© C. Debnath, Published by EDP Sciences 2025

1 Introduction

Aquaculture provides nearly half of all fish consumed worldwide (Verdegem et al., 2023), with polyculture systems representing a sustainable approach to maximize productivity through efficient resource utilization (Stickney, 2013). These systems leverage complementary feeding habits and behavioral traits of different species to enhance overall production efficiency (Pillay and Kutty, 2005). The success of these systems depends significantly on stocking strategies, as Shrestha et al. (2011) established that species compatibility and proportional stocking represent critical determinants of polyculture success that extend beyond conventional considerations of spatial requirements or carrying capacity.

Channa striata (striped murrel) and Heteropneustes fossilis (stinging catfish) represent two economically important freshwater fish species cultivated throughout South and Southeast Asia (Talwar and Jhingran, 1991; Kumar et al., 2022). C. striata is valued for its therapeutic properties in traditional medicine (Berlian et al., 2023), particularly for wound healing and postpartum recovery (Ab Wahab et al., 2015), while H. fossilis is recognized for its high iron content and medicinal value (Hossain et al., 2015). Both species command premium prices in markets across Asia and exhibit air-breathing capabilities, making them resilient candidates for aquaculture in oxygen-constrained environments (NBFGR, 2011).

The north-eastern region of India, particularly Meghalaya, presents unique opportunities and challenges for aquaculture development due to its distinctive hill climate characterized by temperatures ranging from 18−25°C, abundant rainfall, high dissolved oxygen levels, and water bodies with lower mineral content compared to lowland regions (Das, 2018).This region harbors economically valuable indigenous species that remain underexploited despite their market potential (Pandey and Rameshori, 2024).

The physiological and behavioral characteristics of these species create complex dynamics for polyculture applications. C. striata exhibits predominantly carnivorous feeding behavior with strong predatory tendencies and territorial aggression (Khan et al., 2023), while H. fossilis functions as an omnivorous bottom-dweller equipped with venomous pectoral spines for defense (Reenamole and Ambili, 2016). Although their complementary ecological niches suggest potential for efficient resource utilization through spatial and trophic separation, their interaction dynamics under the distinctive environmental conditions of hill climates remain poorly understood. This knowledge gap necessitates systematic investigation of optimal species proportions to maximize production efficiency while minimizing aggressive interactions and stress-induced losses in polyculture systems.

Stocking density and species ratios represent critical factors determining the success of polyculture operations by influencing competition, predation, resource utilization, and physiological stress (Shrestha et al., 2011; Li et al., 2021). Suboptimal stocking strategies trigger stress-induced physiological dysfunction, characterized by elevated cortisol levels, altered feeding behavior, and compromised immune responses, which manifests through hematological alterations, metabolic disruptions, and behavioral changes that reduce both fish welfare and production efficiency (Tort, 2011; Dara et al., 2023). While these stress-production relationships are well-established in conventional aquaculture systems, their expression under the distinctive environmental conditions of hill regions remains poorly understood.

This knowledge gap is particularly pronounced for air-breathing fish species, where research specifically addressing polyculture under hill climate conditions remains notably scarce in scientific literature. This research scarcity reflects the convergence of multiple specialized domains—hill climate aquaculture, air-breathing fish species, and polyculture optimization—that have traditionally been studied separately, creating a knowledge gap at their intersection. The distinctive environmental parameters of hill regions—including temperature fluctuations, water chemistry, and altitude effects—potentially modulate fish physiological responses, interspecies dynamics, and overall production outcomes in ways that differ significantly from lowland tropical systems (Debnath et al., 2025; Debnath, 2025). This research limitation constrains the development of regionally optimized aquaculture protocols for mountain regions worldwide. While high-altitude aquaculture operations exist in various mountain regions, including recent rainbow trout cultivation at 3000 meters above sea level in the Chilean Andes (Pepe-Victoriano et al., 2025) and aquaculture development in Ethiopian highland systems (Alemayehu and Getahun, 2017), each region presents distinct environmental conditions that require specific management approaches. The limited research on stocking optimization under these specialized environmental conditions indicates a need for systematic investigation to establish region-specific management strategies for sustainable mountain aquaculture development.

