Analysis of growth performance and haematological parameters of Oreochromis niloticus fed on a varying diet of Moringa oleifera Lam . leaf meal as an additive protein source

The objective of the current study was to investigate the growth performance and haematological parameters of Oreochromis niloticus fed on four different diets at different Moringa oleifera leaf meal inclusion levels over a period of 33 weeks. The results indicate that Moringa leaf meal causes depressed growth in fish but may increase the immunity of the fish to fight infections and diseases. The four inclusion levels used were: 0% (No Moringa control), 5, 10 and 15%. There was a significant (p<0.05) difference in the final weight of fish in the control diet versus the other three treatments. The Specific Growth Rate (SGR), Mean Weight Gain (MWG) and Condition Factor showed no significant (p>0.05) difference among the fish fed with the four diets. However, the SGR and MWG were highest in the control fish followed by 15% then 10% and lastly 5%. The Gonadal Somatic Index, Hepatosomatic Index and Cardiosomatic Index were not significantly (p>0.05) different among the four treatments. The mean values of haematological parameters showed no significant (p>0.05) difference among the fish fed on the four diets. However, the Mean Cell Haemoglobin in 15% Moringa diet was significantly different from the control diet. White Blood Cells were not significantly different among the treatments (p>0.05) though high values were observed in treatments with Moringa inclusion. The study has shown that inclusion of Moringa to the fish diet results in depressed growth but it improves the immunity of the fish because of an increase in White Blood cells. Further studies to explore the use of Moringa in fish diets at rates which do not cause depressed growth but improve immunity are recommended.


INTRODUCTION
Aquaculture production in Africa has been on a steady increase growing more rapidly in Sub-Saharan countries than the rest of Africa (FAO, 2012).In 2010, Africa contributed 1.2 million tonnes of fish from aquaculture production representing 2.2% of global aquaculture production (FAO, 2012).One major constraint in aquaculture production has been the cost of fed ingredients.Fish meal has been a major dietary protein source in compounded tilapia feeds (Olsen and Hasan, 2012).Protein requirement in tilapia feeds under intensive culture can represent about 50% of total costs (El-Sayed, 2006).As a result of the high cost and limited amount of fish meal available, research in fish nutrition has focused on developing alternative cost effective tilapia feeds using plant based sources (El-Sayed, 2006;Olsen and Hasan, 2012).
One plant receiving a lot of attention as a possible replacement of fish meal is Moringa oleifera.This is a fast growing plant and it is widely available in the tropics and subtropics and it has several economically important uses for industry and medicine (Richter et al., 2003).The leaves are rich in proteins, vitamins, fatty acids and minerals (Moyo et al., 2011).It has being nicknamed as a 'wonder plant'.Various studies have been conducted to replace fish meal with Moringa leaf meal in diets of mainly Oreochromis niloticus, Clarias gariepinus and other species with varying results (Madalla et al., 2013;Olaniyi et al., 2013;Hlophe and Moyo, 2014;Ncha et al., 2015;Mehdi et al., 2016).However, most studies have shown depressed growth on fish species fed with diets containing Moringa.
The haematological parameters of fish are important for evaluating the physiological conditions, disease as well as determining the effect of diet and other environmental factors in cultured fish (De Pedro et al., 2005).Apart from diet composition and environmental factors, reproductive cycles and variations in fish activity can also affect their haematological parameters (Rehulka, 2003).Several studies have reported changes in blood parameters indices of fish as a result of feed (Gabriel et al., 2011;Keri et al., 2012;Ayoola et al., 2013;Ozovehe and Nzeh, 2013;Dieyl and Olumuji, 2014).Therefore, determining the basal parameters of blood is important in order to monitor the health status of fish.For example, packed cell volume (PCV) is a useful indicator of anaemia, hypoproteinemia and leukocytosis of fish (Keri et al., 2012).
The high cost of fish meal which is the major protein source for fish diets has resulted in high feed costs.This has made it difficult for small scale producers to be productive because they cannot afford to acquire the fish feed.Thus alternatives of mainly plant based protein sources which can replace wholly or partly the fish meal is under consideration.Most of the studies conducted on the effect of Moringa on fish growth have been done over a shorter period of time compared to the current study.The haematological analysis on fish fed with Moringa have mainly been on other species especially Clarias.This study focused on O. niloticus which is one of the most cultured fish species in the world.This study evaluated the growth performance and the effect of M. oleifera leaf meal on the haematological parameters of O. niloticus in order to ascertain the impact this mineral rich plant has on the well -being of fish.

