Effect of Supplemental Phytase on Phosphorus Digestibility and Mineral Composition in Nile Tilapia (Oreochromis niloticus)  

L. C. Nwanna1 , S. E. Olusola1,2
1. Department of Fisheries and Aquaculture Technology, Federal University of Technology, Akure, Nigeria
2. Department of Aquaculture and Fisheries Management, University of Ibadan, Ibadan, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2014, Vol. 4, No. 15   doi: 10.5376/ija.2014.04.0015
Received: 27 Mar., 2014    Accepted: 28 Apr., 2014    Published: 02 Jun., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Nwanna and Olusola, 2014, Effect of Supplemental Phytase on Phosphorus Digestibility and Mineral Composition in Nile Tilapia (Oreochromis niloticus), International Journal of Aquaculture, Vol.4, No.15: 89-95 (doi: 10.5376/ija.2014.04.0015)

Abstract

In this study, the effect of supplemental phytase on phosphorus digestibility and mineral composition of Oreochromis niloticus fingerlings by dietary intake was investigated. Six experimental diets: 0 (control), 2,000units, 4,000units, 6,000units, 8,000units and 10,000units phytase/kg diets were formulated and replicated twice at 30% crude protein. Fish were fed twice daily at 5% body weight for 63 days. The Apparent Digestibility Coefficient (ADC) for protein, gross energy and lipid and mineral composition (Ca, Mg, P, Fe, Zn, Mn) of the fish were investigated using standard methods. Data were analyzed using descriptive statistics and ANOVA at p= 0.05. Results of apparent digestibility coefficient for protein (60.50±0.04), gross energy (64.30±0.02) and lipid (63.70±0.05) were best in fishes fed diet 5 (8,000 units phytase/kg diet) compared with the same value in fishes fed all the other diets. The mineral composition (Ca, Mg, P, Fe, Zn, Mn) of the fishes were significantly different (p < 0.05) among the treatments. However, the group of fishes fed diets that contained phytase had better mineral composition compared to the control. Also, the fish faecal showed a downward trend in the value of the minerals with increase in the level of phytase in the diets. These results indicate that using phytase as a supplement in plant – based diets may be useful in improving feed acceptability, efficiency and mineral composition of cultured O. niloticus.

Keywords
Oreochromis niloticus; Mineral composition; Soybeans; Phytase; Digestibility

Fish depend on protein and minerals supplied through feed and from the pond environment for fast and health growth (Bello et al., 2012). Recently, the increasing popularity of aquaculture feed constitutes one of the highest operating expenditure in intensive practices (Marimuthu et al., 2010). Several attempts have been made to reduce the cost by increasing the feed efficiency and growth by employing novel and functional feeds that meet the nutrients requirement of O. niloticus in order to maximize utilization of supplied nutrients to cultured fish.
The main source of plant protein in terrestrial and aquatic feed includes soybean meal, corn (gluten), sunflower meal, canola/rapeseed meal, peas and lupins. Soybean meal represents the highest proportion of plant protein in fish diets owing to high yield, relatively high crude protein content and easy and round the year availability (Kumar et al., 2012). Soybean meal is a plant protein source and considered to be the most nutritious and used in many fish diets as partial or total replacement for expensive fishmeal. Soybean meal contains anti-nutritional factors notably; phytic acid, protease inhibitors, haemagglutins, tannins and gossypols, also anti-vitamins and anti-enzymes which reduce their biological value and nutrient utilization and often result in histopathological abnormalities in fish (Goda, 2007).
Phytate is free form of inositol hexakisphosphate (IP6) and a polyanionic molecule with six phosphate groups that can strongly chelate with cations such as calcium, magnesium, zinc, copper, iron and potassium to form insoluble salts. This adversely affects the absorption and digestion of these minerals in fish (Papatryphon et al., 1999). Phytate is found in potentially usable plant derived ingredients of fish feed such as soybean, wheat, maize, groundnut, sesame and rapeseed (Makkar and Becker, 2009). As a consequence of low digestibility of phytate by fish, most of the phytate-P ends up being excreted into the water and may cause algal bloom pollution (Baruah et al., 2004). Moreover phytate can also integrate with cation groups of protein, amino acids, starch and lipids in feedstuff reducing the digestibility of these nutrients in fish, poultry and pig. The ideal approach to maximise the nutritive value of plant-based diet is through hydrolysis of undigestible phytate by use of exogenous phytase enzyme.
Phytase, chemically known as myoinositol (1, 2, 3, 4, 5, 6) - hexaphosphate phosphohydrolase, catalyses the hydrolysis of phytate rendering P available for absorption (Kumar et al., 2012). When phytase is added to fish feed has proved to increase bioavailability of nutrients in plant protein by increasing phosphorus availability in grains and oil seeds by dephosphorylation of myo – inositor hexakisphosphate (phytate) and limited information is available on incorporation of digestive enzyme (phytase) in the diet of O. niloticus. There is need to know what inclusion is optimal, both nutrients utilization and mineral composition. The present study aims to assess the effects of supplemented phytase on the digestibility and mineral composition of O. niloticus fingerlings.
1 Results
1.1 Proximate composition of the experimental diet
The crude protein of the diets was similar and ranged between 30.8 and 31.5%. The values of the ether extract, ash content, crude fibre and nitrogen free extract were similar and ranged between 16.31 and 16.96%, 8.1 and 8.3%, 10.9 and 12.05 and 31.69 and 33.79% respectively (Table 1).

