Supplementation of Pumpkin Cucurbita maxima seed powder as a feed additive on immune response of rohu, Labeo rohita (Hamilton, 1822)

This study investigates the impact of incorporating pumpkin (Cucurbita maxima) seed powder (PSP) into the diet of Labeo rohita fingerlings over 60 days. Pumpkin seeds, known for their high antioxidant and vitamin E content, are used as immuno-nutrients due to their nutritional profile and 24.12% protein content. However, they also contain antinutritional and potentially toxic components such as polyphenols, flavonoids, tannins, and cyanogenic glycosides. PSP was included in fish diets at varying levels: T0 (0%), T1 (1.5%), T2 (3%), T3 (4.5%), and T4 (6%). Immune responses were evaluated, revealing that a diet containing 4.5% PSP (T3) significantly improved immunity. By the end of the 60-day trial, immune parameters such as erythrocyte count, leukocyte count, and respiratory burst activity were notably enhanced in the T3 group. Proximate composition analysis indicated higher protein and fat content in the T3 treatments. Therefore, a diet with 4.5% PSP enhanced fish immunity optimally.

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Introduction

The aquaculture industry plays an essential role in ensuring a steady fish supply amid the ongoing decline of the capture fisheries sector. Over the past decades, the production of aquatic animals has witnessed remarkable growth, rising from 19 million tonnes (Live weight equivalent) in 1950 to an unprecedented 185 million tonnes in 2022, with an average annual growth rate of 3.2% (FAO, 2024). Notably, global aquaculture production in 2022 reached 130.9 million tonnes, reflecting an increase of 8.1 million tonnes compared to the 122.8 million tonnes reported in 2020. Rohu (Labeo rohita) is the most prominent among the three Indian major carp species commonly used in carp polyculture systems. Its popularity in aquaculture stems from its rapid growth rate, adaptability to diverse environmental conditions, and high market demand. The growth of fish at various stages is largely determined by factors such as feed type, feeding frequency, feed intake, and nutrient absorption capacity (Abid & Ahmed, 2009) [1] . As an omnivorous species, farmed rohu thrives on a diet with a protein content of 30-35% and It is high in protein, vitamin A, Omega-3 fatty acids and essential amino acids (Rasal & Sundaray, 2020) [12] . Medicinal plants have been proven as growth promoters. The introduction has the properties of growthpromoting ability, a tonic to improve the immune system, antimicrobial capability, and stimulating appetite and anti-stress characteristics (Citarasu et al., 2010) [3] . Pumpkin is a well-known edible plant with substantial medicinal properties due to its unique natural edible substances (Yadav et al., 2010) [17] . Pumpkin belongs to the genus Cucurbita, family Cucurbitaceae, and economically important species are Cucurbita moschata, Cucurbita maxima, Cucurbita ficifolia, Cucurbita pepo, Cucurbita mixta and Telfairia occidentalis (Singh & Kumar et al., 2023) [15] . Pumpkin seeds have been used traditionally for herbal, medicinal, and clinical purposes. They contain several useful functional components. Safe antibiotic and deworming medicines have been made from pumpkin seeds (Dubey and Dubey, 2012) [7] . Pumpkin seeds may be small, but they are rich in beneficial nutrients and nutraceuticals, including amino acids, phytosterols, unsaturated fatty acids, phenolic compounds, tocopherols, cucurbitacins, and essential minerals (Dotto et al., 2020) [6] . However, pumpkin seeds also contain antinutritional and potentially toxic compounds such as polyphenols, flavonoids, tannins, and cyanogenic glycosides (Del Vechio et al., 2005) [5] . Despite this, they are rich in nutrients, containing approximately 30% crude protein (Sharma et al., 1986; Esuoso et al., 1998) [14,8] . Utilizing cost-effective, readily available, and nutrient-dense terrestrial resources like Cucurbita maxima seeds is essential for the efficient formulation of fish feed.

