Functional Feed Development from Alternanthera philoxeroides for Herbivorous Fish: Effects on Growth Performance, Feed Efficiency, and Hematological Response

2.2 Feed preparation

Fresh A. philoxeroides was harvested from local aquatic habitats, washed, sun-dried, and ground into fine powder. The powder was used to substitute plant-based ingredients in isonitrogenous and isolipidic diets formulated to meet the nutritional requirements of gourami. Feed was pelletized using a manual extruder and dried at 50℃ for 24 hours before storage at 4℃.

2.3 Growth performance

Fish in each aquarium were batch-weighed at the beginning and end of the trial. The specific growth rate (SGR) was calculated using the following equation:

$S G R=\left(\frac{\operatorname{Ln} W_t-\operatorname{Ln} W_0}{t}\right) \times 100$                           (1)

where, W_t is the final mean weight of the fish (g), W₀ is the initial mean weight of the fish (g), and t is the duration of the feeding trial (days).

2.4 Feed conversion ratio (FCR)

Feed intake was recorded daily, and FCR was calculated at the end of the experiment using the following equation:

$F C R=\frac{F}{W_t+D-W_0}$                     (2)

where, F is the total feed consumed by the fish (g), W_t is the final mean weight of the fish (g), W₀ is the initial mean weight of the fish (g), and D is the total weight of dead fish during the experimental period (g).

2.5 Hematological analysis

At the end of the feeding trial, blood samples were collected from the caudal vein of three randomly selected fish per replicate using 1 mL syringes pre-loaded with EDTA as an anticoagulant. The blood samples were then analyzed to determine hematological parameters. The total erythrocyte count was measured using a Neubauer hemocytometer following dilution with Hayem’s solution, while the total leukocyte count was assessed using the same type of hemocytometer, with Turk’s solution serving as the diluent. Both erythrocyte and leukocyte counts were expressed as the number of cells per cubic millimeter (mm³) of blood.

2.6 Statistical analysis

All data were expressed as mean ± standard deviation (SD). One-way ANOVA was performed to detect significant differences among treatments, followed by Duncan’s multiple range test at a significance level of p < 0.05 using SPSS v26.

4.1 Growth performance

The results of this study demonstrate that the inclusion of A. philoxeroides in the diet significantly improved the growth performance of gourami, as reflected by the increased specific growth rate (SGR). The highest growth was observed in fish fed with the P2 diet (20% inclusion), indicating that this level of substitution provides an optimal balance of nutritional benefit and palatability.

The enhancement in SGR can be attributed to the nutritional composition of A. philoxeroides, which is known to contain considerable amounts of crude protein, dietary fiber, and bioactive compounds such as flavonoids, saponins, and phytosterols [19, 20]. These compounds may contribute to improved feed utilization and digestive efficiency by enhancing enzymatic activity and gut morphology in herbivorous fish.

The findings align with previous studies on other plant-based feed additives. For instance, Armando et al. [21] reported that aquatic plants such as Lemna sp. and Azolla increased growth rates in herbivorous fish due to their nutrient density and fiber digestibility. Moreover, Islamy [10, 22] emphasized the potential of aquatic macrophytes as cost-effective protein sources for aquaculture feed, capable of reducing reliance on traditional plant or animal meals.

Interestingly, while all experimental groups (P1-P3) outperformed the control in terms of SGR, the increase plateaued beyond 20% inclusion. The slightly lower growth rate in P3 (30% inclusion) may reflect diminishing returns at higher substitution levels, possibly due to increased levels of antinutritional factors such as tannins, saponins, and oxalates. Tannins are known to form insoluble complexes with proteins, thereby reducing digestibility, while saponins can damage intestinal mucosa and impair nutrient absorption. Oxalates, on the other hand, may chelate essential minerals such as calcium and magnesium, limiting their bioavailability. The combined effects of these compounds likely contributed to the reduced growth performance observed in the P3 group, consistent with previous findings in other herbivorous fish fed high levels of unprocessed plant material [23]. This trend is consistent with prior findings indicating that excessive inclusion of unprocessed plant material in fish diets may lead to reduced feed intake and slower growth [24].

