Dietary effect of moringa (Moringa oleifera; Lamarck, 1785) leaf powder on growth response of tilapia (Oreochromis niloticus; Linnaeus, 1758)

Management of fish health is one of the main considerations in aquaculture and different plant compounds are being used for supporting fish health to minimize the negative impacts of synthetic aqua drugs. In the present experiment, potentiality of moringa (Moringa oleifera; Lamarck, 1785) leaf as a nutritious dietary source for tilapia (Oreochromis niloticus; Linnaeus, 1758) fish was tested and the duration was two months from 30 September to 30 November, 2020. The moringa leaves were brought, cleaned, dried, and finally crushed into powder. Three experimental diets were formulated using the processed moringa leaf powder (MLP) at the rate of 0% (MLP0%) as control, 10% (MLP10%) and 20% (MLP20%) as treatment mixing with rice bran, wheat bran, mustard oil cake, fish meal, soya oil and vitamin-mineral premix. Fifteen tilapia fingerlings having average initial length 10.88±0.11 cm and initial weight 29.06±0.50 g was stocked in each tank with 90 L water. Sixty days feeding trial was performed with three replications of each treatment. The fishes were fed with formulated feeds twice daily at 9 am and 4 pm at a rate of 3% of their body mass. Sampling of fish and water quality parameters were carried out at twelve days interval. Moreover, the blood glucose and cholesterol of tilapia were measured monthly. Final length, final weight, weight gain, percent weight gain, feed conversion ratio (FCR), specific growth rate (SGR) and production of tilapia were significantly different among the treatments. The highest FCR (3.17±0.25) and SGR (1.33±0.12 %) values were in MLP20% and MLP10%, respectively. In the experiment, the highest and the lowest tilapia production were 9.21±0.39 and 7.39±0.35 kg m in MLP10% and MLP20%, respectively. The blood glucose values were significantly different among the treatments (p< 0.05) and the highest value was in MLP0% (48.00±2.00 mg dl ). Moreover, the highest and the lowest blood cholesterol was found in MLP0% (177.67±2.52 mg dl ) and MLP20% (148.33±1.53 mg dl ), respectively whereas the values were highly significantly different among the treatments (p≤ 0.01). Water quality parameters were statistically similar among the treatments (p> 0.05) and the values were within acceptable range for tilapia culture.


Introduction
Fish is a cheap and easily digestible animal protein source significantly contributing to the national food basket of Bangladesh. Aquaculture sector is a major contributor towards fish supply and its rapid development leads to the intensification of the culture practices Srichaiyo et al., 2020). The continuous and uncontrolled intensification of aquaculture creates several health risks of fish such as poor growth, infectious and non-infectious diseases (Hussain et al., 2018). Till now chemotherapy is the only option for prevention and management of aquaculture disease outbreaks which negatively affect the environment and human health Jahan et al., 2021). Hence, in recent years attention is given towards eco-friendly and sustainable methods of aquaculture disease management practices using different plants as immune stimulants (Srichaiyo et al., 2020;Stratev et al., 2018). Many products from the plant origin have been reported to stimulate appetite, promote growth performances, and act as immune-stimulants, antibacterial, antiviral, and anti-parasitic agent in aquaculture (Jahanjoo et al., 2018;Muniruzzaman and Chowdhury, 2008). An application of immune stimulants for the prevention of fish diseases are considered as an attractive and promising area as plants are safer, much more effective and cheaper than the synthetic substances (Yao et al., 2020). Scientists are working to develop low-cost, nutrient-dense feed and to use plant extracts, and natural plant compounds as potential alternatives to synthetic chemicals for stimulating immune responses and disease tolerance in fish (Abdel-Hakim et al., 2010;Reverter et al., 2014). Herbal phytochemicals can boost the innate immune system and have antimicrobial properties that could be very useful in fish farming without causing any environmental or health risks (Jahanjoo et al., 2018;Chakraborty and Hancz, 2011). Such ingredients extracted from plants have antioxidant properties those can upgrade fish's physiological state (Hussain et al., 2018;Zhou et al., 2016). Nutritious diet not only promotes growth of fish but also enhances survival rate which is profitable in case of aquaculture. Moringa oleifera (Lamarck, 1785) or moringa is one of the most well-known plant species and is the only genus in the Moringaceae family with high crude protein content in the leaves and low tannin and other anti-nutritional compounds (Shourbela et al., 2020;Puycha et al., 2017). Its n-3 fatty acids in the form of linolenic acid account for about 67 percent of total fatty acids in the crude lipid (Hussain et al., 2018). Leaf of Moringa contains significant amount of iron, carotenoids, ascorbic acid, and minerals (Elabd et al., 2019). Moringa leaves are also an excellent source of nutrients such as protein, lipid, and gross energy. According to Puycha et al. (2017), moringa can be considered as a potential source of protein introducing at low amount in fish diet to make aquaculture production cost effective. On the other hand, tilapia (Oreochromis niloticus; Linnaeus, 1758) is considered as the second most important farmed fish in the world, after carps (Toutou et al., 2018;Ronald et al., 2014;Reda et al., 2013). Tilapia culture is practiced in most tropical, subtropical, and temperate regions because it grows quickly, requires little oxygen, can withstand a wide range of temperatures, is disease tolerant, and results in high yield Srichaiyo et al., 2020;Makori et al., 2017). One of the main considerations in aquaculture industry is to reduce feed and production cost. Preparation of fish feed mixed with cheap plant ingredient like moringa leaves can result in low-cost nutrient rich feed and also can reduce the necessity of synthetic drugs. So, the present study is aimed to assess growth parameters of tilapia fed with moringa leaves mixed feed.

