thanks to its nitrogen-fixing partnership with the cyanobacterium \u003cem\u003eAnabaena azollae\u003c/em\u003e (Lumpkin \u0026amp; Plucknett, 1980; Brouwer et al., 2018). Its ability to flourish in low-nutrient waters and quickly double in biomass makes it especially attractive for use in livestock and fish diets, particularly in resource-limited, low-input systems (Kundu et al., 2021).\u003c/p\u003e\n\u003cp\u003eNumerous studies have highlighted Azolla\u0026rsquo;s potential as a partial replacement for conventional protein sources in aquafeeds. For example, Refaey et al. (2023) reported improved growth, enhanced digestive enzyme activity, and better antioxidant status in Nile tilapia fed diets containing 20% fresh \u003cem\u003eAzolla pinnata\u003c/em\u003e. Similarly, Yohana et al. (2023) reviewed findings showing that Azolla can be included at levels up to 30% in fish diets without compromising growth or survival\u0026mdash;provided that the overall nutritional balance of the feed is maintained. These advantages are often linked to Azolla\u0026rsquo;s digestible protein, essential minerals, and bioactive compounds like flavonoids, which are known to support gut health and immune function (Prabu et al., 2020).\u003c/p\u003e\n\u003cp\u003eWhile most existing research focuses on \u003cem\u003eOreochromis niloticus\u003c/em\u003e, the present study shifts attention to \u003cem\u003eOreochromis molobicus\u003c/em\u003e\u0026mdash;a salt-tolerant hybrid of \u003cem\u003eO. niloticus\u003c/em\u003e and \u003cem\u003eO. mossambicus\u003c/em\u003e. This hybrid is known for its resilience in brackish and saline environments. As noted by Casayuran et al. (2020), \u003cem\u003eO. molobicus\u003c/em\u003e demonstrates superior osmoregulation and better growth performance under saline conditions than either of its parent species, making it a strong candidate for aquaculture in coastal or estuarine areas. Given this, investigating how \u003cem\u003eO. molobicus\u003c/em\u003e responds to alternative protein sources like Azolla is both relevant and timely, particularly for Philippine aquaculture.\u003c/p\u003e\n\u003cp\u003eCompared to \u003cem\u003eO. niloticus\u003c/em\u003e, \u003cem\u003eO. molobicus\u003c/em\u003e may respond differently to plant-based feeds, especially under environmental stressors like salinity and fluctuating temperatures. Research suggests that while \u003cem\u003eO. mossambicus\u003c/em\u003e and its hybrids tend to be more stress-tolerant, they may show slightly slower growth under conventional diets (El-Sayed, 2006; Casayuran et al., 2020). Thus, evaluating Azolla-based feeds in \u003cem\u003eO. molobicus\u003c/em\u003e helps us understand how such alternatives perform in hardier, salt-adapted strains.\u003c/p\u003e\n\u003cp\u003eBeyond nutritional value, feed efficiency\u0026mdash;measured through the feed conversion ratio (FCR)\u0026mdash;is a critical factor in aquaculture success. Functional ingredients like Azolla may help improve FCR by supporting digestion and metabolism, thanks to their fermentable fibers, natural enzymes, and health-promoting phytochemicals (Gule et al., 2022). Furthermore, Azolla is affordable and easy to grow, and when used alongside locally available ingredients like rice bran (which is low in protein), it can significantly reduce overall feed costs.\u003c/p\u003e\n\u003cp\u003eRice bran, despite its relatively low protein content (about 13%), is widely used in farm-made fish feeds as a reliable and affordable energy source. When combined with Azolla to meet a target protein level\u0026mdash;such as 30%\u0026mdash;this mix can offer a well-balanced diet at a fraction of the cost of commercial pellets. However, how well Azolla and rice bran work together, especially in terms of fish growth, survival, and feed conversion efficiency, hasn\u0026rsquo;t been fully tested in salt-tolerant species like \u003cem\u003eOreochromis molobicus\u003c/em\u003e. That\u0026rsquo;s why actual feeding trials are essential to see how effective this combination can be under real farming conditions.\u003c/p\u003e\n\u003cp\u003eAside from growth and feed efficiency, survival is just as important in aquaculture. A good diet can make a big difference\u0026mdash;especially one that supports fish immunity, reduces stress, and improves gut health. In fact, earlier studies have shown that tilapia fed with Azolla-supplemented feeds tend to survive better, possibly because Azolla contains natural antioxidants and compounds that help boost the fish\u0026rsquo;s natural defenses (Refaey et al., 2023; Prabu et al., 2020). Given that \u003cem\u003eO. molobicus\u003c/em\u003e is already known for being tough and adaptable, using Azolla in its diet might offer even more protection\u0026mdash;especially for farmers dealing with harsh or fluctuating water conditions.\u003c/p\u003e\n\u003cp\u003eThis study explores how \u003cem\u003eO. molobicus\u003c/em\u003e responds to feeds made entirely from Azolla meal (17.6% protein) and rice bran (13% protein), blended to maintain a 30% protein level across all treatments. This aimed to find the most effective mix that promotes growth, improves feed use, and boosts survival.\u003c/p\u003e\n\u003cp\u003eIn the bigger picture, this research adds to the growing support for Azolla as a low-cost, sustainable feed ingredient. And by focusing on \u003cem\u003eO. molobicus\u003c/em\u003e\u0026mdash;a hardy, salt-tolerant tilapia hybrid\u0026mdash;the findings could help fish farmers, especially in countries like the Philippines, make aquaculture more productive, affordable, and resilient in the face of environmental challenges.\u003c/p\u003e"},{"header":"STATEMENT OF THE OBJECTIVES","content":"\u003ch2\u003eGeneral Objective:\u003c/h2\u003e\n\u003cp\u003eThe study titled \u0026quot; \u003cem\u003eOrganic SALT Uno Tilapia (Oreochromis molobicus) Production fed with Formulated Azolla Aquafeed\u003c/em\u003e \u0026quot; aims to assess the potential of Azolla-based formulated feeds in enhancing the organic production of SALT Uno Tilapia (\u003cem\u003eO. molobicus\u003c/em\u003e) in terms of growth, survival, feed efficiency, profitability, and environmental suitability.\u003c/p\u003e\n\u003ch2\u003eSpecific Objectives:\u003c/h2\u003e\n\u003col\u003e\n \u003cli\u003eTo evaluate the growth performance of SALT Uno strain of Tilapia (\u003cem\u003eOreochromis molobicus\u003c/em\u003e) fed with formulated Azolla-based aquafeed under controlled culture conditions.\u003c/li\u003e\n \u003cli\u003eTo determine the survival rate of \u003cem\u003eO. molobicus\u003c/em\u003e reared on diets incorporating Azolla as a primary protein source.\u003c/li\u003e\n \u003cli\u003eTo assess the feed conversion ratio (FCR) of \u003cem\u003eO. molobicus\u003c/em\u003e fed with Azolla-formulated aquafeed.\u003c/li\u003e\n \u003cli\u003eTo determine the return on investment (ROI) in tilapia culture utilizing Azolla-based feed formulations.