1. INTRODUCTION

Fish Aquaculture has become essential globally for meeting the world's food requirements because it now provides fish to more than half of the people worldwide (Ahmadu et al., 2024; Ceccotti et al., 2019; Fred-). The global protein requirement is fully provided by this fast-growing industry (fish aquaculture), and this industry is now also creating jobs for workers, particularly in developing countries where many family-run businesses and small fish farms are present (Hassan et al., 2025a ; Ragasa et al., 2022; Verdegem et al., 2023;). This developing industry has also introduced serious problems such as environmental damage, dependence on artificial growth enhancers, and more frequent disease outbreaks (Abdelkarim et al., 2025; Fachri et al., 2024; Hassan et al., 2025b).

Furthermore, these emerging problems have urged scientists to discover ecological solutions that improve yields without harming the environment or fish health (Owais et al., 2025 ; Singh et al., 2024). One species at the forefront is Pangasius hypophthalmus, which has become a major global commodity - Vietnam's annual exports exceed 1.4 million tons of this freshwater fish (Lingam et al., 2025). Farmers favor this species because it grows remarkably fast (achieving 1-1.5 kg in half a year), makes efficient use of feed, and adapts well to different water environments (Mechaly et al., 2025; Tynchenko et al., 2024). Yet crowded farming conditions have increased its susceptibility to dangerous bacteria (including Edwardsiella ictaluri and Aeromonas hydrophila) and environmental pressures that stunt growth and increase mortality (Das et al., 2024; Haenen et al., 2023). The conventional approach of using antibiotics to control diseases has grown ineffective and controversial, as drug-resistant bacteria emerge and governments tighten regulations. This situation has forced the industry to seek safer, more sustainable health management solutions (Muteeb et al., 2023).

Plant-based feed supplements are gaining attention as effective alternatives, providing multiple health benefits without the negative effects of artificial additives (Biswas et al., 2024; Kirubakaran et al., 2025; Yousefi et al., 2025). The natural compounds that are well-designed plant-based feed additives work in numerous ways that can enhance growth, boost fish farm by up to 12-18% while reducing the death rates by up to 20-30%, increase immunity, and fight cellular damage (Direito et al., 2024; Fan et al., 2022; Sumana et al., 2025).

Aloe vera's active compounds in the fish farming industry reveal varying benefits depending on dosage levels, such as 1% Aloe vera in Nile tilapia produces important advancement in such it increases 28% stronger immunity and19% faster growth performance than untreated fish (Abiri et al., 2021; Abdel-Latif et al., 2024; Habib et al., 2025; Tok et al., 2025). In the rainbow trout 2% Aloe vera in their diet produces central advancement, such as it protects the fish from bacterial contaminations nd shows less signs of cellular stress (Cordiano et al., 2025; Darzi et al., 2021; Dang et al., 2024). Aloe vera sugar-based compounds works through numerous balancing mechanisms that activate immune sensors in fish digestive tissue and then start natural defense systems (Al-Kattan and Al-Bassam, 2024; Kumaresan et al., 2024; Krupa et al., 2025). It also helps to maintain the fish gut microbiota, that is leading to improved digestion mechanisms and intestinal structure (Gupta et al., 2021; Hafeez et al., 2024; Shen et al., 2024). However, different types of fish respond differently to ideal dosage levels varying between 0.5-3% Aloe vera it depending upon various factors such as water quality parameters, fish size, and fish health (Raza et al., 2024). Moreover, it shows potential in various fish species, but its effects on P. hypophthalmus remain poorly understood (Thema et al., 2024; Bagheri et al., 2024). Therefore, the research is required to understand how Aloe vera works under various levels and how it works in high-density culture and fluctuating water parameters. The current study was designed to understand these gaps by thoroughly testing different Aloe vera levels in P. hypophthalmus feed.

2. MATERIALS AND METHODS

Experimental Fish Rearing and Environmental Conditions:

The present was conducted at the Saline Water Aquaculture Research Center (SWARC) Muzaffargarh, Punjab, Pakistan. 300 catfish fingerlings with an average weight were purchased from the Tawakal Fish Hatchery, Muzaffargarh. After the arrival of the experimental fish to the site, the fish were acclimatized upto two weeks by maintaining the fish in cemented tanks with having water capacity of 1,500 1,500-liter. Before shifting the fish, the tanks were treated with chlorine to remove any pathogens and the tanks were equipped with an aeration system. During the acclimatization period 30% Crude protein diet was given to the fish at 3% of their total body weight (Al Sulivany &, Hoseini 2024).

