Introduction

Increasing the global aquaculture depends on fish meal protein which related with increasing the production of aqua feeds (Naylor et al., 2009). Tilapia considered the greatest species for culture because of their high tolerance to adverse environmental conditions, their fast growth and they could be stocked easily (El-Sayed, 1999). The increasing request of fish meal and diminishing fish capture have resulted in increasing the price of fish meal. Thus, it is essential to determine the evaluability of different protein sources as cheaper substitutes to replace fish meal and/or expensive plant protein concentrates as a dietary protein source in aquafeeds (Takeuchi et al., 2002). Moringa oleifera is a natural developing plant used for several industrial and medicinal purposes offered in tropics and subtropics, especially the moringa leaves. Depending on some reports of the health benefits of moringa as natural plant, there are confounding foundations of “beneficial” food stimulating moringa as a medicine (Yuangsoi and Masumoto, 2012).

Moringa oleifera hold attention as protein source to use in fish diets at low levels (Chiseva, 2006) because it is natural and cheap. Valuable effects of bioactive plant constituents in animal nutrition may include the inspiration of appetite, feed consumed, the development of endogenous digestive enzyme excretion, stimulation of immune responses and antibacterial, antiviral and antioxidant actions (Citarasu, 2010). So, the further study was carried out to determine the influence of moringa leaves as feed additive in the formulated diets for Nile tilapia on the growth performance, body composition and the histology examination. Much more, this study states possibilities of using plant leaves as growth promoters to the fishes in future at farm.

1 Materials and Methods

The study was directed at the fish farming in Faculty of Agriculture, poultry production department, Minufiya University, Egypt during the period from May to August 2015.

1.1 The formulated diets

The composition of basal diet formulated of 25% crude protein and gross energy 17 kcal/g dry matter. All ingredients were first ground to a small particle size (approximately 250 mm). The basal diet composition was showed in Table 1. Four experimental diets were formulated including the control as basal diet (D1) without any supplementation, followed by three diets supplemented with moringa leaves meal which added simply to basal diet at 2, 4 and 6 g kg^-1 (D2, D3, D4, respectively). Dehydrated components were carefully mixed before adding water at rate of about 40% moisture. The experimental diets were formulated by a mincer with die into 1-mm diameter spaghetti-like strands, sun dried and stored in airtight containers. The composition analysis of the experimental diets was determined according to AOAC methods (2000).

1.2 The experimental fish and facility

Nile tilapia, Oreochromis niloticus fingerlings were obtained from a local fish hatchery (Saft Khaled, Bahira, Egypt). Fish were randomly distributed in twelve 80-L glass aquaria with 15 fish each with an initial average weight 2.1 ± 0.1 g. The aquaria were supplied with aerated and chlorine free fresh water at a rate of 250 mL/min. The experimental diets were fed to three replicates of fish three times per day at 9.00 a.m., 11 a.m. and 13.00 p.m. at rate of 5% of body weight for 12 weeks feeding. Every two weeks the fish from each aquarium was counted and weighed. The aquaria were cleaned daily and two thirds of the water replaced before feeding. Every other day water temperature and dissolved oxygen was measured using oxygen meter. PH was monitored twice weekly using pH meter (pH pen; Fisher Scientific, Cincinnati, OH).  During the 12-week feeding trial, the water-quality parameters averaged (± SD): water temperature, 28 ± 0.9°C: dissolved oxygen, 6.5 ± 0.5 mg ^–l; pH, 7.5 ± 0.2.

1.3 Analytic procedure

Fish body composition was analyzed according to standard procedures (AOAC, 2000). Counting and weighing fish at the end of the feeding study to calculate percent weight gain (PWG; [BW – initial BW] × 100/initial BW), feed conversion ratio (FCR; dry feed consumed/WG), feed efficiency ratio (FER; WG/ dry feed consumed), protein efficiency ratio (PER; WG/protein intake), specific growth rate (SGR; [ln final BW – ln initial BW] × 100/days), and survival ([no. of fish at the end of the experiment/no. of fish at the beginning of the experiment] × 100). Initial sample of 15 fish was collected randomly and frozen at the beginning of the study at -18°C for analysis of whole body composition. By the end of the feeding trial, six fish from each tank were sampled for biochemical analysis. Fish were homogenized individually for whole body composition and frozen at-18°C for proximate chemical analysis at the laboratory of the faculty of Agriculture at Minufiya University. Samples were analyzed as follows: dry matter after desiccation in an oven (105°C for 24 h), crude protein (micro kjeldahl, N x 6.25), and crude lipid (ether extraction by soxlhet method).