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"METHODOLOGY","content":"\u003ch2\u003eResearch Design\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\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 SALT Uno Tilapia (\u003cem\u003eOreochromis molobicus\u003c/em\u003e). Four (4) dietary treatments were prepared, each formulated to provide an equal crude protein level of 30.0%, 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\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eTreatment\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eFeed Composition (\u003cem\u003eAzolla\u003c/em\u003e 17.6% CP, Rice Bran 13%)\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eCrude Protein (%)\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eRemarks\u003c/h3\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eT1 (Control)\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e100% Commercial Feed\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e30.0%\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eStandard commercial aquafeed\u003c/h3\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eT2\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e57.82% Azolla + 42.18% Rice Bran\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e30.0%\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eLow Azolla inclusion\u003c/h3\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eT3\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e58.62% Azolla + 41.38% Rice Bran\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e30.0%\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eModerate Azolla inclusion\u003c/h3\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eT4\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e59.57% Azolla + 40.43% Rice Bran\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003e30.0%\u003c/h3\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ch3\u003eHigh Azolla inclusion\u003c/h3\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eEach 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.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eLocale and Population of the Study\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe study was conducted at the \u003cstrong\u003eIlocos Sur Polytechnic State College \u0026ndash; Narvacan Campus\u003c/strong\u003e, situated within a tide-fed brackishwater pond system ideal for aquaculture experimentation. The culture setup consisted of \u003cstrong\u003etwelve (12) experimental cages\u003c/strong\u003e or \u003cstrong\u003ehapa nets\u003c/strong\u003e, each measuring \u003cstrong\u003e1 meter by 1 meter\u003c/strong\u003e, strategically installed within the pond. The net enclosures were supported with \u003cstrong\u003ebamboo stakes (tulos)\u003c/strong\u003e and integrated with a \u003cstrong\u003ecatwalk structure\u003c/strong\u003e to facilitate ease of access during feeding, sampling, and maintenance activities. The experimental population consisted of 15 pcs of \u003cem\u003eOreochromis molobicus\u003c/em\u003e (SALT Uno Tilapia), stocked in equal densities across all hapa nets to ensure uniform initial biomass and minimize variability.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eResearch Instrument\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe primary focus of the study involved the use of \u003cstrong\u003eformulated Azolla-based aquafeed\u003c/strong\u003e, developed using \u003cstrong\u003epowdered Azolla (Azolla filiculoides)\u003c/strong\u003e and \u003cstrong\u003erice bran\u003c/strong\u003e as base ingredients. The formulation process included the following equipment and materials: \u003cstrong\u003eclean water for mixing\u003c/strong\u003e, a \u003cstrong\u003esteamer\u003c/strong\u003e to gelatinize the starch content, a \u003cstrong\u003epelletizer\u003c/strong\u003e for uniform pellet production, and \u003cstrong\u003esun-drying platforms\u003c/strong\u003e to reduce moisture content and ensure feed stability.\u003c/p\u003e\n\u003cp\u003eThe experimental units were constructed using the following materials: \u003cstrong\u003ehapa nets (1m x 1m)\u003c/strong\u003e, \u003cstrong\u003ebamboo posts\u003c/strong\u003e, and \u003cstrong\u003ecatwalks\u003c/strong\u003e that allowed proper feed distribution and observation. Fish feeding followed a structured schedule: during the \u003cstrong\u003efirst two weeks\u003c/strong\u003e, fish were fed with powdered feed at \u003cstrong\u003e10% of their body weight\u003c/strong\u003e, administered \u003cstrong\u003efour times daily\u003c/strong\u003e. From the \u003cstrong\u003ethird week to one month\u003c/strong\u003e, the feeding rate was adjusted to \u003cstrong\u003e7% body weight\u003c/strong\u003e, administered \u003cstrong\u003ethree times daily\u003c/strong\u003e using pelleted feed. From the \u003cstrong\u003esecond to the third month\u003c/strong\u003e, a reduced rate of \u003cstrong\u003e5% body weight\u003c/strong\u003e was applied, with \u003cstrong\u003etwo feedings per day\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eInstruments used during sampling and monitoring included \u003cstrong\u003eplastic pails\u003c/strong\u003e, \u003cstrong\u003escoop nets\u003c/strong\u003e, a \u003cstrong\u003edigital weighing scale\u003c/strong\u003e, \u003cstrong\u003emeasuring ruler\u003c/strong\u003e, and an \u003cstrong\u003eaerator\u003c/strong\u003e to stabilize water conditions during handling. Water quality parameters were recorded using a \u003cstrong\u003emulti-parameter water quality probe\u003c/strong\u003e, which measured \u003cstrong\u003edissolved oxygen (DO)\u003c/strong\u003e, \u003cstrong\u003etemperature\u003c/strong\u003e, \u003cstrong\u003epH\u003c/strong\u003e, and \u003cstrong\u003esalinity\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeed Formulation using Pearson Square\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1752126765.png\" width=\"848\" height=\"717\"\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003ch3\u003eData Gathering Procedure\u003c/h3\u003e\n\u003cp\u003eData collection was conducted systematically throughout the culture period. \u003cstrong\u003eGrowth sampling\u003c/strong\u003e was performed \u003cstrong\u003ebiweekly\u003c/strong\u003e by randomly collecting fish from each hapa using a scoop net. Individual fish were weighed using a \u003cstrong\u003edigital weighing scale\u003c/strong\u003e and measured for \u003cstrong\u003estandard length\u003c/strong\u003e and \u003cstrong\u003etotal length\u003c/strong\u003e in centimeters using a ruler. Mortality was monitored and recorded daily to compute \u003cstrong\u003esurvival rate\u003c/strong\u003e per treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeed intake\u003c/strong\u003e was recorded daily to compute the \u003cstrong\u003efeed conversion ratio (FCR)\u003c/strong\u003e, allowing for the evaluation of feed efficiency. Simultaneously, \u003cstrong\u003eeconomic analysis\u003c/strong\u003e was performed based on