The observed optimal growth performance and feed utilization in the P2 group support the assertion that a 20% inclusion level of can constitute the most effective diet formulation among those evaluated in this study. This interpretation aligns with existing literature indicating that plant-based inclusions can yield diminishing returns or even detrimental effects at higher levels due to anti-nutritional factors (ANFs) that can increase with dosage [25, 26]. In particular, the 20% inclusion appears to provide a substantial supply of crude protein, dietary fiber, and bioactive compounds such as flavonoids and saponins, which may collectively enhance digestive enzyme activity and gut morphology, thereby improving nutrient absorption and growth performance [27]. Similar patterns have been reported in other plant-based substitution studies where moderate replacement levels supported favorable feed efficiency and growth relative to higher substitution levels that led to reduced intake and impaired feed conversion efficiency, nutrient digestibility, and overall growth performance [25, 26, 28].

At the 30% inclusion level (P3), the decline in growth is consistent with a dose-dependent increase in ANFs that can compromise nutrient utilization. Tannins and saponins, present in many plant tissues, are known to bind dietary proteins and disrupt intestinal integrity, respectively, which can reduce protein digestibility and impair nutrient absorption when concentrations are elevated [26, 29]. Moreover, oxalates present in certain plants can chelate minerals, reducing mineral bioavailability and further restraining growth under high inclusion conditions [30]. This pattern aligns with evidence that excessive unprocessed plant material in aquafeeds can suppress feed intake and growth, likely due to reduced palatability and impaired nutrient bioavailability [31, 32]. Collectively, the P2 level appears to offer an optimal balance by capitalizing on the nutritional benefits of A. philoxeroides while mitigating the adverse effects of ANFs that intensify with greater dietary inclusion.

These findings are consistent with prior research across various fish species showing that plant-based diet components can support growth and feed utilization up to an optimal threshold, beyond which performance declines due to anti-nutritional and mineral-chelating effects, as well as potential impacts on gut morphology and enzyme activity. For instance, studies in tilapia and other teleosts have noted reduced growth and feed efficiency when high levels of plant-based ingredients substitute major portions of fish meal, especially when ANFs are not sufficiently mitigated (e.g., through processing or supplementation) [32-34]. Moreover, moderate plant inclusion can maintain or even enhance digestive capacity and growth when bioactive compounds positively influence gut physiology [27]. The current results, therefore, fit within this broader framework, supporting the 20% inclusion as the optimal threshold for balancing nutritional benefits against anti-nutritional drawbacks in diets based on A. philoxeroides.

Furthermore, the consistent alignment of our interpretation with analogous findings from plant-based replacement studies in diverse aquaculture systems highlights the generalizability of the principle that there exists an optimum inclusion level maximizing performance while mitigating ANF-related risks. This principle is reflected in meta-analytic syntheses of fishmeal replacement by plant proteins, emphasizing that performance outcomes depend critically on the degree of replacement, plant material quality, processing methods, and the inclusion of anti-nutritional mitigants or supportive minerals and amino acids. Thus, the present analysis indicating P2 as the optimal inclusion level is well-supported by cross-study evidence demonstrating that moderate plant inclusion can sustain superior growth and feed efficiency, while higher inclusions are associated with performance penalties driven by ANFs and limitations in mineral bioavailability. Overall, the growth response suggests that 20% inclusion of A. philoxeroides is a promising level for functional feed formulation in gourami culture, offering a balance between nutrient supply and fish acceptance. Beyond that threshold, the benefits may be offset by potential digestive challenges or reduced feed efficiency. 

4.2 Feed conversion ratio (FCR)

The feed conversion ratio (FCR) is an essential parameter in aquaculture that quantifies feed efficiency by indicating the feed required to achieve a unit of biomass gain in fish. Research highlights that the incorporation of specific feed materials can significantly alter FCR outcomes. For instance, a study demonstrating improved FCR in Nile tilapia with marine microalgae inclusion noted that diets supplemented with microalgae resulted in higher growth rates and better nutritional profiles due to enhanced nutrient digestibility [7]. This aligns with findings related to A. philoxeroides, where a substantial inclusion in the diet can yield notable improvements in growth performance and FCR.

The beneficial effects on FCR attributed to A. philoxeroides can be partially explained by its nutrient profile, particularly its phytosterols and flavonoids, which are acknowledged for their roles in enhancing digestive enzyme activity and promoting better nutrient absorption. These compounds may support gut health, which is vital for optimizing feed utilization, especially within herbivorous species [35]. Similar mechanisms have been documented where other plants, such as Ocimum sanctum, resulted in improved FCR due to better nutrient uptake and palatability, albeit their specific contributions are less conclusive for tilapia [36].