The present study addresses this research gap by investigating how different stocking ratios influence growth performance, physiological responses, and injury patterns in polycultured C. striata and H. fossilis under the hill climate conditions of Meghalaya. C. striata (carnivorous) and H. fossilis (omnivorous) occupy different trophic levels, enabling the complementary resource utilization that characterizes polyculture systems. This investigation has significant implications for global aquaculture development in similar hill regions, potentially informing stocking strategies that optimize production while minimizing stress-induced adverse effects on fish health and welfare. By elucidating the complex interactions between stocking ratios, species characteristics, and environmental conditions, this study aims to establish foundational knowledge for developing sustainable polyculture systems in hill regions globally.

2 Materials and methods

2.1 Experimental site and environmental conditions

The experiment was conducted at the ICARResearch Complex for North Eastern Hill Region, Ri-Bhoi district, Meghalaya, India (25.6°N, 91.9°E). The facility is situated at an elevation of 950 m above sea level, characterized by a hill climate with moderate temperatures ranging from 18−25°C during the experimental period (March-April 2024). The region received approximately 450mm of rainfall during the study period, with relative humidity ranging from 65–85%.

2.2 Experimental design

2.2.1 Experimental units and water quality

Nine outdoor concrete tanks (3m × 2m × 1m) with a water depth of 0.8m were used for the experiment. Each tank was filled with dechlorinated tap water and equipped with ten broken PVC pipes to provide ‘hideouts’ for fish. Continuous aeration was supplied using air stones connected to an air blower.

Water exchange was performed at 1/3rd of water column volume every three days to maintain water quality, a regimen established through preliminary trials that demonstrated effective maintenance of ammonia and nitrite levels below critical thresholds while minimizing handling stress. All water quality measurements were conducted 24 hours after water exchange using APHA (2017) protocols to ensure parameter stabilization and represent typical conditions experienced by the fish during the culture period. Parameters were maintained within optimal ranges throughout the experimental period: temperature ranged from 18−25°C (average 21.6 ± 1.8°C), dissolved oxygen (DO) between 6.5–7.8 mg/L, pH values of 7.2−7.8, ammonia nitrogen below 0.05 mg/L, nitrite-nitrogen below 0.01 mg/L, total alkalinity ranging from 45−65 mg/L as CaCO₃, and total hardness between 35−55 mg/L as CaCO₃. These parameters reflect the distinctive water quality profile of the hill climate and were consistently maintained across all experimental treatments.

2.2.2 Experimental fish and acclimatization

Juvenile specimens of Channa striata (15.3 ± 0.4 g) and Heteropneustes fossilis (15.1 ± 0.5 g) were procured from the institute’s fish rearing facility and subjected to a seven-day acclimatization period in separate fiberglass tanks. These tanks were maintained under continuous aeration with regular water exchange, replicating the conditions of the experimental system to minimize environmental stress. During acclimatization, fish were fed a high-protein commercial feed (ABIS India, 40% crude protein) at 3% of body weight, split into two equal feedings per day. Uneaten feed was removed 30 minutes post-feeding by siphoning, and the removed volume was replenished with clean water of equivalent quality. To ensure fish health and minimize disease risk, a five-minute prophylactic bath with 2% potassium permanganate solution was administered on alternate days throughout the acclimatization period.

2.2.3 Experimental treatments

A total of 270 fish were used in the experiment, distributed across three treatment groups with three replicates each, maintaining a consistent stocking density of 30 fish per tank (5 fish/m²). Preliminary pilot observations indicated that ratios beyond 70:30 resulted in excessive stress responses while ratios closer to 50:50 showed minimal competitive interactions, leading to the selection of these specific proportions to generate measurable physiological responses. Fish were randomly assigned to experimental tanks to eliminate selection bias:

• T1: 1:1 ratio (15C. striata : 15 H. fossilis per tank) − This balanced ratio was selected to assess equal representation of both species.

• T2: 3:7 ratio (9C. striata : 21 H. fossilis per tank) − This ratio, with higher proportion of H. fossilis, was chosen to examine the effects of dominance by the omnivorous bottom-dwelling species.

• T3: 7:3 ratio (21C. striata : 9 H. fossilis per tank) − This ratio, with higher proportion of C. striata, was selected to investigate the effects of dominance by the predatory species.