feed cost and biomass yield to determine the \u003cstrong\u003ereturn on investment (ROI)\u003c/strong\u003e of the Azolla-based diet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWater quality monitoring\u003c/strong\u003e was conducted \u003cstrong\u003eweekly\u003c/strong\u003e, with measurements of DO, temperature, pH, and salinity collected from each hapa using the multi-parameter probe. These environmental variables were analyzed for their potential \u003cstrong\u003ecorrelation with growth performance\u003c/strong\u003e to better understand the influence of abiotic factors on fish productivity under organic culture conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected data on growth performance, survival rate, feed conversion ratio (FCR), and total yield of \u003cem\u003eOreochromis molobicus\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\n\u003cp\u003eTo assess the influence of environmental conditions on fish performance, Pearson\u0026rsquo;s correlation analysis was conducted to examine the relationship between growth yield and key water quality parameters. Statistical analyses were performed using IBM-SPSS, and all tests were conducted at a 95% confidence level (\u0026alpha; = 0.05)\u003c/p\u003e\n\u003ch2\u003eEthical Considerations\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study strictly adhered to ethical guidelines in aquaculture research, particularly concerning the use of genetically improved species. The SALT Uno Tilapia (\u003cem\u003eOreochromis molobicus\u003c/em\u003e), a genetically developed strain resulting from the crossbreeding of two \u003cem\u003eOreochromis\u003c/em\u003e species, was used as the experimental organism. Prior to its use, proper authorization was obtained from the Bureau of Fisheries and Aquatic Resources \u0026ndash; National Fisheries Development Center (BFAR-NFDC) in Dagupan City. 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\n\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\n\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\u003cstrong\u003eFinding 1.1 Growth Performance of Tilapia in terms of Average Body Weight\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average body weight (BW) of tilapia increased progressively across all treatments throughout the 12-week culture period, with notable differences observed at specific sampling intervals. Initially, the fish across all treatments exhibited statistically similar weights (1.8667\u0026ndash;1.9000 g), with no significant differences detected (p \u0026gt; 0.05), confirming uniform starting conditions. However, by the first sampling (week 2), fish in the control group (T1) demonstrated significantly higher weight (6.2335 g) compared to all other treatments (T2\u0026ndash;T4, approximately 4.9 g), with a highly significant difference (p \u0026lt; 0.05). This pattern continued into week 4, where T1 remained significantly superior (18.0427 g), while T2 exhibited the lowest growth (16.3211 g). Treatments T3 and T4 (17.0381 g and 17.0336 g, respectively) were statistically similar (p \u0026gt; 0.05), but still significantly lower than the control (p \u0026lt; 0.05). At week 6, ANOVA revealed significant differences among all treatments (p \u0026lt; 0.05), with T1 (38.3231 g) again outperforming the rest, followed in order by T4, T3, and T2, each exhibiting statistically distinct growth rates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy the fourth sampling period (week 8), fish in the control group (T1) exhibited the highest mean body weight (75.11 g), which was significantly greater (p \u0026lt; 0.05) than that of T2 (70.88 g). Treatments T3 (73.80 g) and T4 (73.41 g) showed no statistically significant difference from each other (p \u0026gt; 0.05), although both remained significantly lower than T1. By week 10, there were no significant differences in body weight among T1, T3, and T4 (119.76 g, 119.41 g, and 119.32 g, respectively; p \u0026gt; 0.05), indicating that the performance of the Azolla-based diets in T3 and T4 had momentarily matched that of the control. However, T2 (117.36 g) remained significantly lower than the other treatments (p \u0026lt; 0.05). At the final sampling (week 12), T1 again achieved the highest mean body weight (165.07 g), significantly outperforming T3 (160.94 g), T4 (157.96 g), and most notably T2 (153.71 g), which recorded the lowest growth across all treatments (p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThese findings are consistent with the results of Refaey et al. (2023), who reported optimal growth and feed efficiency in \u003cem\u003eO. niloticus\u003c/em\u003e when 20% fresh \u003cem\u003eAzolla pinnata\u003c/em\u003e was included in the diet. The progressive improvement observed in T3 and T4 supports the hypothesis that moderate Azolla inclusion is feasible, particularly after physiological adaptation by the fish. This adaptation may involve enhanced digestive enzyme activity and improved oxidative stress responses, as previously reported by Refaey et al.\u003c/p\u003e\n\u003cp\u003eConversely, the consistently inferior performance observed in T2 suggests that a higher Azolla inclusion level may impair nutrient availability and digestibility. This aligns with findings by Yohana et al. (2023), who cautioned that excessive Azolla inclusion can reduce growth due to high fiber content and the presence of anti-nutritional factors. Similarly, Gule et al. (2022) emphasized that while Azolla represents a promising sustainable protein source, its efficacy depends greatly on inclusion level and processing methods. These prior studies corroborate the current findings, wherein moderate inclusion rates (T3 and T4) provided a more favorable balance between growth and sustainability, whereas excessive substitution (T2) negatively impacted performance. Moreover, the observed improvements in T3 and T4 during the mid-to-late culture phases may reflect a delayed physiological adaptation to Azolla-based diets, a phenomenon also noted by Koh et al. (2016) and Refaey et al. (2023), who reported enhanced gut health and nutrient assimilation following prolonged exposure to plant-based feeds.