Experimental conditions and design
The experiment was conducted at the National Aquaculture esearch and evelop ent entre outh ast in itwe a bia outhern frica.welve hapas measuring 1 m x 1 m x 1 m were placed in a 750m 2 semi-concrete pond (Figure 1).The Moringa leaf meal was obtained from local Moringa farmers who prepared it by collecting fresh green leaves, washing them to remove dirty, drying them in the shade and grounding into meal form.The treatments were: Control (0%) -no Moringa, 5% Moringa diet, 10% Moringa diet and 15% Moringa leaf meal diet.These treatments were replicated thrice and assigned in a Complete Randomized Design.The experiment was conducted for a period of 231 days (33 weeks) .The experimental fish were handled according to the Canadian Council guide to the care and use of experimental animals (CCAC, 1989).

Stocking and sampling
A total of 15 fish weighing 42.0±2.50g were stocked according to sex in each hapa.Initial measurements of weight (g), standard and total lengths (mm) were taken at stocking.Sampling to measure weight, standard length and total length were then done fortnightly.

Feeding
The fish for the first 7 days before the introduction of the experimental diets were feeding on natural food in water.This was done to prepare the fish for the new diets and eliminate variations in weight.The fish were then fed with an isonitrogenous (32% crude protein) feed comprising the following ingredients; fish meal, maize meal, mineral mix, vitamin premixes and soya gold oil.The feeding was done at 3% live body weight at 09:00 hours and 14:00 hours daily.The feed was formulated using Winfeed 2.8 feed package software (Winfeed, 2006).The feed was administered in form of pellets and the feed ration was adjusted fortnightly upon sampling of the fish.The composition of the diet for each treatment is shown in Table 1.

Water quality
Water quality parameters were taken once per day using a water checker (YSI professional plus) in each hapa.The following parameters were been measured; Temperature, pH, Dissolved Oxygen, Ammonia and Nitrates.

Measurement of growth performance
The growth parameters were calculated as follows: i) Specific Growth Rate: ((InWf -InWi)/t)*100 Where InWf is the natural logarithm of the final body weight, InWi is the natural logarithm of initial body weight of fish and t is the final *Corresponding author.E-mail: ibbole@gmail.comTel: +260 966 491256.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License   Silva and Anderson, 1995).
ii) Mean weight gain: Where Wf is the mean final weight and Wi is the mean initial weight of fish (De Silva and Anderson, 1995).

Determination of haematological parameters
A total of 12 fishes were randomly selected from each treatment and blood samples collected following the procedures by Wedemeyer and Yasutake (1977) at termination of the experiment.Two 2ml of blood was collected using a syringe and needle by piercing the vein located on the caudal peduncle.Each blood sample was placed separately in each sterile vacuum tube, containing Ethylene Diamine Tetraacetic acid (EDTA) as an anticoagulant and sent to the laboratory at Ndola Central Hospital for haematological analysis.
After the blood samples for haematological analysis were collected the fish were preserved in 10% formalin for 10 days.Then the gonads, liver and heart were collected from each fish for: i) Gonadosomatic index (GSI) = ((weight of the gonads (g)/ weight of fish (g))* 100 (Bolger and Connolly, 1989).

Statistical analysis
The data was analyzed using a Statistical Package for Social Scientists (SPSS) version 15.0.(SPSS Inc., Chicago, IL, USA).Tests among the treatments were done using the ANOVA test and uncan's Multiple ange tests uncan 55 was e ployed were differences occurred at a significance level of 0.05.

RESULTS
The growth trend of O. niloticus revealed a higher final weight in the fish fed with control diet (no Moringa) which was significantly (p<0.05)different to the other three diets.The Specific Growth Rate (SGR) and condition factor were not significantly (p>0.05)different among the four diets although the SGR was highest in the fish fed on control diet followed by 15% Moringa then 10% Moringa and 5% Moringa diet was least.The mean weight gain was similarly highest in the control diet followed by 15% then 10% and 5% was least (Table 2 and Figure 2).The GSI, HSI and CSI were all not significantly (p>0.05)different among the four treatments.Furthermore, the final weight of male fish was significantly (p<0.05)higher than that of the female fish among the four treatments but there were no differences between the sexes in each treatment.
The haematological parameters in fish fed on the four different Moringa diet levels showed no significant (p>0.05)difference except for MCH were 15% Moringa diet was significantly different from both the control and 5% diet (Table 3).The highest blood indices were observed in 10% Moringa diet for all the parameters measured except Haematocrit.At 15% Moringa diet blood parameter indices reduced in all the parameters taken.
The second order polynomial regression described the best relationship between the blood parameters and the level of Moringa inclusion in the diet.The regression coefficient (R 2 ) obtained for RBC and Hb were 0.97 and 0.82 respectively.Those for MCV, MCHC, MCH were 0.83, 0.83 and 0.76 while for WBC and BASO were 0.98 and 0.85 respectively (Figure 3).