Materials and Methods

The experiment was conducted at the Department of Aquaculture, College of Fisheries Science, Veraval to "Supplementation of Pumpkin Cucurbita maxima seed powder as a feed additive on growth, survival and immune response of rohu, Labeo rohita (HAMILTON, 1822)". The experiment was conducted in 20 rectangular plastic aquarium tanks, each measuring (size 2 x 1 x 1 feet). All tanks were thoroughly cleaned and disinfected with potassium permanganate (KMnO4) before use. They were then filled with 40 liters of filtered and disinfected freshwater. Adequate aeration was provided in all 20 experimental tanks. The Labeo rohita seed was sourced from a commercial IMC hatchery and transported to the experimental facility in polythene bags. Upon arrival at the college, the seed was acclimatized in a 500-liter FRP tank with continuous aeration and feeding for 7 days. Fish with an average weight of 1.310±0.009 grams were selected for the experiment.

Experimenatal diet preparation

Five distinct experimental diets were formulated based on the different treatment groups. Treatment groups include T 0 (control, no Pumpkin seed powder (PSP)), T 1 with (1.5% PSP), T 2 (3% PSP), T 3 (4.5% PSP) and T 4 (6% PSP). The required ingredients were collected and precisely weighed according to the feed formula in Table 2. The feed mixture was then subjected to heat treatment at 121 °C under 15 lbs of pressure for 15 minutes. The resulting mixture was then pressed into uniform pellets using a mechanical pelletizer.

Ingredients	Diet (30% Protein)	—	—	—	—
	T0 (Control)	T1 (1.5% PSP)	T2 (3.0% PSP)	T3 (4.5% PSP)	T4 (6% PSP)
Pumpkin seed powder	0	15	30	45	60
Sterilized fish meal	25	24	24	24	24
Wheat flour	15	14	14	13	13
Tapioca powder	13	13	12	12	12
Gnoc	41	41.5	41	40.5	39
Plant oil	2	2	2	2	2
Fish oil	2	2	2	2	2
Vitamin and minerals	2	2	2	2	2
Total	100	100	100	100	100

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Composition of Experimental Diet (in gms)

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To prevent spoilage, the pellets were dried until their moisture content was less than 10%.

Proximate Analysis of Ingredients

The proximate composition of the ingredients was analyzed using the standard method of AOAC (2019). The composition of the experimental diet was formulated using Pearson's square method at a 30% protein level. The formula for the experimental diets, including their percentage composition, is detailed in Table 2 based on the treatment specifications. During the experimental period, Labeo rohita was fed twice daily at 5% of its body weight. The feed used contained 30% protein. Key parameters measured include Total erythrocyte, leukocyte counts and respiratory burst activity across all five treatment groups.

Immune Response

The total erythrocyte count (TEC) was conducted following the method outlined by Lal (2010) [10] . The total leucocyte count (TLC) was carried out using the method of Shah & Altindag (2005) [13] . The respiratory burst activity of phagocytes was measured using the nitroblue tetrazolium (NBT) assay. Carcass composition was measured at last. Completely Randomised Design (CRD) experimental design will be used in the experiment and estimate the arithmetic mean, standard deviation, and coefficient of variation using standard statistical Methods (Snedecor & Cochran, 2014) [16] . The collected data was analyzed using a one-way analysis of variance (ANOVA) to identify significant differences between the means. The statistical analysis was performed using IBM SPSS version 22 software.

Results

The study examines Immunological parameters in L. rohitafed diets with varying levels of pumpkin seed powder.

Effect of dietary pumpkin seed powder on immune response of Labeo rohita

In this experiment, pumpkin seed powder's impact on Labeo rohita immunity was examined by evaluating factors like respiratory burst activity, total erythrocyte count (TEC), total leukocyte count (TLC) and respiratory burst activity. Blood samples were taken using a 26-gauge needle and mixed with an anticoagulant to conduct the immune assessment.

Effect of Pumpkin seed powder on Total erythrocytes counts (TEC) of L. Rohita

At 60 days, the highest Total erythrocyte count was in T 3 (2.10±0.127), and the lowest in T 0 (1.36±0.012). T 3 and T 4 had significantly higher counts (p>0.05). Musthafa et al.