Table 1 Proximate composition of the experimental diet (DM)


1.2 Apparent digestibility coefficient of Nile tilapia fed phytase diets
Apparent digestibility coefficient for lipid was the highest in the fishes fed diet 5, followed by the fishes fed diet 6 and the lowest value was recorded in the fishes fed diet 4. Similarly apparent digestibility coefficient for gross energy followed the same trend as for the apparent digestibility for protein (Table 2).

Table 2 Digestibility coefficient of Nile Tilapia fed phytase diets at different inclusion levels


1.3 Mineral composition of Nile tilapia fed phytase diets
The mineral composition of the fishes after the experiment is presented in Table 3. The fishes fed diets that contained phytase had marginally higher composition than the fishes fed diet without phytase. The mineral composition of the fishes Ca, Mg, P, Fe, Zn and Mn shows significant differences (p < 0.05) among the treatments.

Table 3 Mineral composition of Nile tilapia fed phytase diets (mg/L)


1.4 Mineral composition of the fish faeces after the experiment
The mineral composition of the fish faeces after the experiment was presented in Table 4. The result showed a general increase in the value of the minerals in the faeces of the group of the fish fed diet without phytase compared with the values from other fishes fed diets with phytase. The Table also showed a downward trend in the value of the minerals with increase in the level of phytase in the diets.