Experimental design
The experiment was carried out on a rooftop in Rajbari Sadar sub-district under Rajbari District, Bangladesh (Latitude: 23°36'22.03" N; Longitude: 89°50'26.30" E). The duration of the study was two months from 30 th September to 30 th November, 2020. In case of fish rearing, 9 fish holding tanks containing 90 L of water were used. Throughout the experiment, two air-pump of 18 W (Resun, Model ACO-001, China) with six nozzles were used to provide dissolved oxygen to the fish tanks, each with two nozzles with perforated stones whereas the aeration was maintained for 24 hours. Three experimental diets were made for the fish using moringa leaf powder that was processed at the rate of 0%, 10% and 20% denoting as MLP 0% , MLP 10% and MLP 20% , respectively ( Table 1). The experiment was designed out with three replications for each treatment.

Feed preparation with moringa leaf powder
Moringa leaves were collected from moringa plant and after collection; the leaves were separated from the stems and dried under sunlight. When the moringa leaves were crispy, they were crushed into small pieces by hand and then finely powdered in blenders. The ingredients for the fish feed were purchased from local market including rice bran, wheat bran, mustard oil cake, fish meal, soya oil and vitamin-mineral premix. All the dietary ingredients were crushed finely and manually sieved thoroughly to obtain a homogenous mixture following Rana et al. (2020). To adjust the ingredients amount, rice bran and wheat bran were partially replaced with moringa leaf powder (MLP) in case of MLP 10% and MLP 20% . Using an electronic balance (Electronic Precision Balance, EK 600i), all ingredients, including vitamins and minerals premixes were weighted according to the composition of experimental diets ( Table 1). The ingredients were mixed well, and water was used to moisten the mixture. The resulting dough was pressed through pelleting machine using 1 mm meshed sieve. The formulated pellets were sun dried, broken into small pieced and stored in airtight plastic container. Here, MLP 0% = Feed with 0% moringa leaf powder (control); MLP 10% = Feed with 10% moringa leaf powder and MLP 20% = Feed with 20% moringa leaf powder.

Tilapia stocking and rearing
Tilapia fingerlings were brought from the Elem Fish Farm which is located in the Rajbari Sadar sub-district under Rajbari district, Bangladesh. After collection, fishes were acclimatized for 7 days in the culture tank to release the stress during transportation and for adapting with new environmental conditions. Fifteen tilapia fingerlings with average weight 29.06±0.50 g was allocated randomly in each tank. Throughout the experiment, fishes were fed with experimental diets twice a day at 9 am and 4 pm at a rate of 3% of their body weight. The uneaten feed and feces were removed with ¼ th water exchange daily through siphoning and the entire water was changed fortnightly with fresh underground water.

Sampling of tilapia
Fishes were caught using a scope net for sampling and the fishes were sampled at twelve days interval during study. The length and weight of fish from each tank were measured using a measuring scale and an electronic balance. To test the growth efficiency of tilapia, several parameters were calculated namely length gain, weight gain, percent length gain, percent weight gain, specific growth rate (SGR), feed conversion ratio (FCR), survival rate and production according to Moniruzzaman et al. (2015).

Blood parameters
Blood glucose and cholesterol of tilapia were measured thrice during experiment at 30 days interval and initiated from 30 th September, 2020. The values of blood glucose and cholesterol were determined using an 'Easy Touch GCHb Meter, Bioptik Technology, Taiwan' where the minimum requirements for blood analysis are 4 μl and 15 μl, respectively. Aketch et al. (2014), Zang et al. (2013) and Beecham et al. (2011) also utilized handheld portable blood meter for testing fish blood. During blood test, five fishes from each tank were randomly caught and blood was collected through caudal venipuncture. Green test stripe of the blood meter was used for glucose determination while blue colored test stripe was for cholesterol test. The stripes were attached to the stripe slot of the device during test. Collected blood was put on the mark of stripe and the result came out on the screen. After blood collection through injection, the blood test was done immediately as clotted blood does not provide result and then the fishes were released in their specific tanks.

