Effect of Moringa olifera leaves on growth and gut microbiota of Nile tilapia (Oreochromis niloticus)

The study was conducted to evaluate the effect of Moringa olifera on the growth and gut health of Tilapia ( Oreochromis niloticus ). The feed having 30% crude protein was prepared as an experimental diet with 4%, 8% and 10% M. olifera leaf supplementation, respectively. The control diet was devoid of M. olifera leaves . The 10 weeks feeding trial was carried out on 60 fish in aquaria. Fish was fed @ 3% of body weight twice a day. Diet with the high level of inclusion of M. olifera leaves significantly increased the growth rate, Survival Rate (SR), Specific Growth Rate (SGR) and Feed Conversion Efficiency (FCE) in all treatment groups compared to the control group. Similarly, Feed Conversion Ratio (FCR) gradually decreased and found highly-significant. To check the gut health of the Tilapia, random samples were selected and dissected. Nutrient agar was used as culture media to check the growth of bacteria. Pour Plate Method was used for viable colonies count by colony counter. Through staining method, the different bacteria such as Escherichia coli, Salmonella , Shigella and Pseudomonas aeruginosa were identify abundantly in the intestine of control diet fish but less number present in treatment diets groups. These results showed that M. olifera leaves up to 10% of dietary protein can be used for Nile tilapia for significant growth and healthy gut microbiota of fish.


Effect of Moringa olifera leaves on growth and gut microbiota of Nile tilapia (Oreochromis niloticus)
Efeito das folhas da Moringa olifera no crescimento e na microbiota intestinal da tilápia do Nilo (Oreochromis niloticus)

Experimental design
The present study was conducted at the hatchery, Department of Fisheries and Aquaculture, the University of Veterinary and Animal Sciences, Ravi Campus, Pattoki. The experiment was conducted in glass aquariums and the experimental fish species was tilapia (Oreochromis niloticus). Sixty (60) Tilapia fish, of 10 grams were collected from the pond and were placed in four different glass aquaria. Each aquarium contained 15 fish and categorized in three different treatments and one control group. The Control group was fed on a normal diet, devoid of M. oleifera leaves. Whereas Treatment 1 was (4%), Treatment 2 (8%) and Treatment 3 (10%) M. oleifera leaves supplementation. Water quality parameters; dissolved Oxygen and pH were monitored during the whole trial respectively.

Feed formulation
A feed with 30% CP was prepared by 4%, 8% and 10% M. oleifera leaves supplementation with different feed ingredients as shown in Table 1. The control diet was free from M. oleifera leaves. Fish were fed 3% of body weight twice a day during the entire trial. Feed formulation is below in Table 1.

Evaluation of growth parameters
Growth performance and diet nutrients consumption were evaluated in terms of body weight gain (BWG), length gain, daily body weight gain (DWG), feed conversion ratio (FCR), survival rate (SR) and specific growth rate (SGR).

Weight gain %
The increased body weight was figured as difference between final and initial weight by using the Formula 1:

Weight gain Final body weight g Initial body weight g =
(1)