While the inclusion of A. philoxeroides at a certain level may enhance FCR, it is important to note that higher inclusion levels (such as 30%) could lead to diminished digestibility or the introduction of antinutritional factors [37]. This observation has been supported by studies showing that excessive plant material can negatively impact growth performance due to compounds such as tannins and saponins, which can impair protein assimilation [38]. This reinforces that a balance in dietary composition is crucial to maximizing feed efficiency without compromising fish health or growth performance.

Overall, the insights gained from various studies underscore the potential of A. philoxeroides and similar plant materials as functional feed ingredients to enhance FCR in herbivorous fish. Identifying optimal inclusion levels not only improves the economic viability of aquaculture practices but also supports sustainable feed strategies that leverage plants rich in beneficial phytochemical constituents [39, 40].

4.3 Hematological response

Hematological parameters such as erythrocyte and leukocyte count serve as key biomarkers in evaluating fish health and immune function. Recent studies indicate that dietary supplementation with various herbal materials and functional feeds can enhance these parameters, suggesting improved health and immunity in fish. Enhanced metabolic activity is crucial as it supports growth and maintains efficient oxygen delivery to tissues, especially in fish subjected to active feeding regimes or stressors [41].

The elevated leukocyte count, particularly in the 20% inclusion group, suggests immune-modulating properties of A. philoxeroides. Bioactive components such as flavonoids, phenolics, and saponins found in this plant may relate to improved immune responses. Compounds in these plants can stimulate the production and activation of various white blood cells, thereby bolstering nonspecific immunity [42]. Previous studies have indicated that dietary supplements, including herbal mixes, can induce increases in leukocyte counts and overall immune function across different fish species. For example, increases have been observed in Helostoma temminckii when administered herbal-based diets [43].

Interestingly, the P3 group did exhibit slight reductions in hematological values compared to the P2 group, suggesting a threshold beyond which the addition of plant material might interfere with digestibility or nutrient absorption, potentially due to the presence of antinutritional factors. Increased inclusion of certain herbs may lead to adverse effects, such as lower nutrient bioavailability or immune response modulation, underscoring the importance of optimal inclusion levels in aquaculture feeds [44].

Overall, these findings support the potential of A. philoxeroides as a functional feed ingredient, indicating it not only enhances growth but also contributes to better blood health in fish, thereby promoting overall resilience and well-being within aquaculture systems. Such insights advocate for the incorporation of this plant in sustainable aquaculture, as its immunomodulatory effects could lead to healthier fish populations.

4.4 Environmental implications

Utilizing A. philoxeroides, commonly known as alligator weed, in aquaculture represents a strategic approach that can benefit fish growth and health while addressing the ecological impacts of this invasive species. The integration of A. philoxeroides into fish feed may enhance aquatic ecosystems while simultaneously managing the overgrowth issues associated with this plant in freshwater environments. By converting a problematic species into a resource, aquaculture can contribute to ecological sustainability goals.

Studies indicate that A. philoxeroides exhibits significant biomass accumulation in nutrient-rich environments, particularly when nitrogen and phosphorus are available [45]. This growth potential allows for large-scale harvesting and incorporation into fish feed, which can mitigate its ecological burden. Moreover, previous findings suggest that leveraging invasive species like A. philoxeroides in aquaculture could offset their negative ecological impacts while redistributing their nutritional value into fish diets [46].

Research by Harms et al. [47] highlights that A. philoxeroides demonstrates plasticity in response to nutrient availability, offering a competitive advantage in various environments. This adaptability implies that management strategies using A. philoxeroides can be refined to optimize fish growth while controlling its invasiveness in freshwater ecosystems. The nutrients provided by incorporating A. philoxeroides into fish feeds not only support fish health but also contribute to sustainable ecosystem management by controlling the growth of this invasive plant through strategic harvesting.

The large-scale adoption of A. philoxeroides in aquaculture shows promise, as it facilitates dual benefits—enhancing aquaculture productivity while contributing to ecological management. This approach aligns with broader sustainability goals, transforming invasive plant species from ecological threats into valuable resources [48]. By repurposing A. philoxeroides, it is plausible to alleviate its negative impacts on native flora and fauna, thus benefiting biodiversity conservation efforts in freshwater ecosystems.

