Effect of Substituting Soybean Meal with Moringa oleifera Meal on the Growth and Body Composition of Labeo rohita Fingerlings

Daniel Masood1, Noor Khan1*, Khalid Javed Iqbal2, Sadaf Dogar1, Abdul Hanan1, Sadia Nazir1, Sheeza Bano1, Azra Anwar2, Sameul A.M. Martin3 and Chris J. Secombes3 1Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore 2Department of Zoology, The Islamia University of Bahawalpur, Pakistan 3School of Biological Sciences, University of Aberdeen, United Kingdom Article Information Received 15 August 2019 Revised 12 September 2019 Accepted 25 September 2019 Available online 13 May 2020


INTRODUCTION
F ish is an excellent source of vitamins, essential minerals, high quality protein and unsaturated fatty acid (Petricorena, 2014). The demand for fish production via aquaculture is increasing to fulfill the protein requirement in human diets across the globe. The principal input in fish production is the feed, with high prices and limited supply of fish meal, major constraints in the aqua feed industry (Gabriel et al., 2007). Therefore, fish culturists are currently focusing on replacement of high cost animal ingredients with natural high quality plant proteins for fish feed formulation to reduce the cost (Hashem et al., 2017). Plant based protein source for fish feed, especially of soybean meal (SBM), have been extensively used in aquaculture production (Storebakken et al., 2000;Barros et al., 2002). However, excess reliance on SBM may increase its price. Therefore, exploitation and consumption of other low-cost plant based protein sources for fish feed will help is used for the treatment of many human health conditions including malnutrition and cardiovascular disease, and possessed diuretic properties, is hepatoprotective, has antiulcer effects and lowers cholesterol levels (Luqman et al., 2012).
The M. oleifera leaves contain a high proportion of crude protein, varying from 25% to 32% (Makkar and Becker, 1996;Soliva et al., 2005). This protein is easily digestible due to the presence of a low content of acid detergent insoluble protein (1 to 2%) and high content of pepsin soluble protein (82 to 91%) (Makkar and Becker, 1996). The leaves of M. oleifera have been reported previously to be an excellent fish meal substitute in the diet of north African catfish Clarias gariepinus and roho labeo Labeo rohita fingerlings up to a 10% inclusion level (Ozovehe et al., 2013;Ezekiel et al., 2016;Arsalan et al., 2016;Mehdi et al., 2016). Furthermore, seed meal of M. oleifera has also been explored as a good alternative plant based protein feed in Nile tilapia (Oreochromis niloticus) fingerlings (Hashem et al., 2017). With this in mind the present study was designed to evaluate the potential of M. oleifera meal to replace soybean meal in diets for L. rohita and also to analyze its effects on growth, body composition, liver and gut health of L. rohita fingerlings.

Study site, experimental fish and procedure
The experiment was conducted at Fish Seed Hatchery of the Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences (UVAS), Ravi Campus, Pattoki. The fish L. rohita (average weight 190.25±00g) were collected from fish ponds at UVAS Ravi Campus Pattoki, Punjab, Pakistan and stocked randomly in 8 aquaria (60.96 × 55.88 × 40.64 cm) at15 fish/aquarium. Before stocking fish net body weight and length were measured and recorded. The feeding trial lasted for 90 days.
Collection of Moringa oleifera and processing M. oleifera leaves were collected from Bahawalpur and washed with freshwater to remove all contaminants, properly drained and dried under shade for 1 week. Thereafter, dried leaves were crushed into a fine powder with an electric grinder and stored in opaque, air tight plastic zipper packets.