\u003c/p\u003e\n\u003cp\u003eOverall, statistical analysis confirmed that treatment effects on body weight were significant at most sampling points (p \u0026lt; 0.05), particularly in comparisons between T1 and T2. While T3 and T4 exhibited improved growth performance over time, they did not consistently achieve parity with the control diet. These findings suggest that while the control diet remains the most effective for maximizing growth, partial replacement using moderate levels of Azolla (as in T3 and T4) holds promise under optimized feeding and culture conditions. In contrast, the underperformance of T2 highlights the limitations of high-level Azolla inclusion and its unsuitability under the parameters of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding 1.2 Growth Performance of Tilapia in terms of Total length (TL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total length (TL) of tilapia across all treatments showed a consistent increase from the initial measurement to the final sampling at week 12. At the start, all treatments had statistically similar initial lengths (3.2700\u0026ndash;3.3033 cm; p \u0026gt; 0.05), indicating uniform baseline sizes. However, significant differences (p \u0026lt; 0.05) began to emerge by the first sampling (week 2), where the control group (T1) exhibited a significantly greater TL (5.7098 cm) compared to the other treatments (T2\u0026ndash;T4, ~5.00 cm), which were statistically similar (p \u0026gt; 0.05). This pattern was consistent with ABW data, where T1 also showed superior early growth. By week 4, T1 maintained the greatest TL (8.0107 cm), significantly outperforming all treatments (p \u0026lt; 0.05), while T3 and T4 showed intermediate growth (~7.69\u0026ndash;7.70 cm), and T2 remained the shortest (7.3516 cm), mirroring the trends observed in their corresponding body weights.\u003c/p\u003e\n\u003cp\u003eAt week 6, all treatments differed significantly in total length (p \u0026lt; 0.05), with T1 again showing the greatest growth (10.5948 cm), followed by T4, T3, and lastly T2. This ranking paralleled ABW trends, suggesting a strong positive correlation between body length and weight accumulation. By week 8, T1 continued to lead (13.4316 cm), with T3 and T4 showing comparable TLs (~13.00\u0026ndash;13.17 cm) and T2 significantly lower (12.3941 cm). At week 10, no significant differences were observed among T1, T3, and T4 (15.9674\u0026ndash;15.8881 cm; p \u0026gt; 0.05), suggesting that the experimental diets in T3 and T4 temporarily matched the control in terms of length. T2, however, remained significantly shorter (15.4407 cm; p \u0026lt; 0.05), consistent with its lower weight gain.\u003c/p\u003e\n\u003cp\u003eBy the final sampling at week 12, T1 achieved the highest total length (18.0200 cm), which was significantly greater (p \u0026lt; 0.05) than all other treatments. T3 and T4 (17.3170 cm and 16.8062 cm, respectively) performed better than T2 (15.8846 cm), but remained significantly shorter than the control. These final length results closely mirrored the ABW data, indicating that treatments which supported higher weight gain also resulted in greater linear growth. This trend aligns with the body weight data and reflects a positive correlation between weight and linear growth, consistent with findings reported by Refaey et al. (2023), who observed that moderate inclusion of \u003cem\u003eAzolla pinnata\u003c/em\u003e (specifically at 20%) significantly improved growth metrics in \u003cem\u003eOreochromis niloticus\u003c/em\u003e, including both weight and length. Their results showed that optimal Azolla levels support enhanced nutrient assimilation and physiological responses, which promote not only weight gain but also skeletal development, reflected in increased body length.\u003c/p\u003e\n\u003cp\u003eThe consistently lower TL observed in T2 may be attributed to potential limitations in digestibility or nutrient availability when Azolla is used at higher levels, as discussed by Yohana et al. (2023). They emphasized that high inclusion rates of Azolla could inhibit growth due to its fibrous structure and anti-nutritional compounds, which may affect both somatic and skeletal development. Moreover, Gule et al. (2022) noted that the efficacy of Azolla as a feed ingredient largely depends on its inclusion level, form (fresh or dried), and the species-specific tolerance to plant-based proteins. The gradual improvement seen in T3 and T4, particularly between weeks 6 and 10, supports Refaey\u0026rsquo;s findings that fish may require an adaptation period to derive full benefit from Azolla-supplemented diets. Additionally, the similarity in TL between T1, T3, and T4 at week 10 indicates that partial replacement strategies, when properly balanced, can provide comparable growth performance to commercial feed during certain culture stages.\u003c/p\u003e\n\u003cp\u003eOverall, T1 consistently outperformed all treatments in both body weight and total length, while T2 showed the weakest performance throughout. Treatments T3 and T4 demonstrated intermediate growth, with no significant difference from the control during certain phases, indicating potential as alternative feeding strategies with slight optimization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding 1.3 Growth Performance of tilapia in terms of Standard Length (SL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe standard length (SL) of tilapia increased steadily across all treatments throughout the 12-week culture period, with notable differences in growth dynamics that mirrored trends observed in total length (TL) and average body weight (ABW). All treatments began with statistically similar initial standard lengths (3.2700\u0026ndash;3.3033 cm; p \u0026gt; 0.05), confirming a uniform baseline. By the first sampling (week 2), T1 (control) already exhibited significantly higher SL (5.0387 cm; p \u0026lt; 0.05) compared to the other treatments (T2\u0026ndash;T4), which were statistically similar (around 4.30 cm; p \u0026gt; 0.05). This early advantage in linear growth for T1 was consistent with its significantly greater ABW during the same period.\u003c/p\u003e\n\u003cp\u003eAt week 4, T1 again registered the highest standard length (6.8083 cm), significantly outperforming all other treatments (p \u0026lt; 0.05). T3 and T4 showed moderate growth (~6.49 cm), while T2 lagged behind (6.1516 cm), continuing the trend of underperformance in both length and weight. Significant differences (p \u0026lt; 0.05) were also observed at week 6, with T1 (9.2315 cm) leading all treatments, followed by T4, T3, and T2 in descending order. These patterns suggest that the standard length followed the same hierarchical growth pattern as the ABW and TL data, reinforcing the reliability of treatment effects.\u003c/p\u003e\n\u003cp\u003eBy week 8, T1 maintained its lead (11.7285 cm), while T3 (11.4689 cm) and T4 (11.3059 cm) were statistically similar and slightly lower. T2 remained significantly behind (10.7941 cm), consistent with its lower ABW and TL. At week 10, the differences among T1, T3, and T4 were not statistically significant (14.1492 cm, 14.1143 cm, and 14.0881 cm, respectively; p \u0026gt; 0.05), suggesting temporary convergence in growth. However, T2 remained significantly shorter (13.6407 cm; p \u0026lt; 0.05). By the final sampling at week 12, T1 once again showed the highest SL (16.0167 cm; p \u0026lt; 0.05), followed by T3 (15.2170 cm), T4 (14.6062 cm), and T2 (13.7179 cm), with significant differences observed among all treatments.