2.2.4 Feeding and management

Fish were fed a commercial floating pellet feed (ABIS India) formulated with 40.2 ± 0.3% crude protein, 5.1 ± 0.2% crude fat, 6.2 ± 0.3% crude fiber, and 10.3 ± 0.4% moisture. Feeding was carried out twice daily at 08:00 and 16:00 hours, at a rate of 5% of the estimated biomass. Feed rations were adjusted weekly based on the average weight of 10 randomly sampled fish per tank (5 fish per species to maintain proportional representation) to ensure appropriate feeding levels for both species. Uneaten feed was removed 30 minutes post-feeding by siphoning, and the extracted water was replaced with clean water of similar quality and temperature. Routine daily maintenance included the removal of fecal matter and settled debris via bottom siphoning, followed by proportional water replenishment to maintain optimal water quality and consistent water levels.

2.3 Sampling and analytical procedures

2.3.1 Growth performance

Sampling was performed on Days 1, 7, 14, 28, and 56 of the experimental period. At each time point, 5 fish of each species were randomly netted from every experimental tank using a scoop net. To minimize handling stress—particularly for H. fossilis, which possesses venomous pectoral spines—fish were anesthetized using clove oil at a concentration of 50 µL/L. Complete anesthetization, characterized by loss of equilibrium and reduced opercular movement, was achieved within 3−5 minutes for both species. Once anesthetized, individual fish were weighed with a precision digital balance (Shimadzu, ± 0.01 g) and measured for total length using a measuring board (± 0.1 cm accuracy). After data collection, fish were placed in aerated recovery tanks for 5–10 minutes until they resumed normal swimming behavior, and were then returned to their respective tanks. At the end of the 56-day trial, all tanks were fully drained, and the remaining fish were collected, identified by species, and counted to determine final survival rates.

The following growth parameters were calculated (Hopkins, 1992):

• Weight gain (WG) = Final weight (g) − Initial weight (g).

• Specific growth rate (SGR, %/day) = [(ln final weight − ln initial weight) / experimental days] × 100.

• Feed conversion ratio (FCR) = Dry feed intake (g) / wet weight gain (g).

• Survival rate (%) = (Final number of fish / Initial number of fish) × 100.

2.3.2 Blood collection and hematological analysis

Blood samples were collected from three randomly selected fish of each species from each replicate tank (nine fish per treatment) on sampling days. Fish were anesthetized in a separate container with clove oil solution (50 µL/L) until loss of equilibrium and reduced opercular movement were observed. Blood was drawn from the caudal vein using 2 mL heparinized syringes equipped with 23G needles by inserting the needle at a 45° angle just below the lateral line and posterior to the anal fin. For each fish, 0.5 to 1 mL of blood was collected and divided into two portions: one with anticoagulant (EDTA) for hematological analysis and one without anticoagulant for serum separation. After blood collection, fish were placed in aerated recovery tanks for 5–10 minutes until they resumed normal swimming behavior, and were then returned to their respective tanks.

Hematological parameters were assessed following the methodologies of Witeska et al. (2022). The red blood cell (RBC) count was determined using the traditional manual counting method with a Neubauer hemocytometer after diluting blood samples with Hayem's solution. White blood cell (WBC) count was similarly quantified using the Neubauer hemocytometer, but with Turk's solution as the diluent. Hemoglobin (Hb) concentration was measured using the cyanmethemoglobin method, where hemoglobin is converted to cyanmethemoglobin by addition of Drabkin's reagent, followed by spectrophotometric determination at 540 nm wavelength.

2.3.3 Biochemical analysis

Serum samples were prepared through centrifugation of collected blood at 3,000 rpm for 10 minutes and preserved at −20°C until analysis. Serum glucose concentration was quantified via the glucose oxidase-peroxidase method (Shaker and Swift, 2023). Total protein concentration was determined using the Biuret method (Lubran, 1978). Additionally, three key antioxidant enzymes were analyzed to assess oxidative stress status. Catalase (CAT) activity was measured by monitoring the rate of hydrogen peroxide decomposition (Hadwan et al., 2024). Superoxide dismutase (SOD) activity was determined through the enzyme's ability to inhibit pyrogallol auto-oxidation under alkaline conditions, with one unit of activity defined as the amount causing 50% inhibition (Gao et al., 1998). Glutathione peroxidase (GPx) activity was assessed using the NADPH oxidation method described by Sattar et al. (2024), which measures the rate of NADPH consumption during the reduction of oxidized glutathione in a coupled enzymatic reaction.