Table 4 Mineral composition of the faeces after the experiment


2 Discussion
The effect of toasting and incubation of soybean meal supplemented with phytase on the nutrient digestibility and minerals deposition in Nile tilapia, Oreochromis niloticus was investigated. These results indicated that phytase supplementation to the experimental diets had improved the Apparent Digestibility Coefficient (ADC) of protein, gross energy and lipid. The improvement in the ADC of protein and gross energy agreed with the findings of Portz and Liebert (2004) who reported similar improvement in O. niloticus fed diets with 1000 and 2000 FTU kg-1 phytase supplementation. Also, this results support the report of Goda, 2007 who reported higher ADC (p < 0.05) values for crude protein, ether extracts (lipid) and gross energy were observed in O. niloticus fed diet supplemented with 1000 FTU kg-1 phytase. The inclusion rate of phytase in present study may play an important role in releasing phytate-phosphorus in soybean meal-based diets. Lie et al., (1999) reported improve digestibility of crude protein by 6.6% in Crucian carp, fed phytase supplementation of 500 FTU/kg diet.
Also, inclusion of soybean phytase in diet of Atlantic salmon improved protein utilisation parameters, ADC, and body levels of Ca, Mg and Zn and retention of Phosphorus (P) (Storebakken et al., 1998; Vielma et al., 1998, 2000). Protein digestibility in rainbow trout was significantly increased when fed a practical diet supplemented with 2000 FTU/kg phytase and also when reared with soybean meal-based diets sprayed with phytase (Vielma et al., 2001, 2004). Biswas et al., (2007) reported that protein digestibility was significantly influenced by phytase supplementation in red sea bream fed soybean meal based diets supplemented with graded doses of phytase, this report support this present study. Phytase supplemented diet in pangus increased apparent protein digestibility and were significantly (p < 0.01) higher at a minimum supplement of 500 FTU/kg or higher in contrast to diet without phytase. Baruah et al. (2007a) reported that maximum apparent digestibility of P and crude protein was recorded when phytase-supplemented diets contained 750 FTU phytase/ kg diets in feed of rohu, agastic fish.
From the result of the mineral composition of the fishes after the experiment, it could be observed that the minerals increased with addition of phytase in the diets. Phosphorus (P) is a critical element for fish and other livestocks. It plays a major role in the structure and function of living cells. It is an integral component of adenosine triphosphate (ATP), nucleic acids, nucleotides, phospholipids, proteins, and a key component of many coenzymes. These compounds function in energy releasing cellular reaction, cellular division and growth, in the transport and metabolism of fats, and in the absorption and utilization of carbohydrates, fatty acids, and proteins. Thus P is an essential nutrient for growth, skeletal development and reproduction in fish (Asgard and Shearer, 1997). Phosphorus deficiencies induce skeletal deformities such as curved spines and soft bones in Atlantic salmon, cephalic deformities in common carp, scoliosis in haddock and halibut (Lall and Lewis-McCrea, 2007).
However, Baruah et al. (2007b) reported that addition of 3% citric acid activated microbial phytase in feed of rohu increased absorption of Na, P, K, Mn, Mg, Fe, Zn and N in whole body and plasma. Masumoto et al., (2001) observed that P concentrations in whole body and plasma were higher in Japanese flounder fed a phytase supplemented compared to the control which is in the support with this present study. Similarly, Jackson and Robinson (1996) reported that inclusion of phytase in the diet of fish at 1,000 units per kg or higher significantly increased the mineral content of bone-especially the concentration of calcium, magnesium, phosphorus and zinc. Bransden and Carter, 1999 reported a significant increase in nitrogen and mineral digestibility of flat fish, Greenback flounder (Rhombosolea tapirina) fed soybean phytase. The findings of the reports were in agreement with the present study.
The minerals deposition in the faeces of the fishes after the feeding trials showed that the deposition were significantly higher in the faeces fishes of the fed diets without phytase than in the feaces of the fish fed diet with phytase. The result showed that phytase inclusion in the diets of O. niloticus could successfully reduce the minerals availability in faeces. Discharge of high levels of soluble P from fish culture systems into open water environment stimulate phytoplankton growth, resulting in wide fluctuations in dissolved oxygen concentrations (Li et al., 2004). Many studies have reported a clear effect of phytase supplementation in reducing P excretion from fish. Ai et al., (2007) showed that the total P effluent was significantly lowered when fish reared with a diet supplemented with phytase (200 FTU/kg). Similarly, soybean meal based diets supplemented with phytase decreased the excretion of P from red sea bream and maximum reduction was reported at 2000 FTU/ kg feed (Biswas et al., 2007). Similar results were also observed for rainbow trout (Sugiura et al., 2001).