Fish feed formulation
Four types of experimental feeds containing 26% crude protein were formulated (Table I), with differing inclusion levels of M. oleifera meal, with other nutrients from soybean meal, fish meal, rice polish, wheat bran, sunflower meal and nutrimix. The M. oleifera meal was incorporated into each diet to give levels of 0% (control), 10%, 20% and 30%, with the fish fed these diets designated as T 0 , T 1 , T 2 and T 3 , respectively. Experimental diets were analyzed using proximate analyses. All analyses followed the procedure described by AOAC (2006). Feed ingredients were mixed thoroughly and a sufficient amount of water was added for smooth pelleting. Pellets (2mm) were then formed using a local pelleting machine and stored in the air tight packaging until use.

Feeding protocol
The L. rohita fingerlings were fed twice a day at 7:30 -8:30 AM and 2:30 -3:30 PM at a rate of 3% of total body weight per day. Feed quantity was recalculated every fortnight.

Determination of growth parameters of fish
Growth parameters of fish, such as initial weight and length, were measured before stocking. At the end of trial other parameters such as final weight, feed conversion ratio (FCR), percentage weight gain, net gain in weight and specific growth rate (SGR) were also recorded.

Physico-chemical parameters of water
All physico-chemical parameters of aquarium water such as dissolved oxygen (DO), temperature, salinity, electrical conductivity, pH and total dissolved solids (TDS) were recorded on a daily basis using a dissolved oxygen meter and multi meter, except nitrates which were recorded on a fortnightly basis using HANNA Nitrate Test Kit HI3874.

Statistical analysis
The results of body composition, growth and physicochemical parameters were analyzed using one way analysis of variance (ANOVA) using statistical software SAS version 9.1.

RESULTS
Statistically significant (P ≤ 0.05) differences were recorded in growth parameters of all treatment groups (Table II). The maximum weight gain (254.00±4.24g) was observed in T 3 followed by T 1 (127.50±9.19g), T 2 (198.50±20.50g) and control T 0 (80.97±17.80g). Highest length increase (85.10±0.98cm) was found in T 2 , followed by T 0 (78.10±1.83cm), T 1 (75.95±4.59cm), and T 3 (74.20±3.95cm). Significant differences (P < 0.01) were observed in the feed conversion ratio (FCR) between T 3 (1.81±0.06) and T 1 (2.70±0.13) and specific growth rate (SGR %) of T 3 (0.42±00) and T1 (0.25±0.01). The lowest (0.25±0.01) specific growth rate was recorded in fish fed   Proximate analysis of the fish fed on the four diets formulated for the experimental trial is presented in Table  III. A significantly higher proportion of crude protein (62.45±0.15%) was observed in T 3 that was notably different from the control group (T 0 ). The maximum fat content (8.67±0.03%) was found in T 2 exhibiting significantly greater content than T 1 (6.53±0.04%) and control T 0 (5.70±0.05%) fish. Ash content in T 0 (6.80±0.08%) was significantly higher than all other diets. No difference in moisture content was found in fish fed the different diets. The dry matter of the fish sampled in T 2 and T 3 showed significantly higher values (P < 0.05) than T 1 .
Physico-chemical parameters of the water exhibited non-significant results in all treatments (Table IV). A favorable temperature (21-22°C) was recorded in all the treatment groups. The DO remained in the favorable range throughout the experiment (5.72±0.91mg/L, 5.60±0.80mg/L, 5.52± 0.87mg/L and 5.81±0.29mg/L for T 1 , T 2 , T 3 and T 0 groups respectively). During the whole experimental period the pH was stable at 8 in all the treatments. The maximum electrical conductivity (2640.86±965.67µS/cm) was observed in T 2 . No significant differences were found in salinity, TDS and nitrates among all the treatments (Table IV).