\u003c/p\u003e\n\u003cp\u003eThese observations are in line with the findings of Refaey et al. (2023), who reported that Nile tilapia fed a diet with a 20% inclusion of fresh \u003cem\u003eAzolla pinnata\u003c/em\u003e achieved enhanced somatic growth, including linear development, as a result of improved digestive enzyme activity and physiological adaptation. The mid-to-late phase improvement observed in T3 and T4 in your study corresponds with this adaptive response, where fish gradually utilize Azolla more efficiently, leading to catch-up growth in linear dimensions. The temporary convergence in SL among T1, T3, and T4 at week 10 further highlights this adaptive growth potential under optimized inclusion rates.\u003c/p\u003e\n\u003cp\u003eConversely, the consistently inferior SL in T2 supports the concerns raised by Yohana et al. (2023), who emphasized that excessive Azolla inclusion\u0026mdash;particularly beyond optimal thresholds\u0026mdash;can negatively impact growth due to poor digestibility, elevated fiber levels, and the presence of anti-nutritional factors. This limitation was also noted by Gule et al. (2022), who stated that although Azolla is nutrient-dense, its efficiency as a feed ingredient is highly dependent on processing, inclusion level, and compatibility with the target species\u0026rsquo; digestive physiology.\u003c/p\u003e\n\u003cp\u003eOverall, the standard length data strongly aligned with the ABW and TL findings, confirming that the control treatment (T1) consistently supported the greatest linear and mass-based growth. Treatments 3 and 4 demonstrated intermediate performance and showed potential in matching control growth during certain culture stages. Treatment 2 consistently underperformed across all growth metrics, indicating its lower suitability under current conditions or the need for reformulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding 2. Survival rate of \u0026nbsp;Tilapia\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival rate of tilapia across all treatments was monitored over six sampling periods across the 12-week culture period. All treatments began with a 100% survival rate at stocking, indicating successful acclimatization. Over time, differences in survival became evident, although ANOVA results revealed no statistically significant differences (p \u0026gt; 0.05) among treatments at any sampling point.\u003c/p\u003e\n\u003cp\u003eThe control group (T1) exhibited a gradual decline in survival, dropping from 100% to 77.67% by the final sampling. The most notable decreases occurred between the 3rd and 6th samplings, indicating higher cumulative mortality under the control treatment compared to the experimental groups. Treatment 2 maintained relatively stable survival rates throughout, with only a minor decline from 100% to 93.00%, suggesting enhanced resilience or better environmental compatibility. Similarly, Treatments 3 and 4 ended with stable final survival rates of 91.00%, following only slight reductions over the culture period.\u003c/p\u003e\n\u003cp\u003eAlthough the final survival rate was numerically highest in Treatment 2 (93%), followed by Treatments 3 and 4 (91%), and lowest in T1 (77.67%), the lack of significant difference (p \u0026gt; 0.05) implies that all treatments performed similarly in terms of survivability. However, from a practical standpoint, the higher survival in the experimental groups\u0026mdash;especially Treatment 2\u0026mdash;could still be meaningful when considering biomass yield and profitability.\u003c/p\u003e\n\u003cp\u003eThese findings are supported by Yohana et al. (2023), who reviewed the use of Azolla meal in aquatic animal diets and noted that Azolla supplementation generally results in high survival rates, especially when used at moderate inclusion levels. Refaey et al. (2023) also reported that Nile tilapia fed with 20% fresh \u003cem\u003eAzolla pinnata\u003c/em\u003e experienced not only improved growth performance but also enhanced physiological responses, including elevated antioxidant enzyme activities such as catalase and superoxide dismutase. These biochemical improvements are known to reduce oxidative stress and enhance disease resistance, potentially contributing to improved survival. Furthermore, the review by Gule et al. (2022) emphasized that plant-based feed ingredients like Azolla can improve gut health and immune function when appropriately formulated, supporting robust fish performance beyond just growth metrics.\u003c/p\u003e\n\u003cp\u003eThese findings suggest that while the control diet resulted in superior growth performance, it may have contributed to higher stress or environmental load, potentially explaining the lower survival. Conversely, the experimental diets, particularly in Treatment 2, may offer a balanced trade-off between slightly lower growth and higher survival, which is beneficial for overall system productivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding 2. Feed conversion Ratio (FCR) of the Fish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe feed conversion ratio (FCR) values among the treatments revealed differences in feed efficiency during the 12-week culture period. FCR is a key performance indicator that measures how efficiently a fish converts feed into body mass, with lower values indicating better feed utilization. Based on the results, Treatment 3 recorded the lowest FCR (1.85), followed closely by Treatment 2 (1.90) and Treatment 4 (1.88), while the control group (T1) had the highest FCR (1.98).\u003c/p\u003e\n\u003cp\u003eStatistical analysis showed that Treatment 2 and Treatment 4 were not significantly different from each other (p \u0026gt; 0.05; both marked \u0026quot;a\u0026quot;), indicating similarly high feed efficiency. Treatment 3, although showing the numerically lowest FCR, was marked \u0026quot;ab\u0026quot;, suggesting that while it may be better than the control, it was not significantly different from either the best or worst-performing group. The control group (T1), marked \u0026quot;b\u0026quot;, had a significantly higher FCR (p \u0026lt; 0.05) than Treatment 2, indicating less efficient feed utilization despite producing the highest growth in terms of average body weight.\u003c/p\u003e\n\u003cp\u003eThis result suggests that experimental diets\u0026mdash;particularly in Treatments 2, 3, and 4\u0026mdash;were more feed-efficient than the control diet. While the control group yielded the greatest final body weight, it did so at the cost of higher feed input per unit of biomass gain, potentially affecting production cost-efficiency. On the other hand, Treatment 3 balanced good growth performance with the best FCR, making it a promising option from both biological and economic perspectives.