Faecal waste of P in rainbow trout was reduced by phytase supplementation in soybean protein concentrate diet (Vielma et al., 1998) and a significant decrease was noticed when practical diet supplemented with phytase at a level of 2000 FTU/kg (Vielma et al., 2001) was fed. Phosphorus concentration in faecal matter was reduced when trout were fed a diet with phytase supplemented at 500 and 1000 FTU/kg compared to non-supplemented feed (Verlhac et al., 2007). Storebakken et al., (2000) observed that phytase treated soy protein concentrate based diet induced significantly lower excretion of P compared to when a fishmeal diet was fed to Atlantic salmon. Phosphorus content of faeces was also reduced in Atlantic salmon fed a phytase supplemented diet (Sajjadi and Carter, 2004). In juvenile catfish, Ictalurus punctatus,
Li and Robinson (1997) reported that microbial phytase supplementation in diets reduced the excretion of faecal P by about 60%.
Exogenous phytase was substantially efficient in enhancing the bioavailability of P and thus reducing the amount of faecal-P. Thereby, inclusion of phytase in aqua feed tends to reduce the phosphate load from fish wastes and thus eventually prevents phosphate induced algal bloom contamination. Any reduction in P excreted by fish and other animals is of benefit to both the environment and sustainable production (Baruah et al. (2007a). Many studies suggest potential environmental benefits to the extent of 30% to 40% reduction in P excretion (Omogbenigun et al., 2003). Sugiura et al., (2001) reported that excretion of P in the faeces of fish fed a low-ash diet containing phytase-treated soybean meal was reduced by 95-98% compared with P excretion by fish that consumed commercial diet without phytase. These ascertain supports the observation from the present study that the total wastes of P effluent were significantly reduced by phytase treatment of the different experimental diets compared to the control diet.
3 Conclusion
Plant ingredients have limitations due to the presence of phytate and other anti-nutritional factors that restrict their inclusion in fish diets. Phytate-rich plant ingredients restrict the bioavailability of P along with other minerals. A great potential exists for using phytase in plant protein based diets, which can enhance the digestibility and bioavailability of P and trace elements, reduce the amount of inorganic-P supplement in the diet to maximize growth and bone mineralization, and markedly decrease P load to aquatic environment. From the results of this study it could be suggested that phytase addition in diets could reduce mineral excretion into the culture environment which invariably means more mineral deposition in the tissues of the fish. The minimization of the minerals effluent into the environment would reduce environmental pollution associated with fish culture effluents.
4 Material and Methods
4.1 Experimental system
The experiment was carried out using twelve glass tanks (60 × 30× 30 cm) for 8 weeks in the Department of Fisheries and Aquaculture Technology Laboratory of the Federal University of Technology, Akure, Nigeria. The water level in each tank was maintained at a depth of 0.45 m throughout the experiment and replaced every three days to maintain relatively uniform physico-chemical parameters and prevent fouling from feed residues. The source of water was from Federal University of Technology, Akure water station. Each tank was well aerated using air stones and aerator pumps (Cosmos aquarium air pump, double type 3500 50Hz, 2.5w-3w) as described by Lawson, 1995.
4.2 Experimental procedure & feeding trials
There were six dietary treatments each having two replicates, with 15 fish each with a mean initial body weight of 6.23±0.1g.The fish were weighed, distributed into experimental tanks and allowed to acclimatize for 14 days before the experiment. The experiment lasted for 8 weeks during which the fish were fed at 5% body weight twice daily. The diet per day was divided into two; 2.5% given in the morning by 8.00 – 9.00 am and 2.5% in the evening by 5.00 pm. Weight changes were recorded weekly and feeding rates adjusted to the new body weights. The fish faeces were collected early in the morning before feeding by siphoning after which they were oven dried. Dried fish faeces were store in polythene bags prior to use.
4.3 Treatment of soybeans
Soybean (Glycine max) bought from a market in Akure, Ondo State was processed by using heat treatment method. The soybean was weighed using electronic weighing balance and toasted for 10 hours at 700C and ground into fine powder to form a meal. Soybeans meal (5.1kg) was mixed with 5.1 litres of water and dried in an electronic electric oven at the rate of 700c for 8 hours. After incubation dried meal was blended again into fine powder packed in plastic bags and stored at ambient temperature prior to use.
4.4 Preparation of experimental diets
Feed ingredients such as fishmeal, soybean, maize, wheat, starch, vegetable oil and vitamin- mineral premix were added and the dry ingredients were mixed thoroughly in a mixer. Water was added and the resulting dough was extruded through a ¼ mm die mincer of Hobart A-200T pelleting machine (Hobart GmbH, Rben-Bosch, Offenbug, Germany) to form noodle-like strands, which were mechanically broken into suitable sizes for the O. niloticus fingerlings. The pelletted diets were sun –dried, and stored in refrigerator (40C) to prevent mycotoxin formation until required (Table 5).