Water quality parameters of fish tanks
Water temperature, dissolved oxygen (DO), pH, electrical conductivity (EC) and total dissolved solids (TDS) were measured at twelve days interval using a thermometer, a Lutron Dissolved Oxygen meter PDO-519, Hanna HI-98107 pH Meter and E-1 portable EC-TDS meter, respectively. The water quality parameters were measured at 10 am of the sampling date throughout the experiment.

Data processing and analysis
The data were statistically analyzed for variance and one-way ANOVA using SPSS 20.00 (Statistical Package for Social Sciences) and significant differences among the mean values of the treatments were compared using Duncan's Multiple Range Test (DMRT) considering 5 and 1% level of probability.

Weight gain and percent weight gain of tilapia
Fish weight during last three sampling showed significant differences among the treatments (Figure 2). The highest and the lowest mean final weight were 60.68±3.05 and 53.90±2.09 g in MLP 10% and MLP 0% , respectively, whereas the mean weight gain of tilapia were 25.63±0.42, 31.89±0.57 and 27.46±0.76 g in MLP 0% , MLP 10% and MLP 20% , respectively and the values were highly significantly different (p≤ 0.01) among the treatments ( Table 2). The highest percent weight gain was in MLP 10% (103.70±4.38 %) followed by MLP 0% (92.53±0.99 %) and MLP 20% (89.22±0.89 %), respectively and the values were lower than the findings of Nadia et al. (2020) and Moniruzzaman et al. (2015). Moreover, Nassar et al. (2021) found 16.92±0.21 to 20.75±0.19 g weight gain after 97 days. Nirmala et al. (2020) opined that, fish growth is influenced by internal and external factors like disease resistance, feed availability and culture system.

Specific growth rate (SGR) of tilapia
In the present experiment, the highest and the lowest SGR were 1.33±0.12 and 1.07±0.03 % in MLP 10% and MLP 0% , respectively (Table 2). Moreover, the values were highly significantly different among the treatments (p≤ 0.01). Makori et al. (2017) found higher SGR (2.03-2.34%) than the present study feeding fish with commercial feed at 10% body weight rearing at pond. It might be due to more favorable water quality in pond than tank culture system. During cage culture of tilapia, Moniruzzaman et al. (2015) noted SGR ranging from 2.02 to 2.35% in 120 days.

Feed conversion ratio (FCR) of tilapia
FCR is useful to determine the fish ability to digest feed and it shows the amount of feed used to increase fish weight by 1 kg. The FCR values of tilapia were 2.73±0.21, 2.60±0.17 and 3.17±0.25 in MLP 0% , MLP 10% and MLP 20% , respectively (Table 2). Higher FCR in MLP 20% might be due to the wastage of feed and less attraction to the formulated feed with 20% moringa leaf powder which might be due to the omnivorous nature of tilapia. According to Nirmala et al. (2020), FCR is influenced by several factors namely species, size, quality, and quantity of feed as well as the water quality that will determine the feed effectiveness. Nadia et al. (2020) found FCR 3.2±0.11 over a period of 106 days where they supplied commercial floating feed at the similar rate of the present study. However, low FCR value in MLP 10% in the present study indicated high fish efficiency in utilizing the feed consumed. Akter et al. (2020) reported similar FCR (2.98 to 3.68) to the present study where they reared tilapia in tank.

Survival rate of tilapia
In the current study, the highest survival rate of tilapia was in MLP 10% (91.00±3.46 %), followed by MLP 0% (82.22±13.61 %) and MLP 20% (80.10±7.51 %), respectively ( Table 2). The lowest survival rate in MLP 20% might be due to improper growth and less ingestion of nutritious feed. But the survival rates were not significantly influenced by the treatments (p> 0.05). Survival rate of tilapia reported by Moniruzzaman et al. (2015) (90.93%) and Abdel-Hakim et al. (2010) (85.19%) were in line with the present study. Makori et al. (2017) and Toutou et al. (2018) found survival rate ranging from 78.43±1.96 to 84.31±1.96 % and 72.59±5.20 to 77.50±1.44 %, respectively those were close to the present study. The survival rate of tilapia noted by Akter et al. (2020) (66.66 to 76.66% in 60 days) was lower than the present study where they utilized commercial floating feed. The outcome of the present study indicates that, moringa leaf powder mixed feed is more effective to increase survival rate of fish than the commercial feed.

Tilapia production
Profit from aquaculture mostly depends on fish production which depends on fish growth and suitability of the culture system. The highest fish production was 9.21±0.39 kg m -3 in MLP 10% followed by MLP 0% (7.49±0.63) and MLP 20% (7.39±0.25) kg m -3 (Table 2). Fish production was significantly different among the treatments (p< 0.05) which indicate that addition of 10% moringa leaf powder (MLP 10% ) in feed has high potential than the other treatments. Moniruzzaman et al. (2015) found 12.4 to 18.8 kg m -3 monosex tilapia over a period of 120 days in cage culture system in Kaptai Lake which was higher than the present study and it might be due to the open water condition.