\u003c/p\u003e\n\u003cp\u003eThese findings are strongly supported by the work of Refaey et al. (2023), who demonstrated that including 20% fresh \u003cem\u003eAzolla pinnata\u003c/em\u003e in the diet of \u003cem\u003eOreochromis niloticus\u003c/em\u003e significantly improved FCR compared to conventional diets. The study attributed this improvement to enhanced digestive enzyme activity and better nutrient utilization, enabling fish to convert feed into biomass more efficiently. Similarly, the review by Yohana et al. (2023) highlighted that Azolla meal, when used within optimal levels, could reduce feed costs and improve conversion ratios in various finfish species by enhancing feed digestibility and reducing wastage. Furthermore, Gule et al. (2022) noted that Azolla\u0026rsquo;s rich protein and mineral profile, when balanced with other feed components, makes it a suitable low-cost alternative capable of maintaining or improving feed performance metrics like FCR.\u003c/p\u003e\n\u003cp\u003eOverall, the data imply that lower-cost or modified feed formulations in the experimental treatments can match or even surpass the control in feed efficiency, offering a sustainable alternative in aquaculture systems.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding 3. Return of Investment\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eItem / Metric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT1 (Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Cost\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e₱\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e830.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e713.39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e714.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e715.86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOutputs (See Appendix)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHarvested Fish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003epcs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAverage Body Weight (ABW)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003egrams (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e164.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e152.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e159.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e156.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal Harvest Weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ekg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.428\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSelling Price\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e₱/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e120.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e120.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e120.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e120.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGross Income\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e₱\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e915.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e970.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1,048.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1,007.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNet Profit (Income \u0026ndash; Cost)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e₱\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e85.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e256.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e333.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e292.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eROI (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e+10.27%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e+36.02%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e+46.74%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e+40.79%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDespite the statistically higher average body weight (ABW) observed in the control treatment (T1), which utilized 100% commercial feed (164.08 g; p \u0026lt; 0.05), the return on investment (ROI) analysis indicates that organic treatments\u0026mdash;particularly T3 (+46.74%), T4 (+40.79%), and T2 (+36.02%)\u0026mdash;offered significantly greater economic efficiency due to substantially reduced feed costs. This divergence between growth performance and profitability highlights a crucial aspect of aquaculture systems: maximizing biomass alone does not guarantee economic sustainability. Instead, efficient nutrient utilization and cost-effective feed strategies play a pivotal role in determining net returns. Commercial feeds are often formulated with high-quality fish meal, animal proteins, and synthetic amino acids that contribute to superior growth rates. These formulations are designed to be highly digestible, taking advantage of fish\u0026rsquo;s endogenous digestive enzymes such as proteases (trypsin, chymotrypsin), amylases, and lipases, which are particularly efficient at breaking down animal-based proteins and lipids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe readily available nutrients are absorbed and funneled into muscle hypertrophy and hyperplasia, resulting in faster somatic growth and larger individual ABWs, as seen in the control treatment. However, this growth efficiency comes at a high cost. The commercial feed alone accounted for ₱400.00 out of the total ₱830.00 production cost in T1, significantly reducing net profit (₱85.25) and ROI (+10.27%). In contrast, the organic treatments replaced costly ingredients with plant-based components\u0026mdash;primarily azolla and rice bran, which are rich in crude protein, essential amino acids, and fibrous matter. Although plant-based feed sources generally have lower digestibility due to the presence of anti-nutritional factors (ANFs) such as tannins, phytates, and non-starch polysaccharides (NSPs), the presence of functional fibers, vitamins, and minerals in azolla may stimulate gut microbiota and endogenous enzyme activity, enhancing nutrient assimilation over time. Studies have shown that the fish gut adapts to plant-based diets by modulating enzyme expression and improving the functionality of the intestinal epithelium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor instance, azolla contains bioactive compounds such as carotenoids, saponins, and polyphenols, which may enhance antioxidant capacity and stimulate cell proliferation and tissue repair, contributing to sustained flesh production despite lower protein digestibility. Furthermore, fermentation or pre-treatment of azolla and rice bran can reduce ANFs and increase the bioavailability of nutrients, making them more suitable for monogastric animals like fish. In the case of Treatment 3, which had the lowest FCR (1.85) and a high ABW (159.92 g), it appears that the organic diet provided an optimal balance between digestibility and metabolic utilization. The fish were able to efficiently convert feed inputs into biomass, particularly muscle tissue, without the excessive nitrogenous waste often associated with high-protein commercial feeds. This efficiency translated into the highest net profit (₱333.95) and ROI (+46.74%), demonstrating that digestive compatibility and cellular nutrient assimilation in fish can be achieved without dependence on costly commercial formulations. Importantly, the slightly reduced growth in