After the acclimatization period, the experimental fish were evenly distributed in each fifteen indoor tanks with having capacity water (250-liter, 20 fish per treatment group, each having three replicate groups. All the treatment tanks were equipped with the proper water circulation systems having inlet and outlet pipes to maintain water quality conditions by continuously removing waste and ensuring optimal water quality for fish health. Furthermore, excellent oxygen levels were maintained through air stones connected to the pumping systems, and a fine filter was used at the drainage opening to keep the fish safely inside the tanks. Breathable fabric covers were placed over tank openings to reduce dust and other airborne contaminants. Before the start of the experiment, fish were checked and selected the active fishes for the experiment following the established selection standards (Langi et al., 2024). Whole the period of study, water quality parameters were maintained in optimum conditions for P. hypophthalmus. The water quality parameters were maintained, such as temperature 27-29°C, Dissolved oxygen level 7.5-7.8 mg/L and pH stayed within 7.2-7.6.

Diet Formation and Experimental Arrangement:

Experimental diets of different levels of Aloe vera were prepared to assess its impact on P. hypophthalmus. For making uniform feed formulations, all the dry components were measured accurately and blended by using mechanical mixing equipment. Five different feed formulations were formed by adding increasing amounts of Aloe vera powder viz; 0%, 0.5%, 1%, 2%, and 4%.

warm purified water with an average temperature 30-35°C were mixed with the powders to form a uniform dough, which was then shaped into 2mm pellets using specialized equipment. After that, these pellets at room temperature (28-30°C) were dried naturally for about two days to attain appropriate consistency, then stored in wrapped plastic bags under refrigeration to preserve freshness and nutritional value until feeding time (Ghiasi et al., 2025). Experimental fish were given fish twice per day (8 AM and 4 PM) during the 60-day study trial at a 3% of their total weight. All fish groups were maintained under identical conditions during the experiment. Leftover food was removed half an hour after each feeding, and tanks were cleaned frequently to preserve water cleanliness.

Table 1 details the feed ingredients and nutritional content of each diet formulation.

Note: *The vitamin-mineral premix provided per kg of feed: Vitamin A – 7200 IU, Vitamin D₃ – 1600 IU, Vitamin E – 200 IU, Vitamin C – 90 mg, Vitamin B₁ – 1.5 mg, Vitamin B₂ – 10 mg, Vitamin B₆ – 8 mg, Vitamin B₁₂ – 0.02 mg, Niacin – 55 mg, Pantothenic acid – 40 mg, Folic acid – 0.08 mg, Biotin – 1.8 mg, Copper – 6 mg, Iron – 110 mg, Zinc – 30 mg, Manganese – 18 mg, Iodine – 0.6 mg, Selenium – 0.2 mg, and Cobalt – 0.12 mg.

Ethical Approval and Consent:

All experimental protocols involving live fish were conducted in strict accordance with the ethical guidelines for the care and use of laboratory animals. The study procedures were reviewed and approved by the Animal Ethics Committee of the Institutional Research Committee, Department of Fisheries, Government of Punjab, Pakistan (Approval Code: APF-188; 2023).

Growth Performance Calculation:

At the end of the 60-day feeding trial, all P. hypophthalmus fingerlings were subjected to a 24-hour fasting period to ensure complete evacuation of the digestive tract. Each fish was weighed using a digital balance with 0.01 g precision to record final body weights.

Growth performance and feed utilization efficiency were determined using the following standard formulas (Abdulrahman and Al Sulivany, 2025).

Weight Gain (%) = ((Final Weight – Initial Weight) / Initial Weight) × 100

FCR = Feed intake (g) / WG (g).

SGR (%) = [(ln (FW)-ln (IW))/ t] ×100.

Survival Rate (%) = (Fish Final Number/Fish Initial Number) ×100.