DISCUSSION
The higher growth of fish observed in the control group compared to experimental fish feeding on Moringa diet is similar to the findings by Ritcher et al. (2003).This could be attributed to some anti-nutritional factors such as phenolics, saponin and phytic acids in the Moringa leaves as reported by Richter et al. (2003).Considering the longer growing period the fish was subjected to in this study compared to other studies (Bello et al., 2013;Dienye and Olumuji, 2014), it may be concluded that the inclusion of Moringa leaf meal in the diet of O. niloticus does not result in corresponding somatic growth.
The GSI results obtained among the male fish implies that the reproductive characteristics of the male fish were not affected by the addition of Moringa in the diet though the values were lower at inclusion rate of 10 and 15%.The HSI was also similar among the treatments which indicate that Moringa leaf meal had no hazardous effect on the fish.The HSI is a useful biomarker to detect hazardous effects of environmental stressors (Sadekarpawar and Parikh, 2013).
The haematological parameters obtained were within or close to the values reported by other authors on Oreochromis niloticus and other fish species.The Red Blood Cells (RBC) counts obtained in this study were in the range of 0.92 -0.98 x10  This reduction is similar to the reports on inclusion of Moringa in the diet of C. gariepinus with inclusion levels of above 10% (Ozovehe, 2013;Dienye and Olumuji, 2014).The Haemoglobin (Hb) and Haematocrit (PCV) were in the range 4.16 -4.75 g/dL and 13.42 -15.28% respectively.These values are within the reported ranges for O. niloticus of 3.99 -4.93 g/dL and 15 -45% for Hb and PCV respectively (Bittencourt et al., 2003;Gabriel et al., 2011;Kefi et al., 2013).The Hb and PCV results also compare favourably with results obtained for Clarias species (Ayoola et al., 2013).The Hb and PCV results also decrease at the inclusion rate of above 10% Moringa similar to RBC counts.The decrease in RBCs, Hb and PCV with increased Moringa inclusion could be as a result of higher anti-metabolites which are toxic in the diet of the fish, especially tannin (Ozovehe, 2013;Dienye and Olumuji, 2014).(Bittencourt et al., 2003).The MCV is also within the range of 115-183fL for hybrid tilapia (Hrubec et al., 2000) Figure 3. Relationships between white blood cells and Red blood cells to dietary Moringa levels.and 121.67 -299.33fL for Oreochromis andersonii (Kefi et al., 2013).This implies that addition of Moringa leaf meal does not negatively affect the MCV, MCH and MCHC.
The White Blood Cell (WBC) results obtained ranged from 360 -534 x10 3 /µL.The WBC count obtained in this study was higher than reported by most authors on other species (Ozovehe and Nzeh, 2013;Ncha et al., 2015).The diet with Moringa inclusion had higher values for WBC compared with the control though not statistically different.This is similar to the positive correlation between increase in Moringa inclusion and increase in WBC obtained on studies of Clarias species ( Hlophe and Moyo, 2014;Ncha et al., 2015).The high values of WBC may be an indicator that the fish feeding on Moringa diet have high immunity which will be effective in fighting infections (Hrubec et al., 2000).

Conclusion
Summarizing, it can be concluded that the addition of Moringa to the fish diet could be a viable option.Although it decreases growth it improves the immunity of fish.This is extremely beneficial because of an increase in White Blood Cells which will eventually improve the capacity of the fish to fight infections and diseases.Further studies are recommended to investigate the level at which the Moringa leaf meal does not suppress growth but increase immunity.

Figure 2 .
Figure 2. Growth trend of Oreochromis niloticus feeding on four different diets during 231 days experimental period.

Table 1 .
Diet formulation (DM %) of the four experimental diets.
time of the experiment in days (De

Table 2 .
The growth parameters of O. niloticus fed on four different diets (Means ± Standard error).

Table 3 .
Haematological parameters of Oreochromis niloticus fed on different diets containing Moringa leaf meal (mean ± standard error).Values within the column having different superscripts are significantly different (P<0.05).