2.3.4 Immune response assessment

Lysozyme activity was determined by measuring the rate of lysis of Micrococcus lysodeikticus cells following the turbidimetric method described by Saurabh and Sahoo (2008). Respiratory burst activity was measured through the quantification of intracellular superoxide anion production using nitroblue tetrazolium (NBT) reduction according to the methods of Ellis (1990). Alternative complement pathway activity (ACH50) was assessed by measuring the hemolytic capacity of the fish serum against rabbit red blood cells following protocols established by Ellis et al. (2012).

2.3.5 Injury assessment

External injury assessment was conducted during each sampling interval by the same evaluators who were blinded to treatment groups, with tank identities concealed during scoring procedures to minimize assessment bias. Inter-rater reliability was established through initial training sessions and periodic calibration exercises to ensure consistent scoring. A structured injury classification system was developed based on previous studies of fish injury patterns (Wenzel et al., 2022) and adapted for the distinctive morphological features of the study species. For C. striata, injuries were categorized into two main types: spine injuries (damage or erosion to dorsal and caudal fins resulting from aggressive encounters) and body injuries (scale loss, wounds, or bruises on the body surface typically arising from territorial confrontations). For H. fossilis, injuries were classified as either self-inflicted injuries (resulting from contact with their own venomous pectoral spines during stress-induced erratic swimming behavior) or predatory injuries (distinctive wound patterns caused by C. striata attacks).

To quantify injury severity, a standardized scoring system was implemented on a scale of 0 to 3, where 0 represented no visible injury, 1 indicated mild injury affecting a limited body area with superficial tissue damage, 2 denoted moderate injury with more extensive area coverage or deeper tissue involvement, and 3 represented severe injury characterized by substantial tissue damage, extensive wound area, or compromised physiological function. Injury incidence (%) was calculated as the percentage of fish exhibiting any visible injury (injury score ≥1) among the total number of fish sampled from each treatment at each sampling point.

2.4 Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics version 21.0. Prior to analysis, normality was confirmed using Shapiro-Wilk tests (all p > 0.05, range: 0.089–0.441), and homogeneity of variance was verified using Levene's tests (all p > 0.05, range: 0.094–0.441). After confirming these assumptions were met, differences among treatments were analyzed using one-way ANOVA. Where significant differences were detected, Tukey's HSD post-hoc test was applied to identify specific differences between treatment groups. All data were expressed as mean ± standard error (SE), and statistical significance was established at p < 0.05 for all analyses.

3 Results

3.1 Water quality parameters

Water quality parameters remained within acceptable ranges for fish culture throughout the experimental period across all treatments (Tab. 1). No significant differences were observed among treatments for most parameters, except for ammonia levels which were slightly higher in T3 (p < 0.05).

3.2 Growth performance

Table 2 presents the growth performance parameters for both species across different stocking ratio treatments. Statistical analysis revealed significant differences (p< 0.05) across all treatments for weight gain, SGR, FCR, and survival rate.

The balanced ratio treatment (T1) consistently demonstrated superior performance across all measured parameters for both species. Growth parameters followed a clear pattern with T1 showing the highest values, followed by T2 (higher proportion of H. fossilis), and T3 (higher proportion of C. striata) showing the poorest performance. This trend was consistent across both species, though more pronounced in H. fossilis.

Feed utilization efficiency, as indicated by FCR values, progressively declined from the balanced ratio toward both unbalanced treatments, with T3 showing the least efficient feed conversion. Survival rates followed similar patterns, with C. striata maintaining higher survival percentages across all treatments compared to H. fossilis. The C. striata-dominant treatment (T3) exhibited particularly detrimental effects on H. fossilis survival. Overall biomass production paralleled these trends, with T1 yielding the highest total biomass and T3 the lowest, further demonstrating the production advantages of the balanced stocking ratio.

