Effect of Azadirachta indica supplemented feed on growth performance of Labeo rohita

Effect of neem (Azadirachta indica) on the growth of Rohu (Labeo rohita) was investigated under lab conditions in the Department of Zoology, University of Agriculture Faisalabad. Experiment was performed for twelve-fortnights with three replicates. Fingerlings of L. rohita were acclimatized for one week in glass aquarium at reference diet before initiating the trial. T1 reference diet was administered to control group of fish without neem supplement but treated fish groups T2, T3 and T4 were administered with 5, 10 and 15g/1000g test neem supplemented diet respectively. Growth parameters including total body weight, total body length, fork length, feed conversion ratio and specific growth concentration was calculated. Statistical analysis of growth parameters revealed that 15g/1000g neem test diet showed highly significant (p<0.01) gain in body weight (11g), total body length (9.7cm), fork length (3.6cm) and specific growth concentrations (0.43) of L. rohita as compared to other diet groups but at 5g/1000g test diet of neem showed maximum (2.75) mean value of feed conversion ratio. Neem (Azadirachta indica) is a naturally occurring medicinal herb having a lot of bioactive and biosafety compound that acts as growth enhancer, appetite stimulator and immunomodulator against infectious pathogens. The impact of current study is that higher concentration of neem supplemented feed effectively leads to higher growth of fish’s body parameters such as total body weight, total body length, fork length as well as feed conversion ratio.


Introduction
Aquaculture is the biggest source of earning because of fast growing food production sector in the world [1,2]. In aquaculture, the continuous increasing trend of fish culturing has increased its demand as pivotal component for the health of animals in industry [3,4]. Nutrition in fish diet promotes growth, maintain health of aquatic species of fish to meet the demand of human consumption and enhance activities of immune system to create resistance against diseased pathogens [5]. The danger of food shortage can be minimized by potential of healthy fish as it provides high quality protein, minerals and fatty acids to animal industry [6]. Labeo rohita is commonly well known as Rohu that is cultivated on commercial scale in Asia. Rohu gives approximately 35% contribution to major carp production as it is cultural and abundantly present in Asian continent [7]. Labeo rohita is a vital source of animal protein, mineral, beneficial fat contents, vitamin and essential amino acid that provide healthy diet to animal industry [8]. Due to poor management and non-hygienic conditions of aquaculture, fish breeding leads to higher mortality [9].Vaccination is much more expensive for producer of fish aquaculture or may not be feasible for all diseased species of fish [10,11]. Plants sources are the best alternative because of lower phosphorus contents, no harmful growth depressing entities, low cost and easy availability [12]. Neem (Azadirachta indica) is a naturally occurring medicinal herb having a lot of bioactive and biosafety compounds that acts as growth enhancer, immunostimulant to activate the activity of immune system, antistressor, antifungal, antiviral and anticancerous agent [13][14][15][16]. Neem is known as well defined "Village Dispensary" in India that is an essential part and parcel of human society to control fish diseases and gives valuable growth concentrations and immunity to fish farms [17 -19].
The objective of the present study was to evaluate the medicinal effects of Azadirachta indica supplemented feed on growth performance in Labeo rohita to meet the demand of health status of fish and human consumption by increasing growth rate as well as immunity of fish.

Materials and Methods Fish collection
Fish seeds was collected from Government Fish Seed Hatchery, Satiana Road, Faisalabad and transferred to Fisheries Research Farm, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan. This laboratorial work was settled for the period of six months from Dec, 2018 to May, 2019. Fingerlings of Labeo rohita were acclimatized for one week in glass aquarium at reference diet, 12h light/dark period, continuous aeration and water quality parameters at standard pH value 7, Temperature 26±2°C, DO 5.0±0.3mg/L before initiating the trial. Neem supplemented diet preparation Commercial diet for fishes was taken in the form of small pellet and grinded in powder form with the help of electric grinder. Leaves of neem plant were taken from surrounding area of Department of Zoology, Wildlife and Fisheries and aerated at room temperature for 48h. These dried leaves were grinded with the help of pestle and mortar. Supplemented diet was prepared by the addition of commercial diet in different concentrations of neem powder mixture. T 1 reference diet was without neem diet and considered as control diet. T 2 , T 3 and T 4 test diets were prepared by adding neem 5/1000, 10/1000 and 15g/1000g into commercial diet. Linear formulation method was followed during reference and test diet preparation (Table 1). Feeding protocol Initial weight (g) and length (cm) of fish samples were measured and transferred to glass aquarium covered with nylon net and plastic clip in Laboratory after acclimatization process. Three replicates of fish fingerling were kept in each aquarium. The water was filled in each aquarium up to 65L or 3/4 part of aquarium. Water of all aquariums was recycled once in a day. Diet was given to fish at the concentration of 6% of their body weight.

Growth parameters
Growth parameters of each aquarium such as total body weight (g), total body length (cm) and fork length (cm), FCR (feed conversion ratio) and SGR (specific growth concentrations) were measured fortnightly and compared with controlled fish samples. To calculate FCR (feed conversion ratio) and SGR (specific growth concentrations), the values percent weight gain (g), initial body weight of last fortnight, final body weight of that fortnight was noted. Weight gain of fish was calculated by formula [20]. Specific growth concentrations and feed conversion ratio were calculated as Feed Conservation Ration = ___F___ W f -W i Where F, indicates the weight of supplied feed to Rohu W f , value indicates the final body weight of fish at present fortnight W i, value indicates the initial body weight of fish at last fortnight.