(2017) [11] found that pumpkin seed meal enhanced immune activities in fish, while Dada and Abiodun, (2014) [4] showed that T. occidentalis leaf powder improved growth and erythrocyte count in fish when added to feed.

Effect of Pumpkin seed powder on Total leucocyte count (TLC) of L. Rohita

The Total leucocyte count after 60 days is shown in Table 3, with the highest count in T3 (30.44±0.217) and the lowest in T0 (22.5±0.29). Dada and Abiodun (2014) [4] found that supplementing Nile tilapia with 2.5 g/kg powdered pumpkin extract improved growth, feed utilization, white blood cell counts, and overall hematological health. Fish fed the fluted pumpkin extract had better white blood cell counts and immune protection compared to the control group, suggesting enhanced feed efficiency and immune defense.

Treatments	Total Erythrocytes count 106 cells/mm-3	Total Leucocytes count 103 cells/mm-3	Respiratory burst activity
T0	1.36±0.012c	25.01±0.114e	0.37±0.006c
T1	1.42±0.022c	26.19±0.158d	0.39±0.014c
T2	1.52±0.011c	28.67±0.112c	0.46±0.015b
T3	2.10±0.127a	30.44±0.217a	0.52±0.016a
T4	1.82±0.019b	29.57±0.402b	0.50±0.010a

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Effect of pumpkin seed powder on total erythrocytes count (TEC), Leucocyte count (TLC) and Reparatory burst activity of L.

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Effect of Pumpkin seed powder on Reparatory burst activity of Labeo rohita

After 60 days, the highest respiratory burst activity in Labeo rohita was observed in T 3 and T 4 , with no significant difference (p>0.05). Lower values were recorded in T 0 and T 1 , also without significant differences. Diets with 4.5% and 6% pumpkin seed powder enhanced immunity, consistent with Musthafa et al. (2017) [11] , who reported improved respiratory burst activity and immune benefits from pumpkin seed meal, attributed to its rich essential fatty acid content. Supplementing with 4 g/kg and 6 g/kg of pumpkin seed meal.

Effect of pumpkin seed powder supplemented diet on body composition of Labeo rohita

The proximate composition of Labeo rohita, analyzed after the experiment, is shown in Table 4. The fresh fish meat was carefully minced for accuracy.

Parameters	T0	T1	T2	T3	T4
Moisture (%)	78.56	78.91	79.86	80.34	79.77
Crude Protein (%)	50.21	52.55	53.73	56.37	55.81
Crude Fat (%)	6.92	7.05	7.41	8.53	8.24
Ash (%)	11.76	12.03	11.38	10.61	10.97

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Body composition of Labeo rohita fed with Pumpkin seed powder Supplemented diets after 60 days

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To analyze the protein content in the samples, the micro Kjeldahl technique was used. Treatment group T 3 had the highest protein (50.21%). These results demonstrate how the dietary profiles of the various treatment groups differed.

Physico-Chemical water parameters

Water quality was checked every week during the experiment. It was in the ideal range throughout the experiment. Temperature, pH, dissolved oxygen, hardness and Alkalinity were measured by the standard analytic methods (APHA, 2017).

Conclusion

The study concluded that supplementing the Labeo rohita diet with pumpkin seed powder (PSP) positively immune response. Immune response, assessed through respiratory burst activity, total erythrocyte count (TEC), and total leukocyte count (TLC), was enhanced with 4.5% and 6% PSP, with T 3 showing the highest TEC and TLC. The study concluded that 4.5% PSP supplementation improved immune performance in Labeo rohita, yielding the best outcomes.

Figures and tables from the paper

Ingredients (%)	Crude protein (%)	Crude fat (%)	Ash (%)	Moisture (%)
Fishmeal	55.03	4.20	15.20	7.91
Pumpkin seed powder (PSP)	24.12	22.17	3.31	6.184
Wheat flour	11.94	1.3	5.32	9.81
GNOC	35.56	4.92	8.07	7.19
Tapioca	4.81	0.61	0.14	8.13

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Proximate Analysis of Ingredients

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