The growth trajectory over the 56-day period showed divergence among treatments starting from day 14, with differences becoming progressively more pronounced (Figs. 1 and 2).

Fig. 1 Growth trajectory of Channa striata under different stocking ratio treatments over the 56-day experimental period.

Fig 2 Growth trajectory of Heteropneustes fossilis under different stocking ratio treatments over the 56-day experimental period.

3.3 Hematological parameters

Hematological parameters for both species across treatments are presented in Table 3. Significant alterations were observed across treatments, particularly from day 14 onwards. Both species showed progressive increases in RBC and WBC counts, with T3 exhibiting the highest values, followed by T2 and T1 (p < 0.05). Conversely, hemoglobin concentrations decreased over time in T2 and T3, with the lowest values observed in T3 (p < 0.05).

3.4 Biochemical parameters

Biochemical parameters measured over the experimental period are presented in Table 4. Significant differences were observed in all biochemical parameters across treatments (p < 0.05). Serum glucose levels progressively increased in all treatments, with the highest levels observed in T3 on day 56, followed by T2 and T1. Total protein levels showed an opposite trend, with the lowest values in T3 by day 56. Antioxidant enzyme activities (CAT, SOD, GPx) were significantly elevated in T3 and T2 compared to T1 (p < 0.05) by day 56, with progressive increases observed throughout the experimental period. These biochemical alterations were more pronounced in H. fossilis than in C. striata.

3.5 Immune parameters

Immune parameters measured throughout the experimental period are presented in Table 5. All immune parameters showed significant differences across treatments (p < 0.05), with progressive changes from day 14 onward. Lysozyme activity, respiratory burst activity, and ACH50 were highest in T1 and lowest in T3 for both species throughout the study period. The differences became more pronounced over time, with the greatest disparities observed on day 56.

3.6 Injury assessment

The injury assessment results are presented in Table 6 and Figure 3. Significant differences in injury patterns were observed across treatments for both species (p < 0.05). For C. striata, the highest injury scores were observed in T3, followed by T2 and T1. For H. fossilis, an interesting pattern emerged. Self-inflicted injuries were highest in T2, where H. fossilis was stocked at the highest proportion (70%). Predatory injuries, however, were highest in T3, where C. striata dominated (70%). The overall injury incidence was highest in T2 for H. fossilis and T3 for C. striata (Fig. 4).

Fig. 3 Temporal progression of injury severity scores in Channa striata and Heteropneustes fossilis across different stocking ratio treatments during the 56-day experimental period. Data points represent mean values with error bars indicating standard deviation. Letters above error bars denote statistical significance, where different letters indicate significant differences between treatments at each sampling time point (p < 0.05).

Fig. 4 Temporal progression of injury incidence in Channa striata and Heteropneustes fossilis across different stocking ratio treatments during the 56-day experimental period. Data points represent mean percentage of fish exhibiting any visible injury, with error bars indicating standard deviation. Letters above error bars denote statistical significance, where different letters indicate significant differences between treatments at each sampling time point (p < 0.05).

4 Discussion

4.1 Growth performance and stocking ratios

The balanced stocking ratio (T1) demonstrated superior growth performance for both C. striata and H. fossilis in this polyculture system, highlighting the importance of species proportions in polyculture optimization. The findings align with fundamental ecological principles of interspecific competition and resource partitioning (Roughgarden, 1976; Stewart and Levin, 1973). The significantly higher SGRs and lower FCRs observed in T1 suggest that balanced species proportions create complementary effects through niche complementarity, a phenomenon observed in various polyculture systems globally (Thomas et al., 2021; Kim et al., 2022).

Previous studies on stocking optimization have predominantly focused on conventional warmwater species in tropical conditions. However, Shrestha et al. (2011) established that species compatibility and proportional stocking represent critical determinants of polyculture success that extend beyond conventional considerations of spatial requirements or carrying capacity. This understanding provided the conceptual foundation for the stocking ratios evaluated in the present study, which aimed to identify optimal species proportions under the distinctive environmental conditions of hill climates.

