INTRODUCTION

Protein is an essential nutrient in aquaculture diets for crustaceans because it directly influences growth performance, immune function, and cellular processes such as tissue repair, renewal, and reproduction [1]. In _Macrobrachium _rosenbergii, the recommended dietary crude protein level for achieving optimal growth is approximately 35% of the total diet [2]. Fishmeal remains the predominant protein source in commercial aquafeeds because of its balanced amino acid profile, high digestibility, and low content of anti-nutritional factors (ANFs) [3, 4]. However, increasing aquaculture production and the growing demand for fish-derived products have contributed to rising fishmeal costs and concerns regarding the environmental and ecological consequences of overfishing [5, 6]. Consequently, rising price volatility and limited availability of fishmeal have intensified the search for sustainable, cost-effective alternative protein sources for aquaculture feeds [7, 8]. Plant-derived protein ingredients, including soybean, canola, blue-green microalgae, and cottonseed, have been extensively investigated as potential alternatives to fishmeal in sustainable aquaculture systems because of their favorable protein content and availability [3, 4, 9].

Leucaena leucocephala, a leguminous plant belonging to the family Fabaceae, is widely distributed throughout tropical and subtropical regions of Asia, Africa, and the Americas [10]. The plant contains relatively high protein concentrations (25.25%–30.81%) and provides essential amino acids, including lysine, leucine, proline, and serine, making it a potentially valuable feed ingredient [10]. In marine shrimp (_Penaeus _vannamei), dietary inclusion of 5%–20% L. leucocephala leaf powder did not adversely affect animal health, biochemical composition, or meat quality [9]. Similarly, replacement of fish protein with 25%–50% L. leucocephala leaf meal devoid of mimosine did not negatively affect the health of Nile tilapia [11]. Furthermore, fish fed diets containing 33% L. leucocephala seed powder exhibited enhanced immune responses during infection with _Vibrio _harveyi and Pseudomonas aeruginosa [12]. Beyond its nutritional value, L. leucocephala contains bioactive phytochemicals, particularly flavonoids, which possess antioxidant and immunomodulatory properties [13]. Flavonoids have been reported to induce erythropoietin gene expression and exhibit strong anti-inflammatory and antioxidant activities [14]. In aquatic animals, these compounds can interact with hemocyte receptors and activate intracellular signaling pathways associated with immune defense mechanisms [15]. Collectively, previous studies have demonstrated that partial replacement of fishmeal with up to 20% L. leucocephala leaf meal can be implemented without causing severe adverse effects on animal health or growth performance [9].

Despite these advantages, the application of plant-derived proteins in aquafeeds remains constrained by the presence of ANFs, including phytates, trypsin inhibitors, lectins, and other compounds that may reduce palatability, impair nutrient utilization, and adversely affect growth and immune performance [16]. Fermentation has emerged as an effective strategy for improving the nutritional quality and functionality of plant materials through biochemical transformation and detoxification [17]. Among the microorganisms used for this purpose, lactic acid bacteria (LAB), including species of Lactiplantibacillus, Aerococcus, and Streptococcus, have received considerable attention because of their safety and ability to enhance nutrient bioavailability [17, 18]. Fermented products are generally recognized as safe, nutritionally enriched, and stable during storage. Controlled fermentation using selected LAB strains has therefore been proposed as a valuable approach for producing high-quality functional feed ingredients [17].

Lactiplantibacillus_ plantarum_ is a particularly promising microorganism because of its capacity to biotransform phenolic-rich substrates and generate bioactive compounds with potent antioxidant activity. This bacterium facilitates the conversion of hydroxybenzoic acids, hydrolyzes tannins to gallic acid, and promotes the transformation of hydroxycinnamic acids into biologically active metabolites [18]. In addition to its well-recognized probiotic properties in humans, L. plantarum has demonstrated beneficial effects in aquaculture by enhancing growth performance, improving immune responses, and increasing disease resistance through the production of antimicrobial compounds that suppress pathogenic bacteria [19]. In _M. _rosenbergii, dietary supplementation with L. plantarum has been shown to improve host-associated microbiota, feed utilization, carcass composition, immune status, and survival following infection with _Aeromonas _hydrophila [19].

Although the nutritional and immunological benefits of L. leucocephala and L. plantarum have been independently documented, limited information is available regarding the combined application of these two components as a fermented functional feed ingredient in crustacean aquaculture. Previous studies have primarily focused on direct inclusion of L. leucocephala leaf meal or probiotic supplementation alone, whereas the effects of L. leucocephala fermented with L. plantarum on immune modulation, antioxidant status, intestinal health, and disease resistance have not been investigated in _M. _rosenbergii. Furthermore, the influence of fermented L. leucocephala on hematopoietic activity, phenoloxidase-mediated defense responses, and immune-related gene expression during bacterial infection remains poorly understood. Addressing these knowledge gaps is essential for evaluating the feasibility of fermented plant-based proteins as sustainable alternatives to fishmeal in freshwater prawn production systems.

Therefore, this study was conducted to evaluate the effects of partial replacement of fishmeal with L. leucocephala leaves fermented by L. plantarum TISTR1284 in diets for _M. _rosenbergii. Specifically, the study investigated the effects of dietary inclusion of fermented L. leucocephala on antioxidant properties of the diets, total hemocyte count, hematopoietic tissue proliferation, phenoloxidase activity, immune-related gene expression, intestinal morphology, and resistance to _A. _hydrophila infection. The findings provide insight into the potential application of fermented L. leucocephala as a sustainable functional feed ingredient capable of reducing dependence on fishmeal while enhancing immune competence and disease resistance in freshwater prawn aquaculture.