Tilapia blood parameters 3.2.1. Glucose
In the present study, the glucose concentrations of tilapia blood were 48.00±2.00, 44.33±1.53 and 43.67±1.53 mg dl -1 in MLP 0% , MLP 10% and MLP 20% , respectively and there were significant differences (p< 0.05) in glucose concentrations among the treatments (Table 3). Glucose is one of the most important energy sources used by fish to cope up with physiological stress. Increase in blood glucose levels is responsible for provision of energy that is used by fish to adjust with stress. In the present study, the lowest glucose in MLP 20% (Figure 3) might be due to the utilization of glucose as a result of stress resulting from temperature fluctuation. Toutou et al. (2018) noted that, dietary supplementation of lemon peel decreased the blood glucose level. Jimoh et al. (2015) reported that diet of tilapia containing watermelon seed meal resulted no significant change in blood glucose level.

Cholesterol
Moreover, the blood cholesterol values of tilapia were 177.67±2.52, 152.67±2.08, and 148.33±1.53 mg dl -1 in MLP 0% , MLP 10% and MLP 20% , respectively and there were highly significantly differences (p≤ 0.01) among the treatments (Table 3). The concentration of blood cholesterol is influenced by the fat content in the diet and is an important blood component to evaluate new diets. At the end of experiment, the value of blood cholesterol in MLP 20% was lower than MLP 0% and MLP 20% . The highest cholesterol value was 180 mg dl -1 in MLP 0% on 30 th November, 2020 ( Figure 4). The finding of Nirmala et al. (2020) Yao et al. (2020) opined that, lotus leaf extract in feed lowers the blood cholesterol level significantly in tilapia.

Water quality parameters 3.3.1. Temperature
Water temperature is a very crucial parameter for fish to maintain normal physiological process in body and it strongly affects aquaculture production (Akter et al., 2020;Boyd, 2017). During present experiment, the water temperature was fluctuated throughout the study ranging from 23.54 to 31.17°C ( Figure 5a) and the mean values were statistically similar among the treatments (p> 0.05) ( Table 4). Water temperature in all the treatments increased up to 24 th October, 2020 and after that downward trend of temperature was found up to the 30 th November, 2020 ( Figure 5a). Sudden fluctuation in water temperature might have reduced the survival rate and appeal for feed of the stock. The temperature range of the present study was suitable for aquaculture (Abdelrahman and Boyd, 2018;Boyd, 1990) and the values were close to the finding of Nirmala et al. (2020) whereas they found 28.07-30.60°C during an experiment of tilapia after using phytochemical. According to Handayani et al. (2013), the optimum temperature range for the cultivation of tilapia was 25-30°C.

Dissolved oxygen (DO)
The mean values of dissolved oxygen (DO) were 5.18±0.60, 5.24±0.53 and 4.93±0.53 mg L -1 in MLP 0% , MLP 10% and MLP 20% , respectively (Table 4). During study, the lowest DO (4.27 mg L -1 ) was observed on 30 th November, 2020 in MLP 20% (Figure 5b). The DO concentrations were not significantly different among the treatments (p> 0.05) ( Table 4) which might be due to suitable stocking density in the rearing tanks and regular siphoning of the excreta and uneaten feed from the bottom. Makori et al. (2017) found DO 10.6±8.4 mg L -1 during tilapia culture. Akter et al. (2020) reported that, continuous use of air pump during fish culture enhances dissolved oxygen which increased up to 8.2 mg L -1 .

pH
The highest and the lowest pH values were 7.67±0.42 and 7.42±0.40 in MLP 20% and MLP 0% , respectively and the mean values were statistically similar among the treatments (p> 0.05) (  Figure 5c). A study by Nirmala et al. (2020) showed that, the pH value during the rearing of red tilapia ranged from 6.88 to 7.91. Whereas, Handayani et al. (2013) stated that the optimum pH range for the tilapia culture was 6.5-8.5.

Total dissolved solids (TDS)
The mean values of TDS were 355.17±23.52, 352.50±19.44 and 365.83±14.96 mg L -1 in MLP 0% , MLP 10% and MLP 20% , respectively. The values were statistically similar among the treatments (Table 4) which might be due to the regular siphoning of the waste materials from the tanks. However, the TDS of the present study ( Figure  5e) was lower than the result of Khan et al. (2017) where they reported TDS of 653-740 mg L -1 during pond culture. Roy et al. (2021) observed water TDS during tilapia culture ranging from 188.36±36.45 to 249.51±57.12 mg L -1.