1.4 Blood analysis

After 12-week feeding study 6 fish per treatment were randomly sampled and about 1 ml of fish blood from the caudal vein was drawn and transmitted into a heparinized tube. The plasma was obtained after blood centrifuged at 1500 x g for 5 minutes at room temperature (24°C) and was stored at -18°C for biochemical analysis.

1.5 Histological analysis

Six fish per treatment were randomly selected and sacrificed. The viscera were separated and well-kept in 10% neutral buffered formalin (Thermo Fisher, Kalamazoo, MI) for 48 h. The following day, the viscera were washed with water several times and preserved in 75% ethyl alcohol for further processing. The liver, intestine and gonads were separately dissected and examined according to standard histological techniques. All tissues were longitudinally sectioned. Sections were cut to 5 µm increments, mounted on glass slides and stained routinely with hematoxylin and eosin (H&E) stain for examination through the light electric microscope (Banchfort et al., 1996).

1.6 Statistical procedure

A one way ANOVA test was used to test the differences among dietary treatments. The percentage data of weight gain and specific growth rate were arcsine transformed before the ANOVA analysis. Differences were considered significant at the P < 0.05. The differences among means were determined using Duncan ´s multiple range test (Duncan, 1955).

2 Results

2.1 Growth performance

The performance parameters of Nile tilapia to moringa leaves meal as feed additives in the experimental diets are illustrated in (Table 2). The results showed that there were no significant differences in final body weight (FBW), weight gain (g/fish), weight gain (%), and SGR (% day ^-1) among fish groups fed different diets. However, there were no significant differences (P > 0.05) in survival rates % among the different treatments. Fish fed diet D1 showed the highest growth performance in terms of  final body weight (g/fish), gain in weight (g/fish), gain in weight (%) and specific growth rate (SGR % day^-1) compared with others during feeding trial (Table 2, Figure 1). While fish fed diet D4 showed the lowest results of final body weight, gain in weight, gain in weight (%) and specific growth rate. Also, feed utilization parameters in terms of FCR, FER, FI, PI, and PER of fish fed diet D4 exhibited the same trend.

2.2 Carcass composition

The body composition of the experimental fish at the end of the feeding trial as affected by different levels of moringa leaves meal as feed additive are shown in Table 3. The moisture content among the different treatments did not differ significantly (P > 0.05). The lowest value (72.5%) was observed with fish fed D1 diet without supplementation. The protein, lipid and ash content of fish bodies among treatments are illustrated in Table 3. The highest value of protein content was observed with fish fed diet supplemented with 4 g kg^-1 moringa leaves meal (66.2). The lipid values decreased with increasing the inclusion of moringa leaves meal (Table 3). Whereas, the highest value of ash content was observed with fish fed diet supplemented with 6 g kg^-1 moringa leaves meal (12.2).

2.3 Biochemical parameters

The effects of moringa leaves meal as feed additives in the experimental diets for Nile tilapia on blood components are presented in Table 4. Among diets contained different levels of moringa leaves meal, fish fed diet D3 detected low value of total protein (6.2 g/dl) while fish fed diet D4 obtained the highest value (9.3 g/dl). The concentrations of glucose, cholesterol and triglycerides were varied significantly between all treatments (P < 0.05). Fish fed diet D3 exhibited low value blood glucose (35.3 mg/dl), while the low value of blood cholesterol and triglycerides was resulted in fish fed diets control and diet D4 (41.1 and 52.4 mg/dl respectively).

2.4 Histology

Liver histology from fish fed control and supplemented diets with different levels of moringa leaves meal (2, 4 and 6g kg^-1) for 12 weeks are briefly illustrated in Figure 1. In the control group, congestion was observed in the central veins accompanying with focal inflammatory cells infiltration in the hepatic parenchyma (Figure 2 A). The hepatic parenchyma of fish fed diet supplemented with moringa leaves meal at 2 g kg^-1 showed no alterations but dilatation and congestion were detected in the central veins in the liver structure (Figure 2 B). Also, the hepatic parenchyma showed focal haemorrage as well as focal inflammatory cells infiltration of fish fed diet supplemented with moringa leaves meal at 4 g kg^-1 (Figure 2 C). Additionally, dilatation was detected in the central vein and sinusoids of fish fed diet supplemented with moringa leaves meal at 6 g kg^-1 for 12 weeks (Figure 2 D). Normal structure was observed in the intestine of fish fed the control diet without any supplementation (Figure 3 A). However, the intestine of fish fed diets supplemented with moringa leaves meal at different levels (2, 4 and 6 g kg^-1) showed diffuse goblet cells formation in the lining mucosal epithelium associated with inflammatory cells infiltration in the underlying lamina propria (Figure 3 B, Figure 3 C, and Figure 3 D). Also, normal structure was observed in the ovary of fish fed the control diet without any supplementation (Figure 4 A). There were few primary follicles and multiple mature one in the parenchyma. Also, the same results were observed in the ovary of fish fed diets with different levels of moringa leaves meal (Figure 4 B, Figure 4 C, and Figure 4 D).

