Effect of Moringa oleifera Leaf Meal on Mineral Contents of Labeo rohita Fingerlings

Food production through aquaculture, which has been used for centuries, is currently undergoing rapid growth (Eroldoğan et al., 2022). The global aquaculture production has reached 122.6 million tonnes out of which, 87.5 million tonnes are aquatic fauna (FAO, 2022). One of the major Indian carp farmed extensively in Pakistan is rohu (Labeo rohita). This species is widely cultivated under polyculture system with other members of carp family (Cyprinidae) (Hussain et al., 2011).

One of the exceptional protein source in aqua feed is known as fish meal (FM) which is commonly utilized in this industry owing to its excellent digestibility, palatability and nutritionally balanced quality (Olsen and Hasan, 2012). So, FM is necessary for the aqua feed formulation. However, the demand and price of this meal has increased gradually as the aquaculture increased (Ahmad et al., 2021). These drawbacks compel the researchers to find the substitute for FM to meet the demand as well as to form cost effective feed for aquaculture (Caruso, 2015).

The alternatives such as plants and animal’s by-products, has been widely used in commercial aquaculture for partial replacement (Naylor et al., 2000; Nishshanka et al., 2022). One of the promising alternatives for the fish meal are the agriculture based by-products (Hussain et al., 2011) such as soybean meal (Ye et al., 2011), canola meal (Luo et al., 2012), cottonseed meal (Hassaan et al., 2019) and sunflower meal (Iqbal et al., 2022). Now, the researchers are looking for the unconventional sources of plant by-products i.e., seeds, leaves and other high quality protein products, to obtain eco-friendly as well as viable commercial production (Abo-State et al., 2014). These plant-based substitutes need to be higher in protein and nutrients and lower in fibres, anti-nutritional elements, and carbohydrates. These substitutes include, corn, barley, canola and peas. The replacement of these products also depends upon their costs, palatability and accessibility for the aquaculture species (Naylor et al., 2009). Thus, it has been established that one of the unconventional plant-based by-products, Moringa oleifera leaf meal (MOLM), is the best option to replace FM.

M. oleifera’s leaves, fruits, roots and flowers are used as vegetables in many countries (Siddhuraju and Becker, 2003) because they contain vitamins, phenolic acid, calcium (Ca), iron (Fe), beta-carotene and riboflavin (Shank et al., 2013). However, Fahey (2005) stated that Moringa leaves possess quality proteins compared to milk and egg. It contains more Ca content than milk, high K level compared with bananas, high Fe in contrast to spinach, more vitamin C than oranges and more vitamin A than that of carrots (Rag Tag, 2013). The fiber helps the body to remove some poisonous compounds and eventually stops concentration of excess cholesterol (Sodamade et al., 2013). The mineral makeup of MOLM is 2098 mg of Ca, 406 mg of magnesium (Mg), 1922 mg of potassium (K), 28.3 mg of Fe, 5.4 mg of zinc (Zn), and 351.1 mg of phosphorus (P) per 100 g of dry extracted leaves (Charles et al., 2011).

Our study’s goal is to develop a more sustainable and economically viable aquaculture feed by using different levels of MOLM based diets in L. rohita fingerling’s diet for improving mineral availability.

This investigation was conducted to determine how different concentrations of MOLM based diets affected the mineral digestibility of L. rohita fingerlings. This research was done at Government College University, in Fish Nutrition Laboratory, Department of Zoology, Faisalabad. This particular area may be found at a longitude of 31.4166 degrees North and 73.0707 degrees East.

From the Government Fish Seed Hatchery, fingerlings were purchased and kept in 70 L of V-shaped water tanks. Before the trial began, they acclimatized for 15 days and soaked one to two minutes in 0.5% saline water for the prevention of pathogenic infections (Rowland and Ingram, 1991). During the trial, water quality parameters (pH, 7.5-8.5; temperature, 25-28℃; dissolve oxygen, 5.5-7.5 mg/L) and aeration were checked daily.

M. oleifera leaves were soaked for the removal of anti-nutritional factors and completely dried to prevent from vitamin discharge. To lower the amount of crude fiber in the feed, the leaves were threshed from the stalks. Then the leaves which were dried, crushed into powder and stored in polythene bags at room temperature.

The feed components were crushed and sieved to the appropriate size (3 mm) before the feeding experiment began. These feed components were obtained from a nearby feed mill (Table I) and analyzed for chemical composition according to standard protocols (AOAC, 1995). Chromic oxide was added to the diet to obtain the digestibility. The homogeneous dough was prepared by adding water content (10-15%) and oil (5%) to the ingredients. The dough was pelletized using a lab extruder. Five iso-nitrogenous diets were prepared, with MOLM levels ranging from 0% to 40%. Fingerlings were fed 5% of their body weight two times a day. They were randomly divided into tanks with 15 fingerlings in each replication tank. Feeding trial was conducted for a total of 70 days and 5 g of feces were collected from each replicate. A system of V-shaped tanks was designed for the purpose of collecting feces. Just after a two-hour feeding session, the remaining feed was taken out of each tank. Each tank’s valve was opened at two-hour interval, and the fecal material was emptied from the fecal collecting tube. Nutrient loss could be reduced by carefully collecting the fecal threads. The fecal samples were dried for further chemical analysis.

MOLM*, Moringa oleifera leaf meal.

MOLM, Moringa oleifera leaf meal; Ca, calcium; Na, sodium; K, potassium; Fe, iron; Cu, copper; Zn, zinc; Mn, manganese; P, phosphorus; Mg, magnesium; Al, aluminium and Cr, chromium; PSE, pooled standard error. PSE, √MSE / n (where MSE = mean squared error). Mean within rows having different superscripts are significantly different at p<0.05. Data are means of three replicates (n=3).