The reduced growth performance observed in the C. striata-dominant treatment (T3) can be attributed to heightened intraspecific competition and territorial aggression. This competitive dynamic aligns with the findings of Prashant et al. (2023) research, which established a significant positive correlation (r=0.67, p=0.032) between fish density and aggression in freshwater species. Their study documented that snakehead fish exhibit particularly intense territorial behaviors during territorial disputes, behaviors that substantially intensify in higher-density environments. This behavioral pattern creates a costly metabolic trade-off where energy is diverted from somatic growth toward aggressive interactions, a phenomenon independently confirmed by Zhang et al. (2022) through their systematic documentation of behavioral patterns between territorial fish species in polyculture systems.

The physiological mechanisms underlying these competitive dynamics have been further elucidated by Sloman and Armstrong (2002), who established clear connections between elevated metabolic rates, aggressive behavior, and social hierarchy formation in fish populations. Zhang et al. (2021) expanded this understanding by demonstrating how aggressive behavior triggers cascading physiological effects on stress hormones, appetite regulation, and feeding efficiency. Our findings of optimal performance at balanced ratios (1:1) provide empirical support for Martins et al.'s (2012) assertion that appropriate social conditions significantly impact welfare indicators in farmed fish.

Interestingly, the H. fossilis-dominant treatment (T2) showed better growth performance than T3 but remained inferior to the balanced ratio. This finding suggests that species-specific responses to stocking densities can override general ecological principles, as demonstrated by Gimenez et al. (2023). Their research across gravel pit lakes revealed that stocking intensity and species composition significantly modulated fish community dynamics, producing nonlinear effects on community structure and functional characteristics. Similarly, our results indicate the relationship between stocking density and production outcomes follows complex patterns influenced by species-specific behavioral and physiological responses. This complexity emphasizes the need for context-specific optimization rather than universal prescriptions for polyculture systems.

4.2 Physiological responses

The hematological and biochemical alterations observed across treatments provide important insights into stress physiology beyond the immediate experimental context. The elevated RBC and WBC counts coupled with reduced hemoglobin levels in unbalanced treatments (T2 and T3) indicate classic stress responses consistent with the general adaptation syndrome described by Barton (2002). These responses have been documented across diverse fish taxa facing various stressors, suggesting conserved physiological mechanisms that transcend species boundaries (Skov et al., 2011).

The seeming contradiction between increased RBC counts and decreased hemoglobin levels observed in stressed fish can be explained by impaired erythrocyte function and hemoglobinsynthesis under chronic stress conditions. As demonstrated by Witeska et al. (2022), physiological stress in fish often leads to the production of smaller, less functional erythrocytes with reduced hemoglobin content, despite compensatory increases in total RBC counts. This adaptive response aims to maintain oxygen-carrying capacity under adverse conditions but becomes maladaptive when stress persists.

The elevated glucose levels observed in T2 and T3 treatments represent a well-established neuroendocrine response mediated by cortisol and catecholamines, which mobilize energy reserves to meet increased metabolic demands under stress (Tort, 2011). However, the concurrent reduction in serum protein levels suggests potential protein catabolism for gluconeogenesis, a mechanism that compromises growth and tissue synthesis when stress becomes chronic (Wenzel et al., 2022). This metabolic shift from anabolism to catabolism represents a significant production constraint in aquaculture systems globally, as highlighted by Abdel-Tawwab et al. (2019) in their investigation of stress physiology in cultured fishes.

The enhanced antioxidant enzyme activities (CAT, SOD, GPx) in unbalanced treatments indicate activation of cellular defense mechanisms against oxidative stress, consistent with findings by Lushchak (2016) and Song et al. (2023) across multiple fish models. While these enzymatic responses are protective in the short term, sustained elevation implies continuous oxidative challenge that can eventually overwhelm defense systems and lead to cellular damage. Interestingly, Martínez-Porchas et al., (2009) proposed that moderate, transient oxidative stress might act as a hormetic stimulus in some aquaculture species, potentially enhancing resilience to subsequent stressors—a perspective that warrants further investigation in polyculture systems.