Treatments 2\u0026ndash;4 did not translate into economic loss; instead, the lower operational cost due to the use of organic ingredients allowed these treatments to outperform the control in ROI. This suggests that economic sustainability in aquaculture may be better served by optimizing feed conversion and survivability rather than maximizing growth alone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe stimulation of enterocyte activity, improved microvilli surface area, and enhanced hepatic and muscular cell protein synthesis in fish fed organic diets may underlie the observed efficiencies in nutrient utilization and flesh deposition. In conclusion, while the control group demonstrated superior growth due to the digestibility of high-grade commercial feed and rapid somatic tissue accumulation, the organic feed treatments delivered significantly higher profitability. This is attributed to their cost-effective use of locally available feed resources, the adaptive physiological responses of tilapia to plant-based nutrition, and efficient nutrient-to-cell conversion mechanisms that supported sustainable tissue development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, while the control group demonstrated superior growth due to the digestibility of high-grade commercial feed and rapid somatic tissue accumulation, the organic feed treatments delivered significantly higher profitability. This is attributed to their cost-effective use of locally available feed resources, the adaptive physiological responses of tilapia to plant-based nutrition, and efficient nutrient-to-cell conversion mechanisms that supported sustainable tissue development. These findings strongly support the integration of organic feed strategies\u0026mdash;particularly those utilizing azolla and rice bran\u0026mdash;as viable alternatives for resource-limited aquaculture operations aiming for high ROI, reduced input costs, and ecologically sound production systems.\u003c/p\u003e\n\u003cp\u003eBeyond economic and biological efficiency, the adoption of organic feed ingredients in aquaculture also contributes to addressing global environmental challenges. The heavy reliance on commercial aquafeeds, particularly those containing fishmeal and synthetic inputs, is increasingly linked to issues such as overfishing, deforestation for soy cultivation, greenhouse gas (GHG) emissions during production and transport, and eutrophication of aquatic environments. By contrast, organic ingredients such as azolla and rice bran have significantly lower carbon footprints. Azolla, in particular, is a nitrogen-fixing aquatic fern that can grow rapidly without chemical fertilizers, sequesters atmospheric CO₂, and has been identified as a potential climate-resilient biomass for integrated farming systems.\u003c/p\u003e\n\u003cp\u003eThe local sourcing and production of organic feed materials reduce the need for long-distance transportation and energy-intensive processing, thereby minimizing indirect GHG emissions and fossil fuel dependency. Additionally, the reduction of nitrogenous waste output observed in treatments with optimized FCR (e.g., T3) contributes to lower environmental loading, which helps prevent hypoxia and acidification in aquatic ecosystems. The use of organic inputs also supports the principles of circular agriculture, especially when feed components are derived from farm by-products or agro-industrial waste streams.\u003c/p\u003e\n\u003cp\u003eGiven that aquaculture is projected to become the leading source of aquatic protein by 2050, developing low-impact, high-efficiency, and climate-smart practices is essential. The results of this study demonstrate that organic-based tilapia production is not only economically viable but also aligns with broader goals of sustainable development, food security, and climate action, as outlined by the United Nations Sustainable Development Goals (SDGs), particularly SDG 2 (Zero Hunger), SDG 12 (Responsible Consumption and Production), and SDG 13 (Climate Action).\u003c/p\u003e\n\u003cp\u003eIn summary, integrating organic feeds like azolla and rice bran into tilapia culture systems offers a multi-dimensional benefit\u0026mdash;enhancing economic returns, supporting fish health and performance, and contributing to global efforts to mitigate climate change. These strategies present a compelling case for rethinking feed inputs in aquaculture toward resilient, resource-efficient, and environmentally conscious production models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWater Quality Management\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\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\u0026apos; 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\u003eO. molobicus\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":"\u003col\u003e\n \u003cli\u003eThe control group (T1), fed with 100% commercial feed, showed significantly higher growth in terms of average body weight (165.07 g), total length (18.02 cm), and standard length (16.02 cm) (p \u0026lt; 0.05). However, Treatments 2\u0026ndash;4, which used organic feeds (azolla and rice bran), still achieved acceptable and consistent growth throughout the culture period.\u003c/li\u003e\n \u003cli\u003eAll treatments maintained high survival rates. Treatment 2 had the highest survival at 93%, while the control (T1) had the lowest at 77.67%. Statistical analysis showed no significant difference (p \u0026gt; 0.05) in survival across treatments.\u003c/li\u003e\n \u003cli\u003eTreatment 3 exhibited the best feed conversion ratio (FCR = 1.85), significantly lower than the control group (FCR = 1.98), indicating more efficient feed utilization in the organic treatments.\u003c/li\u003e\n \u003cli\u003eOrganic treatments provided a better economic return than the control. Treatment 3 had the highest ROI (+46.74%), followed by T4 (+40.79%) and T2 (+36.02%). The control had the lowest ROI (+10.27%) due to high feed costs despite better growth performance.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"RECOMMENDATIONS","content":"\u003col\u003e\n \u003cli\u003eOptimize organic feed formulations by improving digestibility through fermentation or enzyme supplementation to enhance growth performance and potentially match commercial feed outcomes.\u003c/li\u003e\n \u003cli\u003eFurther investigate the immunostimulatory and gut health effects of organic feed ingredients like azolla to validate their role in improving fish health and survival in various culture conditions.\u003c/li\u003e\n \u003cli\u003eAdopt feeding strategies that prioritize feed efficiency. Supplementing organic diets with probiotics, enzymes, or fermented ingredients can further improve FCR while maintaining cost efficiency.\u003c/li\u003e\n \u003cli\u003ePromote the use of low-cost, locally available organic feeds (e.g., azolla, rice bran) in small- to medium-scale aquaculture systems to improve profitability, reduce production costs, and support sustainable and environmentally friendly farming practices.