Statistical analysis of data
The statistical analysis of recorded data was done by using Micro Computer IM-PC. Two Factor Factorial Analysis of Variance was used to analyze data of growth parameters (body weight, total length, fork length, FCR and SGR).The analysis of all parameters was done to finds Significant or nonsignificant differences among parameters along with fortnights. For the computation of these data Minitab software was used.

Results Body weight (g)
Results showed that highest body weight was observed in T 4 diet. The maximum and minimum weight gain (g) for T 4 test diet was 2.4g and 0.3g in 9 th fortnight (April) and 1 st fortnight (December). The maximum and minimum weight gain (g) for T 1 reference diet was 0.6g and 0.1g in 9 th fortnight (April) and 1 st fortnight (December), respectively. The maximum and minimum weight gain (g) for T 2 test diet was 0.8g and 0.1g in 9 th fortnight (April) and 1 st fortnight (December), respectively. The maximum and minimum weight gain (g) for T 3 test diet was 1.4g and 0.2g in 9 th fortnight (April) and 1 st fortnight (December) respectively (Fig. 1).

Body length (cm)
Results showed that highest body length was observed in T 4 diet. The maximum and minimum length gain (cm) for T 4 test diet was 2.2cm and 0.2cm in 8 th fortnight (March) and 1 st fortnight (December). The maximum and minimum length gain (cm) for T 1 reference diet was 0.3cm and 0.1cm in 8 th fortnight (March) and 1 st fortnight (December), respectively. The maximum and minimum length gain (cm) for T 2 test diet was 0.6cm and 0.1cm in 8 th fortnight (March) and 1 st fortnight (December), respectively. The maximum and minimum length gain (cm) for T 3 test diet was 0.7cm and 0.1cm in 8 th fortnight (March) and 1 st fortnight (December) respectively (Fig. 2).

Fork length (cm)
Results showed that highest fork length gain was observed in diet T 4 . The maximum and minimum fork length gain (cm) for T 4 test diet was 0.7cm and 0.1cm in 9 th fortnight (April) and 1 st fortnight (December). The maximum and minimum fork length gain (cm) for T 2 test diet was 0.4cm and 0.1cm in 9 th fortnight (April) and 1 st fortnight (December), respectively. The maximum and minimum fork length gain (cm) for T 3 test diet was 0.5cm and 0.1cm in 9 th fortnight (April) and 1 st fortnight (December), respectively (Fig. 3).

Feed conversion ratio
Results showed that feed conversion ratio was observed maximum in diet T 2 . The maximum and minimum feed conversion ratio for T 2 test diet was 6.6 and 0.91 in 5 th fortnight (February) and 9 th fortnight (April). The maximum and minimum feed conversion ratio for T 1 reference diet was 3.0 and 0.42in 12 th fortnight (May) and 9 th fortnight (April), respectively. The maximum and minimum feed conversion ratio for T 3 test diet was 6.03 and 0.58 in 3 rd fortnight (January) and 9 th fortnight (April), respectively. The maximum and minimum feed conversion ratio for T 4 test diet was 7.5 and 0.33 in 7 th fortnight (March) and 9 th fortnight (April), respectively (Fig. 4).

Specific growth concentrations
Results showed that mean value of specific growth concentrations was observed maximum in T 4 diet. The maximum and minimum specific growth concentrations for T 4 test diet was 1.19 and 0.05 in 9 th fortnight (April) and 7 th fortnight (March). The maximum and minimum specific growth concentrations for T 1 reference diet was 0.95 and 0.14 in 9 th fortnight (April) and 11 th , 12 th fortnight (May). The maximum and minimum specific growth concentrations for T 2 test diet was 0.45 and 0.06 in 9 th fortnight (April) and 5 th fortnight (February). The maximum and minimum specific growth concentrations for T 3 test diet was 0.7 and 0.07 in 9 th fortnight (April) and 3 rd fortnight (January), respectively (Fig. 5).

Discussion
The aim of this study was to investigate the effect of neem (Azadirachta indica) on growth performance such as total body weight, total length, fork length, feed conversion ratio, Specific growth concentrations in Labeo rohita (Rohu). The growth of fish was directly proportional to concentrations of diet ratio i.e. higher growth of the fish by increase in the neem supplemented diet ratio. The highest growth concentrations as weight, length and fork length was achieved at higher diet ratio of neem (T 4 ) just as 10.3-21.3g, 9.5-19.2cm and 1.9-5.5cm respectively, as compared to other diet ratio at T 2 , T 3 test diets and T 1 reference diet. The inferences of current study are correlated with [21] who have administered fish with neem inoculated diet and found a great influence on growth parameters of fish along with increasing trend of neem concentration. This study showed the same enhancement trend of fish growth that was observed in our study. A study conducted by Nya and Austin [22] to find out the effect of various diet ratio of ginger for 14 days on growth performance, reduction in mortality of rainbow trout. The results showed that ginger had significant effect on fish growth and immune response. The findings of this study concluded that by increasing the dose concentrations of any medicinal plant extract leads to the increase in growth of fish and their results are similar to our work that showed total body weight gain (11g), total length gain (9.7cm) and total fork length gain (3.

Conclusion
This study showed that growth performance of Labeo rohita directly related to the neem supplemented diet, showed enhancement trend and highly significant (p<0.05) results on growth parameters of Labeo rohita. With increase in neem supplemented diet all Growth parameters including total body weight, total body length, fork length, feed conversion ratio and specific growth concentration were increased. This study will be helpful for the aquaculture industry for the increase in the production of fishes. This study was conducted to evaluate the medicinal effects of Azadirachta indica supplemented feed on growth performance, health status and compensate demand of aquaculture industry by increasing growth rate as well as immunity of fish.