Hematological and Biochemical Assessments:

For blood analysis, researchers collected five fish randomly from each test group. To reduce stress during the procedure, fish were sedated using a 0.1 g/L MS-222 solution (tricaine methanesulfonate) following established protocols (Reverter et al., 2021). Blood was carefully drawn from the tail vein using sterile 3 mL syringes with thin needles (23-gauge). Each sample was immediately separated - some placed in EDTA tubes to stop clotting, others left to naturally coagulate for serum collection. The plain tubes sat at room temperature until the blood clotted, then were spun in a centrifuge (3500 rpm for 10 minutes) to separate the clear serum, which was frozen at -20°C for later testing (García-Márquez et al., 2023).

Haematological parameters viz; Red blood cells, white blood cell counts, were measured by specialized counting chambers after sample preparation with a staining solution, and investigated under high-powered microscopes. Hemoglobin examination involved chemical conversion followed by precise optical measurements at specific wavelengths while for PCV small blood-filled tubes were rotated speedily to distinct cells from plasma, with results calculated as the percentage of stable cells relative to total blood volume (Ahmed et al., 2023; Ahmed et al., 2024).

The study analyzed two key blood components: glucose and total protein levels. Glucose measurements used an enzymatic colorimetric method (GOD-POD), while proteins were assessed through copper-based color change reactions (Biuret method). Both tests employed standardized commercial kits (Randox Laboratories, UK) according to established protocols, and all the tests were repeated three times to attain accurate results (Adewale et al., 2024; Banaee et al., 2024).

Innate Immune and Assays Antioxidant:

The fish were anaesthetised and blood samples were collected to determine the antioxidant enzymes and innate immune parameters.

Antioxidant Enzyme Activities:

The SOD (activity of superoxide dismutase) was measured following the protocol drawn by Marklund and Marklund (1974), with minor modifications adapted for aquatic species by Taysi et al. (2024). Absorbance was verified at 560 nm. Catalase (CAT) activity was measured by the modified method of Aebi (1984). The decline in absorbance was used to quantify enzyme activity (Li et al., 2025). Glutathione peroxidase (GPx) activity was measured following procedures validated for fish tissues (Verdi et al., 2024). The thiobarbituric acid reactive substances (TBARS) assay was used to measure Malondialdehyde (MDA) levels. Serum was reacted with the trichloroacetic acid and thiobarbituric acid in a boiling water bath for 15 minutes then, after cooling, absorbance was measured at 532 nm MDA concentrations were expressed as nmol/mg protein (Moussa et al., 2022). The total protein concentration in each supernatant was estimated using the Bradford assay (Kielkopf et al., 2020), which utilizes Coomassie Brilliant Blue G-250 dye and provides a rapid estimation of protein content. This was used to normalize enzymatic activities.

Innate Immune Parameters:

Serum lysozyme activity was measured by the bacterial breakdown test. The measured amount of Micrococcus bacteria (0.2 mg/mL in buffer) was mixed with the small serum samples (25 µL), and the solution's cleared at 450 nm for five minutes. Based on the rate of clearing activity, levels were calculated in standardized units (Hukić et al., 2018). By using a specialized density gradient solution, Immune cells were separated and mixed with stained, inactive yeast cells at a precise ratio (1 immune cell:10 yeast cells). After incubating at 28°C, the samples were prepared on slides and examined under a microscope to calculate immune activity by determining what percentage of cells absorbed yeast particles (Lulijwa et al., 2019).

Statistical Analysis:

ANOVA (One-way analysis of variance) was performed by using SPSS software (version 26.0) to identify significant differences among treatments, followed by Tukey’s Honestly Significant Difference post hoc test and data were also tested for normality and homogeneity of variance using Shapiro–Wilk and Levene’s tests, respectively.

3. RESULTS

The present 60-day feeding trial was conducted to check the effects of dietary Aloe vera supplementation on growth performance, hematological and biochemical parameters, as well as antioxidant and immune responses in Pangasius hypophthalmus. Statistical significance (p < 0.05) was observed for most parameters, indicating a clear dose-dependent influence of Aloe vera inclusion.