DISCUSSION
Fish demand is continuously rising globally due to its nutritious and healthy characteristics. The aquaculture industry is rising globally as the fastest growing fish producing sector (Ottinger et al., 2016). However, this industry is facing a lot of challenges to provide nutritionally balanced feed to fish. Increasing demands, high prices and limited supply of fish meal turn the attention of fish culturists to replace costly fish feed ingredients with alternatives which are cheaper and that are easily, locally and widely available. The chief protein source ingredient in fish feed is fishmeal. Plant derived protein is therefore a good potential substitute and the leaves of M. oleifera are a rich source of plant based protein (30%) (Arsalan et al., 2016). Hence we have investigated the use of moringa leaf meal, at different inclusion levels, in fish diets.
In the present study the maximum weight gain was observed in T 3 , followed by T 1 , T 2 and T 0 . Highest length increase was seen in T 2 , followed by T 1 , T 3 and control groups. Yuangsoi and Matsumoto (2012) showed that when soybean meal was replaced with M. oleifera leaf meal (MOLM) it affected the growth performance and fish (fancy carp) digestibility. Hence, MOLM could be replaced not up to 20% of plant protein in soybean. Continuous rise in the replacement of fish meal (animal protein) with M. oleifera leaf meal (MOLM) could slow down the growth of aquaculture (Richter et al., 2003). Ritcher et al. (2003) observation was further supported by El-Nadi (2015) when it was shown that growth performance of O. niloticus decreased with the increase of M. oleifera plant meals in the diet. Further, substitution of 55% MOL meal for soybean meal awarded the best growth performance (less than 7.34%) and feed nutrient utilization in O. niloticus was explored by Tiamiyu et al. (2016). While the present study shows contradictory results to those of Ozovehe et al. (2013). These authors reported that growth and feed consumption of fish, Clarias gariepinus decreased with increasing proportion of MOLM in fish feed. Afuang et al. (2003) described that solvent extracted MOLM could replace 30% fishmeal in the feed of O. niloticus. Mehdi et al. (2016) studied the effect of M. oleifera on the growth of Labeo rohita and found that 10% inclusion of this plant is better for the growth of this species, beyond this it has negative effect which may be due to presence of antinutritional factors or amino acid imbalance. However, the results of the present study showed that increase in M. oleifera meal replacing soybean meal increases the L. rohita growth.
The products derived from M. oleifera have been explored as important ingredients for practical feed of L. rohita. The findings of the present study revealed that MOLM could be used as a protein substitute up to inclusion levels of 20-30% in diets for growing L. rohita. The findings of our study are broadly in agreement with that of Ncha et al. (2015) who demonstrated that fish fed 0% MOLM diet exhibited a peak value of 6.59 and lowest value of 5.86 in fish given a 60% MOLM diet. Karina et al. (2015) replaced soybean meal with MOLM to give levels of 0%, 8%, 16%, 24% and 32 %, and found highest SGR in fish fed a diet with 32 % MOLM (1.45 ± 0.09%). However, the lowest FCR was seen in the control group (7.50± 1.10), while the highest value was found in fish given the 32% inclusion (9.96±0.13). Similarly, increment in the FCR values were also recorded in Nile tilapia with the inclusion increment of moringa leaves in diet which is an indication of presence some anti-nutrients (Richter et al., 2003).
The crude fat content was found to increase with increasing moringa meal up to 20%. This data agrees with results from Mehdi et al. (2016) where increase in fat was observed in fish being fed up to 20% moringa meal in L. rohita. Further, the present research supports the results of Ganzon-Naret (2014) where an increased proportion of crude fat was found on addition of MOLM in Asian sea bass (Lates calcarifer) diets. Our results differ from other experiments, reported the highest value of crude fat in different fish species using 10% MOLM in diets (Olaniyi et al., 2013;Thiam et al., 2015;Arsalan et al., 2016).
In the present study, all physico-chemical parameters of water exhibited no significant differences between the treatment groups.

CONCLUSION
In conclusion, the current research shows that M. oleifera meal could be used safely for promoting the growth of fish without any harmful effects on body composition of L. rohita. Our results show that M. oleifera meal inclusion up to 20-30% is suitable to replace soybean meal in fish diets.

Conflict of interest
The authors declare that they have no conflict of interest.

Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.