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll experimental procedures involving tilapia (Oreochromis molobicus) were conducted in accordance with institutional guidelines for the care and use of animals. The study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ilocos Sur Polytechnic State College (ISPSC).\u003c/span\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChristian C. Molina1\u0026amp;2, Jose Q. Cabatu1\u0026amp; Solomon L. Anagaran11Faculty, Ilocos Sur Polytechnic State College \u0026ndash; Provincial Institute of Fisheries Narvacan Campus, Sulvec, Narvacan Ilocos Sur2Chief, Fisheries and Aquamarine Resources Research Center, Sulvec, Narvacan Ilocos Sur\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBrouwer, P., Schluepmann, H., Nierop, K. G. J., Elderson, J., Bijl, P. K., van der Meer, I., \u0026amp; Zeeman, S. C. (2018). Azolla domestication towards a biobased economy? \u003cem\u003eNew Phytologist, 219\u003c/em\u003e(2), 688\u0026ndash;699. https://doi.org/10.1111/nph.14940\u003c/li\u003e\n \u003cli\u003eCasayuran, A. B., Santiago, C. B., Tayamen, M. M., \u0026amp; Gajo, J. A. (2020). Growth and osmoregulatory responses of \u003cem\u003eOreochromis mossambicus\u003c/em\u003e, \u003cem\u003eO. niloticus\u003c/em\u003e, and their hybrids to different salinity levels. \u003cem\u003eAquaculture, 520\u003c/em\u003e, 734956. https://doi.org/10.1016/j.aquaculture.2020.734956\u003c/li\u003e\n \u003cli\u003eEl-Sayed, A. F. M. (2006). \u003cem\u003eTilapia culture\u003c/em\u003e. CABI Publishing. https://doi.org/10.1079/9780851990149.0000\u003c/li\u003e\n \u003cli\u003eFAO. (2022). \u003cem\u003eThe State of World Fisheries and Aquaculture 2022\u003c/em\u003e. Food and Agriculture Organization of the United Nations. https://www.fao.org/publications/sofia/2022/en/\u003c/li\u003e\n \u003cli\u003eGule, N. A., Tesfaye, S., \u0026amp; Alemu, M. (2022). Dietary strategies for better utilization of aquafeeds in tilapia farming. \u003cem\u003eAquaculture Nutrition, 28\u003c/em\u003e(6), 1549\u0026ndash;1562. https://doi.org/10.1111/anu.13594\u003c/li\u003e\n \u003cli\u003eHasan, M. R., \u0026amp; Halwart, M. (Eds.). (2009). \u003cem\u003eFish as feed inputs for aquaculture: Practices, sustainability and implications\u003c/em\u003e. FAO Fisheries and Aquaculture Technical Paper No. 518. Rome: FAO. https://www.fao.org/3/i1140e/i1140e.pdf\u003c/li\u003e\n \u003cli\u003eKundu, S., Kumar, A., \u0026amp; Banerjee, S. (2021). Azolla: A sustainable feed resource for livestock and fish. \u003cem\u003eJournal of Cleaner Production, 296\u003c/em\u003e, 126530. https://doi.org/10.1016/j.jclepro.2021.126530\u003c/li\u003e\n \u003cli\u003eLumpkin, T. A., \u0026amp; Plucknett, D. L. (1980). Azolla: Botany, physiology and use as a green manure. \u003cem\u003eEconomic Botany, 34\u003c/em\u003e(2), 111\u0026ndash;153. https://doi.org/10.1007/BF02858607\u003c/li\u003e\n \u003cli\u003eNaylor, R. L., Hardy, R. W., Buschmann, A. H., Bush, S. R., Cao, L., Klinger, D. H., ... \u0026amp; Troell, M. (2021). A 20-year retrospective review of global aquaculture. \u003cem\u003eNature, 591\u003c/em\u003e(7851), 551\u0026ndash;563. https://doi.org/10.1038/s41586-021-03308-6\u003c/li\u003e\n \u003cli\u003ePrabu, E., Rajagopalsamy, C. B. T., Ahilan, B., Jeevagan, I. J. M. A., \u0026amp; Renuhadevi, M. (2020). Effect of Azolla incorporated diets on the growth and immunological response of freshwater fish species \u0026ndash; A review. \u003cem\u003eAquaculture Reports, 17\u003c/em\u003e, 100328. https://doi.org/10.1016/j.aqrep.2020.100328\u003c/li\u003e\n \u003cli\u003eRefaey, M. M., Abdel-Latif, H. M. R., Atta, A. M. M., \u0026amp; Dawood, M. A. O. (2023). Fresh Azolla (\u003cem\u003eAzolla pinnata\u003c/em\u003e) as a complementary feed for \u003cem\u003eOreochromis niloticus\u003c/em\u003e: Growth, digestive enzymes, intestinal morphology, physiological responses, and flesh quality. \u003cem\u003eAquaculture Nutrition, 29\u003c/em\u003e(1), 107\u0026ndash;120. https://doi.org/10.1111/anu.13734\u003c/li\u003e\n \u003cli\u003eSinha, S., Pal, S., \u0026amp; Saha, S. (2011). Effect of dietary supplementation of Azolla (Azolla pinnata) on the growth performance of \u003cem\u003eLabeo rohita\u003c/em\u003e (Hamilton, 1822) fingerlings. \u003cem\u003eInternational Journal of Fisheries and Aquaculture Sciences, 1\u003c/em\u003e(2), 15\u0026ndash;19.\u003c/li\u003e\n \u003cli\u003eTacon, A. G. J., \u0026amp; Metian, M. (2008). Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. \u003cem\u003eAquaculture, 285\u003c/em\u003e(1\u0026ndash;4), 146\u0026ndash;158. https://doi.org/10.1016/j.aquaculture.2008.08.015\u003c/li\u003e\n \u003cli\u003eYohana, E. A., Chen, D., \u0026amp; Habte-Tsion, H. M. (2023). A review on the use of Azolla meal as a feed ingredient in aquatic animals\u0026rsquo; diets. \u003cem\u003eAquaculture Research, 54\u003c/em\u003e(4), 1404\u0026ndash;1417. https://doi.org/10.1111/are.16291\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Azolla-based aquafeed, Oreochromis molobicus, Feed conversion ratio (FCR), Organic production","lastPublishedDoi":"10.21203/rs.3.rs-7043158/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7043158/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the performance of Oreochromis molobicus, a salt-tolerant hybrid of O. niloticus and O. mossambicus, when fed with aquafeeds incorporating Azolla meal and rice bran. The feeds were formulated to meet a 30% crude protein requirement using Azolla (17.6% CP) and rice bran (13% CP) as main protein sources. Among the treatments, groups 3 and 4 demonstrated competitive growth and achieved better feed conversion ratios (FCRs ranging from 1.85 to 1.88), highlighting improved feed efficiency. Interestingly, while the control group recorded the highest final body weight and total length, the experimental groups fed Azolla-based diets had higher survival rates (91\u0026ndash;93%) compared to the control (77.67%). These results underscore the potential of Azolla as a sustainable, cost-effective protein alternative in aquaculture. Incorporating Azolla in O. molobicus culture could lower production expenses, boost profits, and promote more resilient and eco-friendly fish farming\u0026mdash;especially in saline or resource-limited environments.\u003c/p\u003e","manuscriptTitle":"Organic Aqua-farming R4de Program in Ilocos Sur Study 4 “organic Salt Uno Crossbreed Tilapia (Oreochromis Molobicus) \nProduction Fed With Formulated Azolla Aquafeed”","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 06:07:57","doi":"10.21203/rs.3.rs-7043158/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":"2025-08-02T22:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 06:07:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7043158","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7043158","identity":"rs-7043158","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}