For abbreviations and statistical details, see Table II.

The digestion of the feed and feces samples was done by boiling nitric acid and perchloric acid (2:1) (AOAC, 1995). Atomic absorption spectrophotometer was used to assess mineral content after adequate dilutions. Calorimetric analysis of phosphorus at 350nm (UV/VIS spectrophotometer) was performed. Chromic oxide was added to the diet to obtain the digestibility of feed and feces. It was determined at the absorbance of 370 nm following oxidation through perchloric acid via Spectrophotometer (Divakaran et al., 2002).

The mineral content of the experimental diets was evaluated using One-way Analysis of Variance (Steel et al., 1996). Tukey’s Honestly Significant Difference Test was used to compare means, and differences were significant when p<0.05 (Snedecor and Cochran, 1991). These analyses were performed using Co-Stat software (Version 6.303, CA, 93940 USA, Monterey, PMB 320).

Tables II and III show the analyzed mineral composition (%) of MOLM based diets and feces, respectively. The results of analyzed composition of minerals in L. rohita fed MOLM based diets are presented in Table IV. Digestibility of minerals such as Ca, Mg, Na, P, K, Mn, Cu, Zn, Cr and Al showed that different levels

For abbreviations and statistical details, see Table II.

of MOLM based diets were non-significant in L. rohita fingerlings. Analyzed mineral composition of diets, feces and digestibility showed that minimum amount of minerals such as K (0.08%), Ca (1.18%), Mg (0.002%), P (0.18%), Na (0.08%), Cu (0.003%), Mn (0.005%), Al (0.00009%), Cr (0.01%) and Zn (0.01%) were excreted through feces at a 10% level followed by 20% level of MOLM replacement in comparison with control diet and remaining levels of MOLM. Digestibility coefficient values showed that test diet-II (10% level of MOLM based diet) proved best for fingerlings when compared to control and other replacement levels of MOLM based diets. The digestibility values of Na, Cu, Ca, Zn, Mn, P, K and Al were 63.56%, 69.85%, 65.12%, 67.96%, 61.87%, 70.08%, 71.76% and 56.51%, respectively, at 10% MOLM level which were substantially significant (p<0.05) when compared to other levels of MOLM based diets and control diet. However, Mg (71.68%) and Cr (62.72%) showed best digestibility values at 20% MOLM based test diet as compared to control and other reference diets and are substantially different (p<0.05) from all other experimental diets. The Fe digestibility (70.53%) recorded at control level was higher than other levels of MOLM based test diets. According to the findings, best mineral digestibility values were obtained at 10% MOLM replacement level.

The main challenges aquaculture is facing these days is to provide feed that have nutritionally balanced ingredients. One of the most promising sources of protein for various aqua-feeds is M. oleifera (Hussain et al., 2017). MOLM is regarded as a unique substitute for fish meal because of its nutritious amino acid profile (tryptophan, methionine, cysteine and lysine) and its availability around the globe (Tagwireyi et al., 2018). Moreover, it has remarkable mineral content i.e., calcium, potassium, magnesium, zinc, iron and phosphorus (Kou et al., 2018).

In our study, mineral composition of all MOLM based test diets were equal. According to our results, 10% MOLM based diet showed less minerals discharge than control and other levels of MOLM based test diet whereas the maximum mineral excretion was noted in 40% MOLM based level which clearly showed that 10% MOLM based level is better substitute for fish meal. Different researchers partially or entirely replace fish meal with various plants and their by-products (Daniel, 2018) such as soybean meal (Ye et al., 2011), canola meal (Luo et al., 2012), cotton seed meal (Hassaan et al., 2019) and sunflower meal (Iqbal et al., 2022). However, the variable results have been observed with different sources of plants as a substitute.

In our study, maximum mineral digestibility was also recorded in 10% MOLM based diet than all other test diets and control. Digestibility data of minerals like Ca, Mg, Na, P, K, Mn, Cu, Zn, Cr and Al showed significant differences (p<0.05) when rohu fingerlings fed different levels of MOLM based diets. MOLM contains tannins, saponins, and lignins, which are anti-nutritional components. High concentration of these anti-nutrients might negatively impact the assimilation of trace elements and delay the digestion of proteins where as their low concentration is utilized as energy source. Our study is in line with Adeshina et al. (2018), in which 30% of MOLM was proved to be the option to replace soybean meal in Cyprinus carpio. Digestibility data showed that diet-II which was the 10% level of MOLM based test diet, proved best option and showed maximum apparent digestibility coefficient values compared to other replacement levels of MOLM based test diets and control diet. Nowadays, feed additives have been utilized and are being used with MOLM by various researchers to enhance the efficiency of plant by-products (MOLM) and inhibit their negative effects. According to Sarfraz et al. (2023), 35% MOLM supplemented with phytase (600 FTU kg-1) improved the mineral digestibility of Oreochromis niloticus. Variations in outcomes are a result of different species, age, feeding habitats and environmental conditions.

It was deduced that 10% MOLM level is ideal replacement level that improves the mineral digestibility in L. rohita fingerlings. It was also concluded that MOLM based plant by product’s addition by replacing the fish meal is very beneficial in development of affordable and environment friendly aqua-feed.

The authors are thankful to Higher Education Commission, Pakistan for funding Department of Zoology, Government College University, Faisalabad for provision of facilities for this research.

The authors are grateful to HEC Islamabad, Pakistan for its consistent assistance by providing Project # 5649/Punjab/NRPU/RandD/HEC/2016 and Project # 20-4892/NRPU/R and D/HEC/14/1145 to be able to manage this research work.

All applicable institutional, national and international guidelines for the care and use of animals were followed.

All the procedures and methods used in this study followed the ethical guidelines provided by Government College University Faisalabad.

The authors have declared no conflict of interest.