Fish fed diets containing Aloe vera exhibited marked improvements in growth metrics compared to the control group (0%). The FW was highest in the 2% Aloe vera group (72.21 ± 1.54 g; p = 0.021), representing a 23.8% increase over the control (58.30 ± 1.40 g). Intermediate FW were recorded for the 1% (67.52 ± 1.68 g) and 4% (67.89 ± 1.47 g) groups, while the 0.5% group (62.11 ± 1.41 g) showed a more modest improvement. The WG (%) mirrored this trend, with the 2% group achieving the highest gain (371.1 ± 4.5%; p = 0.016), significantly surpassing the control (288.5 ± 4.1%). The SGR was maximized at 2% supplementation (3.21 ± 0.03%/day; p = 0.014), while the FCR was most efficient in the same group (1.54 ± 0.01; p = 0.008), reflecting superior nutrient utilization. Survival rates ranged from 90.2% (control) to 94.0% (2% group), but differences were not statistically significant (p = 0.146) (Table 2).

4. DISCUSSION

The 2% Aloe vera feed produced the best results, showing superior weight gain, faster growth rates, and more efficient feed conversion compared to other tested formulas. These findings indicate that this specific dosage most effectively helps fish utilize nutrients for growth. These results match findings in other fish species - Nile tilapia and rainbow trout showed better growth and feed efficiency when given 1-2% Aloe vera (Albassam, 2022; Li et al., 2023; Purbomartono et al., 2024). Cherukumudi et al. (2024) also describe that Aloe vera enhanced digestive enzyme activity, gut structure and gut microbiota for effective fish growth performance.

The current study indicates notable enhancements in white blood cells, red blood cells, and packed cell volume at 2% fish receiving Aloe vera. These changes show the safe oxygen transport and strong immune defence. Kalaiselvan et al. (2024) and Yousaf et al. (2025) also studied the active compounds in Aloe vera, such as anthraquinones, polysaccharides, which also caused to boost in blood cell formation. Radwan et al. (2024) also studied the effect of Aloe vera in tilapia and African catfish and found increased white blood cells, which indicates stronger immune defences. The present study indicates that all the groups receiving Aloe vera showed increased blood protein levels, especially at the 2% dose level. Similarly, Batoo et al. (2024) also conducted a study on the fish receiving Aloe vera and found better nutrient absorption, increased blood protein levels, and lower stress levels. The present study also describes the decreased blood sugar with the different levels of Aloe vera. Eldamrawy et al. (2024) also found the same result with the Aloe vera inclusion in fish diet and found blood sugar-lowering properties that enhance insulin function or help regulate stress reactions.

In this study Aloe vera presented the increased activity of SOD, CAT, GPx (antioxidant enzymes) with decreased MDA levels that indicates the reduced oxidative damage and it clearly shown the protective function of Aloe vera Hu et al. (2025) and Ghosh et al. (2025) also describe that Aloe vera components such as anthraquinone and flavonoid boost up the antioxidant enzymes activity (fish's natural defense systems). The study found higher lysozyme levels and improved pathogen-clearing ability in treated fish that strengthened immune function. These outcomes confirm previous research that with the Aloe vera's active compounds presented the improvement making of immune enzymes and help identify harmful microbes (Ahmad, 2024; Acar et al., 2025). Furthermore, at 4% Aloe vera indicate the strongest immune activation, antioxidant improvements, which is also noted by Gruber et al. (2025). The study revealed that Aloe vera help to improve immunity, growth and survival rates.

CONCLUSION

This research confirms Aloe vera's effectiveness as a natural feed additive for striped catfish farming, supporting more sustainable aquaculture methods. Using such plant-based supplements can decrease dependence on antibiotics and artificial growth enhancers while improving both yields and food safety. Future studies should examine extended use impacts, effects on gut bacteria, and performance in real farm settings to encourage broader use of Aloe vera in fish diets.

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Parameters	(T1) 0%	(T2) 0.50%	(T3) 1%	(T4) 2%	(T5) 4%	p- value
Initial weight (g)	14.20 ± 0.13	14.17± 0.18	14.13 ± 0.21	14.15 ± 0.20	14.18 ± 0.22	0.023
Final weight (g)	58.30 ± 1.40ᵃ	62.11±1.41ᵃᵇ	67.52 ± 1.68ᵇ	72.21 ±1.54ᵇ	67.89±1.47ᵇ	0.021
Weight gain (%)	288.5 ± 4.1ᵃ	314.5 ± 5.2ᵃᵇ	351.1 ± 5.9ᵇ	371.1 ± 4.5ᵇ	353.0 ± 6.1ᵇ	0.016
SGR (%/day)	2.81 ± 0.04ᵃ	2.94 ± 0.01ᵃᵇ	3.11 ± 0.03ᵇ	3.21 ± 0.03ᵇ	3.11 ± 0.04ᵇ	0.014
FCR	1.83 ± 0.04ᵃ	1.75 ± 0.03ᵃᵇ	1.58 ± 0.04ᵇ	1.54 ± 0.01ᵇ	1.61 ± 0.03ᵇ	0.008
Survival rate (%)	90.2 ± 2.1	91.5 ± 1.4	93.5± 1.3	94.0 ± 1.3	93.5 ± 1.2	0.146