Overall, the potential for A. philoxeroides as a sustainable ingredient in aquaculture addresses both growth and health parameters in edible fish and responds to the urgent need for effective environmental management of invasive species in aquatic systems. With widespread adoption, such strategies could significantly reduce the ecological burden associated with A. philoxeroides overgrowth, fostering its integration into sustainable aquaculture practices.

4.5 Functional feed perspective in sustainable aquaculture

The integration of A. philoxeroides into fish feed presents significant benefits that extend beyond basic nutrition. This invasive aquatic plant demonstrates immunostimulatory effects that underscore its potential as a biofunctional feed ingredient. As consumers and the aquaculture industry increasingly seek alternatives to antibiotics and synthetic growth promoters, the incorporation of A. philoxeroides aligns with sustainable aquaculture principles. Recent research indicates that plant-based functional feeds can enhance fish welfare while promoting consumer safety and health [49, 50].

The immunostimulatory properties of A. philoxeroides stem from its rich bioactive compound profile, which includes flavonoids, phenolics, and saponins. These compounds can enhance immune responses in fish, making functional feeds derived from such plants advantageous for both growth and overall fish health in aquaculture systems that prioritize the reduction of chemical additives [51, 52]. For instance, studies have shown how dietary strategies incorporating natural ingredients can bolster innate immune responses in fish, providing an environmentally friendly approach to disease management.

The movement toward plant-based functional feeds resonates strongly with the One Health approach, which promotes the interconnection between human, animal, and environmental health [49]. By improving fish welfare and reducing reliance on antibiotics, A. philoxeroides showcases its dual role as both a functional feed ingredient and a management strategy for invasive species. This approach can help mitigate environmental impacts associated with A. philoxeroides overgrowth, subsequently enhancing ecosystem management [49].

Moreover, continual improvement in aquaculture welfare practices through the use of biofunctional ingredients can foster consumer confidence and safety in farmed fish products. As aquaculture faces increasing demand from a growing population, alternative feeding strategies that prioritize sustainability will be crucial [53, 54]. Thus, the development and adoption of functional feeds with A. philoxeroides not only meet the nutritional needs of farmed fish but also align with broader ecological sustainability goals, offering a holistic pathway for responsible aquaculture [54, 55].

Overall, A. philoxeroides stands out as a promising candidate for functional feed applications in aquaculture, representing a practical solution that encapsulates health, sustainability, and ecological management.

4.6 Practical recommendations for application

To effectively utilize A. philoxeroides as a functional feed ingredient in aquaculture, it is recommended that incorporation rates not exceed 20%. Such limitations are proposed based on research findings, indicating potential antinutritional factors that could arise from excessive inclusion rates. Current studies emphasize various pre-treatment methods—including drying, fermentation, and enzymatic hydrolysis—that may help mitigate these antinutritional effects and enhance the bioavailability of nutrients present in A. philoxeroides [56].

Research demonstrates that A. philoxeroides has a high-water content (over 90%), and understanding its biochemical composition is crucial for its optimal incorporation into fish diets. The main constituents of its dry matter, which include cellulose, hemicellulose, and lignin, suggest potential barriers to nutrient absorption that pre-treatment methods could alleviate, ultimately improving efficacy in feeding applications [56]. Implementing such methods can encourage aquaculture farmers to safely and efficiently utilize this underutilized biomass while avoiding the pitfalls related to antinutritional factors.

Additionally, studies focusing on submergence and nutrient availability have revealed that A. philoxeroides can thrive under submerged conditions and responds positively to nutrient-rich environments. This adaptability highlights its potential for sustainable application within aquaculture systems [57]. Beyond addressing the nutrient needs of fish, adopting sustainable practices involving A. philoxeroides could foster environmental management strategies targeting invasive species, further supporting ecological balance in aquatic systems [58].

Overall, continued exploration of treatment methods for A. philoxeroides will reinforce its viability as a functional feed ingredient. By enhancing nutrient bioavailability and ensuring safe feeding practices, aquaculture systems can benefit from this approach while contributing to broader sustainability and ecological goals.

The authors would like to express their sincere gratitude to all institutions and universities involved in supporting this research. Special thanks are extended to Universitas Brawijaya for providing the facilities, administrative support, and research infrastructure that enabled the successful completion of this study. The authors also acknowledge the contributions of academic colleagues, laboratory staff, and students whose assistance was invaluable throughout the research process. This study is a collaborative effort that reflects the shared commitment to advancing sustainable aquaculture practices.