3 Discussion

Moringa leaves effects and their mechanisms of action have been widely studied in different animals. Established reports of the healthful natural plant pointed out that moringa hold potential for animal and fish diet ingredients (Yuangsoi and Masumoto, 2012). However, there is no information regarding the utilization of moringa leaves in fish diets as feed additive. Certain plant derived protein sources have shown promise in order to substitute fish meal in fish feeds. The replacement of moringa in conventional diets did not affect the growth performance of Nile tilapia (Oreochromis niloticus) (Afuang et al., 2003; Richter et al., 2003). In this study, there were no significant differences in terms of growth performance and feed efficiency (P > 0.05) among all treatments. This result may be due to the fiber content of the plant-based diets which causes reduction of the nutrients reduces digestibility, resulting in growth depression. These results agree with Afuang et al., 2003 who found that diets supplemented with methanol-extracted moringa leaf meal containing 11, 22 and 33 g kg^-1 did not affect the growth of Nile tilapia Oreochromis niloticus. Using raw moringa leaf in the diets for tilapia showed that the fish meal protein replacement at rate of 10% did not affect the growth performance (Richter et al., 2003). These results are in contrast with (Karpagam and Krishnaveni, 2014) who found significant increase in weight and specific growth rate in Oreochromis Mossambicus fed Moringa oleifera leaves as growth promoter at 5% concentration supplemented feed. In our study, the feed consumed was poorer in fish fed diets with moringa leaves meal. Ozovehe (2013) reported that the depression in growth performance relates to decrease in feed consumed. The same results were found with Afuang et al., 2003 who supposed that because of the comparative concentration of various anti-metabolic constituents present in moringa leaves. No significant differences were observed in the whole body composition in terms of moisture and protein (Table 3). The body lipid values were decreased and ash content increased with increasing moringa leaves meal in the diets. El-Nadi and Khames (2015) found that the results of body composition in terms of moisture, protein, lipid and ash content did not differ significantly except protein among all treatments when fed diets contained different levels of moringa leaves (0, 5, 10 and 15 g/ kg). In the present study, there were significant differences (P < 0.05) in lipid and ash content of fish body fed the experimental diets. Fish fed D4 exhibited the highest value (12.2) of ash content and the lowest value (16.3) of lipid content. The same results were observed on Nile tilapia and common carp respectively when fish fed diets contained plant-based proteins (Pouomogene et al., 1997; Siddhuraju and Becker, 2001; Afuang et al., 2003). The significant differences in blood components of fish fed the experimental diets were observed (Table 4). Fish fed D3 exhibited the lowest value (6.2) of total protein and glucose (35.3) of the blood. The lowest value (41.1) of the blood cholesterol was observed with fish fed D1, however, increased the inclusion of moringa leaves meal decreased the cholesterol. It has been reported that Moringa leaves and pods also have a positive effect in reducing blood cholesterol (Ghasi et al., 2000), while the lowest value (52.4) of triglycerides was observed with fish fed D4. Favier et al. (1997) stated that the reduction in blood cholesterol level after consuming plant protein diets may be related to the binding or trapping of bile salts in the gut due to a relatively high viscosity. These results are in accordance with (Hossain et al., 2001; Siddhuraju and Becker, 2001). No histopathological effect of moringa leaves meal supplemented diets on fish body tissues (liver, gonads and intestine) (Figure 2; Figure 3; and Figure 4). Normal structure of fish tissues among all treatments. These results proved that the inclusion of moringa leaves in the diets did not affect the fish tissues and had no harmful effect on it.

4 Conclusion

It can be concluded that moringa leaves powder can be used as feed additive in the formulated diets for Nile tilapia, Oreochromis niloticus with improving the performance without cause any adverse effect. More studies are recommended to investigate the high levels effects of moringa leaves supplemented diets on fish performance.

5 Acknowledgments

The financial support provided by the Minufiya University College of Agriculture, Shebin El-Kom, Egypt, for our fish research laboratory is greatly acknowledged.

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