Parameters	(T1) 0%	(T2) 0.50%	(T3) 1%	(T4) 2%	(T5) 4%	p-value
RBC (×10⁶/μL)	1.83 ± 0.05ᵃ	2.01±0.04ᵃᵇ	2.24±0.07ᵇ	2.32±0.06ᵇ	2.27±0.05ᵇ	0.012
Hemoglobin (g/dL)	7.2 ± 0.2ᵃ	7.9 ± 0.3ᵃᵇ	8.5± 0.3ᵇ	9.0 ± 0.3ᵇ	8.7 ± 0.2ᵇ	0.015
WBC (×10³/μL)	55.4 ± 2.2ᵃ	58.8 ±2.2ᵃᵇ	64.1 ± 2.3ᵇ	66.5 ± 2.1ᵇ	66.1 ± 2.3ᵇ	0.019
PCV (%)	23.7 ± 1.1ᵃ	25.5 ± 1.2ᵃᵇ	28.3 ± 1.1ᵇ	30.2 ± 1.2ᵇ	29.3± 1.2ᵇ	0.017
Glucose (mg/dL)	71.5± 3.1^a	67.2 ± 2.3^b	64.1 ± 2.2^c	64.7 ± 2.3^c	64.5 ± 2.1^c	0.089
Total Protein (g/dL)	3.4± 0.1ᵃ	3.7 ± 0.1ᵃᵇ	4.2 ± 0.1ᵇ	4.4 ± 0.2ᵇ	4.3 ± 0.1ᵇ	0.013

Parameters	(T1) 0%	(T2) 0.50%	(T3) 1%	(T4) 2%	(T5) 4%	p-value
SOD (U/mg)	7.1 ± 0.4ᵃ	8.1 ± 0.5ᵃᵇ	9.6 ± 0.6ᵇ	10.8 ± 0.5ᵇ	10.7± 0.5ᵇ	0.006
CAT (U/mg)	4.7 ± 0.2ᵃ	5.3 ± 0.1ᵃᵇ	6.2± 0.2ᵇ	6.4 ± 0.3ᵇ	6.2 ± 0.4ᵇ	0.017
GPx (U/mg)	5.4 ± 0.4ᵃ	5.7 ± 0.4ᵃᵇ	6.7 ± 0.5ᵇ	6.9 ± 0.3ᵇ	6.7 ± 0.4ᵇ	0.019
MDA (nmol/mg)	2.2± 0.1ᵃ	1.8 ± 0.2ᵃᵇ	1.6 ± 0.2ᵇ	1.5 ± 0.2ᵇ	1.6 ± 0.2ᵇ	0.020
Lysozyme Activity (U/mL)	10.3± 0.5ᵃ	11.7 ± 0.6ᵃᵇ	13.4 ± 0.7ᵇ	14.3 ± 0.5ᵇ	13.7 ± 0.6ᵇ	0.009
Phagocytic Activity (%)	25.4 ± 1.3ᵃ	29.2 ± 1.5ᵃᵇ	32.5 ± 1.6ᵇ	33.87± 1.3ᵇ	34.0 ± 1.4ᵇ	0.014

Ingredients	(T1) 0%	(T2) 0.50%	(T3) 1%	(T4) 2%	(T5) 4%
Fish meal	190	190	190	190	190
Soybean meal	80	80	80	80	80
Cottonseed cake	110	110	110	110	110
Wheat bran	310	307	304	298	286
Wheat pollard	130	130	130	130	130
Maize germ	100	100	100	100	100
Soybean oil	18	18	18	18	18
Vitamin-mineral premix*	12	12	12	12	12
Aloe vera leaf powder	0	5	10	20	40