4.3 Hill climate modulation of stress responses

The physiological stress responses observed in both species displayed distinctive patterns that appear to have been significantly modulated by the environmental conditions characteristic of Meghalaya's hill climate, presenting both challenges and opportunities for regional aquaculture development. The more pronounced hematological alterations observed in H. fossilis suggest this species experienced greater physiological stress, likely exacerbated by the temperature fluctuations characteristic of hill regions. This observation aligns with findings by Nati et al. (2021), who demonstrated that thermal stability is more critical than absolute temperature for many tropical fish species.

The osmoregulatory responses in both species may have been further influenced by the lower mineral content typical of hill waters, which could increase osmoregulatory load and stress susceptibility. This relationship between water hardness and stress physiology, documented by Romano et al. (2020), represents an environmental factor rarely considered in conventional aquaculture research but appears particularly relevant for hill region operations. These findings provide a novel perspective on water chemistry management in hill aquaculture, potentially informing targeted mineral supplementation strategies in similar environments globally.

The relatively high survival rates observed despite significant physiological perturbations may be partially attributed to the higher dissolved oxygen levels characteristic of hill waters (6.5−7.8 mg/L in this study), which could help offset the metabolic challenges of chronic stress. This potential protective effect aligns with observations by Lushchak et al. (2005) regarding enhanced stress tolerance in goldfish under hyperoxic conditions. This finding highlights an underexplored advantage of hill aquaculture that could inform region-specific production protocols for similar mountain ecosystems globally, including the Andes, Himalayas, Ethiopian highlands, and other temperate mountain regions where comparable environmental conditions create analogous physiological challenges and opportunities for sustainable polyculture development.

4.4 Immunological implications and disease management

The immunosuppression observed in unbalanced stocking treatments has significant implications for disease management in polyculture systems globally. The reduced lysozyme activity, respiratory burst function, and complement system activation in T2 and T3 indicate comprehensive compromise of innate immunity, the primary defense against pathogens in fish (Saurabh and Sahoo, 2008). This stress-induced immunosuppression represents a significant yet often overlooked production risk in intensive aquaculture operations.

The stress-immune relationship observed in this study exemplifies the concept of allostatic load, where chronic stress progressively depletes physiological resources necessary for maintaining immune vigilance (Ellis et al., 2012). This perspective extends beyond conventional productivity metrics to encompass disease resilience as a critical determinant of sustainable aquaculture. Notably, Barton (2002) demonstrated that even subclinical stress can significantly increase susceptibility to opportunistic pathogens that typically remain harmless under optimal conditions.

The differential immune suppression observed between species suggests species-specific immunomodulatory mechanisms that could inform selective breeding programs for enhanced disease resistance. This approach has been successfully implemented in multiple aquaculture species by Yáñez et al. (2015), who demonstrated significant heritability for stress resilience and immune function traits. The identification of stress-resilient populations or strains of C. striata and H. fossilis could substantially improve the sustainability of polyculture operations in hill regions.

4.5 Behavioral dynamics and animal welfare considerations

The distinct injury patterns observed across treatments provide valuable insights into behavioral interactions that extend beyond productivity considerations to encompass fish welfare, an increasingly important aspect of sustainable aquaculture (Browman et al., 2019). The high incidence of predatory injuries in H. fossilis under the C. striata-dominant treatment (T3) reveals aggressive predation attempts despite the defensive spines of the catfish, suggesting that natural defensive adaptations may become insufficient under confined culture conditions.Conversely, the high self-inflicted injury rates in H. fossilis under T2 indicate density-dependent stress responses that manifest as erratic swimming behavior, consistent with observations by Van de Nieuwegiessen (2009) in high-density catfish cultures. This finding challenges conventional stocking recommendations based solely on water quality and spatial requirements by highlighting behavioral compatibility as a critical determinant of stocking optimization.

The temporal progression of injuries over the experimental period suggests cumulative effects of chronic stress and social interactions, highlighting the importance of periodic grading or size sorting in long-term polyculture operations. This practice, while labor-intensive, might significantly improve both welfare and productivity outcomes as demonstrated by Wenzel et al. (2022) in African catfish culture. Future research could explore environmental enrichment strategies proposed by Näslund and Johnsson (2016) for reducing stereotypic behaviors in cultured fish, particularly in high-density treatments to mitigate self-injurious behaviors.