Feed Conversion Ratio (FCR)
Ratio of feed intake to weight gain was evaluated by the following Formula 2: (Shabir et al.,2003).
bring down the use of fishmeal (Tacon and Metian, 2013;Rana et al., 2009). To improve quality and cost effect of fish feed; substitution of fishmeal with topically cheaper plant ingredients (Tacon et al., 2006). Plant can be used as a protein source for fish feed. Conventional and considerable plant sources are soybean, groundnut, cotton seed and rapeseed meal (Jackson et al., 1982;El Sayed, 1990). However, livestock, human consumption and industrial use of these sources make them costly and put far away from the reach of fish farmers, researchers and producers of aqua feed (Fasakin et al., 1999). Therefore, for research purposes such more economical and environment-friendly protein sources are used that are obtained from the plant's leaves, seeds and other agriculture by-products (El Sayed, 1990;Siddhuraju and Becker, 2001). Animals and humans use plant products as a valuable source to maintain their health. Plants have a large number of chemical substances that contain medicinal and curative agents.
Moringa oleifera belongs to family Moringaceae is native to tropical and sub-tropical regions of Pakistan, India, Africa, and Arabia, the western sub-Himalayan region and Asia Minor (Mughal et al., 1999). M. oleifera is cultivated as vegetable (leaves, pods flowers, roasted and seed) spice mainly present in roots and have medicinal uses (Rebecca et al., 2006). These parts are used as antipyretic, antiepileptic and antihypertensive (Dahot, 1988), antiinflammatory (Mehta et al., 2003) anti-ulcerative (Mahajan and Mehta, 2007) anti-diabetics, anti-bacterial, anti-fungal and anti-oxidant. Moreover, M. oleifera is fast growing and have the power to purify water. (Nikkon, 2003).
Tilapia is worldwide preferable aquaculture fish species due to rapidly growing ability, falling to family Cichlidae, has laterally compressed, deep bodies, like other cichlids. Their lower pharyngeal bones are fused into a single toothbearing structure. Beside their temperature sensitivity, Tilapia can exist and adapt wide range of conditions. Tilapia survival has been testified in brackish and mostly, enough salty water cause mortality in fresh water fish (Chapman, 2000;Lind et al., 2019). The aims of the present study were to evaluate the suitability of M. oleifera leaves as a partial protein replacement for fish in practical diets of Nile tilapia, based on its effects on growth performance and total bacterial count (positive or negative) on different culture media such as tryptone soya agar (TSA), Nutrient agar (NA) and Eosin methylene blue (EMB).

Daily feed intake
Daily feed intake was calculated with following Formula 4 as previously described by (Helland et al., 1996) A denoted weight of air-dry feed (g), A DM for dry matter content of air-dry feed (%), W is weight of waste feed collected (g), W DM for dry matter content of waste feed (%), and R for recovery of dry matter of waste feed (%)

Bacterial isolation
Three randomly chosen fish from each treatment were dissected and the intestine was removed from each sample with sterile dissecting instruments. The intestine was longitudinally opened and gently agitated for 2-3 minutes in a 9% saline solution in a flask to remove the other contents. Eppendorf tubes were filled with 900µl, 9% normal saline solution (sterilized). 100µl solution containing intestinal content was transferred to 900 µl normal saline solutions in Eppendorf tubes. This acted as 1 st dilution, then 100 µl solutions from that dilution was added into 900 µl in the next tube. This resulted 100 times second dilution. Bacterial colonies were cultured by pouring and spreading 10 µl the solution in petri plate contained 20 ml media and were placed in an incubator at 37 °C for 24 to 48 hours. Colonies of bacteria were counted after incubation of 24 and 48 hours (Canberra, 1999).

Total viable count
The pour plate method was used for the estimation of viable counts. 15-20ml sterilized Tryptic soy agar was melted and then cooled at 45 °C was settled for solidification. Nutrient agar and Eosin methylene blue (EMB) were employed for the microbial count (Buchanan and Gibbons, 1974) (Formula 5). .

Total Viable Count
Average no of colonies Dilution factor = × (5)

Statistical analysis
Data of growth parameters were put through one-way analysis of variance (ANOVA) (Steel et al., 1996). Means difference were evaluated by Tukey's Honestly Significant Difference Test and considered significant at P<0.05 (Snedecor and Cochran, 1991).

Growth parameter
Growth performance and feed utilization of Tilapia is given in Table 2. Growth performance of Tilapia revealed the body weight, body length, body weight gain and daily body weight gain were significantly (P<0.05) increased in treatments T1, T2 and T3. Body weight was higher in the T3 group as compared to T2, T1, and Control group respectively. A similar pattern was observed for body length, body weight gain and daily body weight gain. In term of feed utilization, the results showed non-significant difference found in FCR, FCE, SGR, FI, SR, FCR and value of mean range cameto1.96 to 2.85. The lowest FCR with high level inclusion of M. oleifera leaves was observed in T3. FI increased in all groups while FCR were decreased. SGR was increased in the all-treatments groups. All fish grow normally but a high mortality rate was found in the control group although no mortality observed in T3. Progressively increased the level of M. oleifera leaves in the diet significantly shown the significant growth and survival rate of Tilapia and indicated the positive effect of M. oleifera leaves on the fish health. Similarly, Figure 1 shows the overall growth performance of the fish after using different treatments. The bar chart clearly showed the maximum weight gain and length gain in treatment 2 and treatment 3. Whereas, weight gain and length gain in control and treatment 1 can be seen as identical. The graph shows that the SR of fish in Treatment 3 remains approximately a hundred percent while others were less.