The integration of behavioral indicators into routine monitoring protocols could enable early intervention before physiological stress escalates to clinical manifestations. This approach aligns with the growing emphasis on welfare-based management practices in aquaculture, potentially improving both productivity and ethical outcomes in polyculture systems (Browman et al., 2019).

4.6 Limitations and future research directions

While this study provides valuable insights into stocking ratio optimization for polyculture of C. striata and H. fossilis under hill conditions, several limitations warrant consideration. The relatively short experimental duration (56 days) may not capture long-term adaptations or compensatory mechanisms that could develop over extended culture periods. Moreover, the fixed total stocking density (5 fish/m²) restricts extrapolation to the higher-intensity production systems common in commercial aquaculture, and the controlled tank environment may not adequately replicate the complex ecological dynamics of earthen pond systems, where natural productivity, benthic communities, and spatial heterogeneity could modify interspecies interactions and stress responses. Future research should explore molecular mechanisms underlying the observed physiological responses, particularly stress-signaling pathways and their epigenetic regulation under fluctuating temperature conditions characteristic of hill climates. Transcriptomic and proteomic approaches could provide valuable insights into acclimation potential. Investigation of gut microbiome dynamics under different stocking conditions might also reveal important mechanisms linking stress, immunity, and growth that could inform probiotic interventions.The economic dimensions of stocking ratio optimization deserve thorough analysis, including cost-benefit modeling under different market scenarios and production scales relevant to hill region economies. Such analyses should incorporate both conventional productivity metrics and emerging sustainability indicators to provide comprehensive decision support for diverse stakeholders.

5 Conclusion

This study establishes that stocking ratios significantly influence growth, physiological responses, and injury patterns in poly-cultured C. striata and H. fossilis under hill climate conditions, with balanced proportions (1:1) yielding optimal outcomes across all parameters measured. The balanced ratio resulted in superior growth performance (SGR: 1.88 ± 0.05 and 1.69 ± 0.06 %/day for C. striata and H. fossilis, respectively), improved feed conversion (FCR: 1.85 ± 0.08), enhanced survival (93.3 ± 1.9% and 90.0 ± 2.2%), and reduced injury rates.Unbalanced ratios triggered progressive physiological stress responses, including hematological alterations, metabolic disruptions, oxidative stress, and immune suppression. These effects were most pronounced in the C. striata-dominant treatment (T3), particularly for H. fossilis, while both species showed distinct injury patterns that reflected their ecological and behavioralcharacteristics.The distinctive environmental conditions significantly influenced fish responses to stocking manipulations, suggesting that aquaculture protocols developed for tropical lowland regions may not be directly applicable to hill aquaculture. These findings provide valuable insights for developing optimized, region-specific aquaculture protocols for hill regions globally, supporting sustainable aquaculture development in these environmentally distinctive areas.

Acknowledgments

The author gratefully acknowledges the support provided by ICAR-Research Complex for North Eastern Hill Region, Meghalaya. Thanks are also extended to the technical staff for their assistance in conducting the experiment and local fish farmers for providing valuable insights into traditional aquaculture practices in the region.

Funding

The study was conducted with the fund available under institute's contingency fund, no external funding was received.

Conflicts of interest

The author declares no conflict of interest.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical statement

All experimental procedures followed the Guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) for Experimentation on Fishes (2021), Government of India.

References

All Tables

All Figures

	Fig. 1 Growth trajectory of Channa striata under different stocking ratio treatments over the 56-day experimental period.
In the text

	Fig 2 Growth trajectory of Heteropneustes fossilis under different stocking ratio treatments over the 56-day experimental period.
In the text

Fig. 3 Temporal progression of injury severity scores in Channa striata and Heteropneustes fossilis across different stocking ratio treatments during the 56-day experimental period. Data points represent mean values with error bars indicating standard deviation. Letters above error bars denote statistical significance, where different letters indicate significant differences between treatments at each sampling time point (p < 0.05).

In the text

Fig. 4 Temporal progression of injury incidence in Channa striata and Heteropneustes fossilis across different stocking ratio treatments during the 56-day experimental period. Data points represent mean percentage of fish exhibiting any visible injury, with error bars indicating standard deviation. Letters above error bars denote statistical significance, where different letters indicate significant differences between treatments at each sampling time point (p < 0.05).

In the text