Bacterial growth
Microbiota in the intestine of sampled fish from each treatment were examined and compared with the control group. The microbiota of the control group was different from treatments. There were present maximum numbers of disease-causing bacteria in the intestine of the control group compared to the treatments.
In the present study Escherichia coli, Pseudomonas aeruginosa, Shigella and Salmonellae were identified in large amounts in the control group as compared to the treatments that contain a different concentration of M. oleifera leaves. There was 80% growth of bacteria in the control group that is devoid of M. oleifera leaves and resistant to control group but in the treatments that contain M. oleifera there were minimum no of bacteria because M. oleifera inhibit the growth of bacteria listed in Table 3.

Discussions
Earlier studies evaluated significantly the effect of Moringa olifera (M. oleifera) on growth parameters (Ozovehe, 2013;Ahmed et al., 2014). Those significant results recognized that Moringa is most effective source of protein, crude fibers, and fats (Francis et al., 2001). Recent study conducted in Egypt revealed that Moringa leaf meal improves the growth rate of Nile tilapia (Elabd et al., 2019). Puycha et al. (2017) concluded that M. oleifera leaf @ 100 g/kg demonstrate significant growth. Another study reported the significant growth rate of tilapia fed with M. oleifera supplements (Tiimub et al., 2020). Protein extracted from M. oleifera seed at 400 mg kg -1 used as growth promoter in Tilapia (Stadtlander et al., 2013). Aqueous extract of M. oleifera is used as growth accentuate for Tilapia (Shourbela et al., 2020) In a previous study M. olifera examined as a better antibacterial effect against Gram-positive bacteria (S. aureus and E. faecalis) than Gram-negative strains (E. coli, Salmonella, P. aeruginosa, V. parahaemolyticus and A. caviae) (Peixoto et al., 2011). Moringa seed contains pterygospermin, moringine, 4-(α-L-rhamnosyloxy)-phenylacetonitrile glycosides and 4-(α-L-rhamnosyloxy) benzylisothiocyanate and constituents inhibit the growth of Streptococcus, E. coli, Shigella and Pseudomonas aeruginosa (Jeon et al., 2014). Another study was conducted on Moringa seed extract to observed the Salmonella enteritidis cultures to be vulnerable to water that is treated by Moringa seed extract and results showed that the extract is not able to inhibit the growth of salmonella (Madsen et al., 1987). In 2010 study held by Oluduro et al. (2010), use M. oleifera, seeds showed strong antibacterial activity against Pseudomonas aeruginosa, E. coli, S. aureus, Penicillium sclerotigenum and Cladosporium cladosporioides. At Sao Paulo study was held by Viera et al. (2010), evaluated the effect of M. oleifera against Gram Positive and Gram-negative bacteria. All the previous studies showed a close resemblance with the present study.

Conclusion
In conclusion, the results obtained from the present study recommended Moringa oleifara leaves could replace with or used with fish meal in Nile tilapia (O. niloticus) feed as a growth enhancer. It is suggested that M. olifera leaves added in cultured fish diet had the best growth performance parameters with minimum cost and maximize profit. M. olifera leaves inhibit the growth of various disease-causing bacteria such as E. coli, P. aeruginosa, Shigella and Salmonella in the intestine of fish. The presence of minerals and vitamins in M. olifera leaves help to enhance the immune system and cure a myriad of diseases. The antimicrobial factors found to present in M. olifera leaves is responsible for antimicrobial properties.