Table 5 Gross composition of experimental diets


4.5 Determination of Acid Insoluble Ash (AIA) and apparent digestibility coefficient
Acid Insoluble Ash (AIA) was obtained by adding 25ml of 10% HCl to known weights of the ash contents of the feed and fish feaces, covered with a wash glass and boiled gently over low flame for five minutes, after which it was filtered through ash less filter paper and washed with, hot distilled water. The residue from the filter paper was returned into the crucible and ignited until it was carbon free and it was re-weighed
% AIA = [(Weight of Ash- Weight of AIA) × 100] / Weight of Ash.
Apparent Digestibility coefficient (ADC) was calculated as.
ADC = 100-100 * (% AIA in feeds x % nutrient in feaces) / (% AIA in feaces % in nutrient in feed).
4.6 Determination of the minerals
Ten grammes (10g) of fish sample were ashed (dry ashing) at 550 for 6 hours in an electric muffle furnace; the ashed sample was diluted in 5ml of 10% HCl. This was filtered and made up to the mark in 50ml volumetric flask, the filtrate was used for the analysis and this was carried out at the Chemistry Laboratory of Federal University of Technology, Akure with the use of an electronic machine-Atomic absorption/emission spectrophotometer Buck scientific, model 200-A. The minerals such as Calcium, Magnesium, Zinc, Iron and Manganese with wavelength 422nm, 285nm, 214nm, 248nm and 279nm were determined respectively in duplicate and the average of the result was found. The absorbance was read off the digital display of the spectrophotometer. The concentration in parts par million (ppm) was calculated by.
ppm=R*X*V / W
Where,
R = value of each mineral, W = weight of sample, V = volume of digested sample used. However, R was calculated with the use of the formula:
R = (10ppm × Sample read) / Standard
4.7 Phosphorus
Phosphorus was determined by the use of the UV visible spectrometer. Ten grammes (10g) were ashed (dry ashing) at 550℃ for 6 hours I an electric muffle furnace, the ashed sample was diluted in 5ml of 10%HCL. The was filtered and made up to the mark in 50ml volumetric flask, the filtrate was used for the analysis and this was carried out in Fisheries and Aquaculture Technology Laboratory, Federal University of Technology, Akure. The wavelength of the phosphorus was 440nm and the standard (stock) solution of the element was prepared and introduced into the instrument. A solution of the sample is then introduced and the readings recorded in duplicate and the average result was found. The concentration in parts per million was calculated by.
A*V*D / Wt.
Where, A = value of each mineral, Wt = weight of sample, V = volume of digested sample used.
4.8 Analytical methods
Apparent digestibility coefficient (protein, gross energy and lipid), mineral composition of fish and faecal were analyzed after the experiment according to the methods of A.O.A.C (2005).
4.9 Statistical analysis
Data resulting from the experiment was subjected to one-way analysis of variance (ANOVA) using SPSS (Statistical Package for Social Sciences 2006 version 15.0). Duncan multiple range test was used to compare differences among individual means.
Authors’ contributions
This work was carried out by two authors; NLC and OSE. Author NLC conceived the idea; NLC and OSE designed the study and wrote the protocol, author NLC supervised the study. Author OSE performed the experiment and the statistical analysis as well as managed the literature searches. Also, Author OSE wrote the first draft of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to OJUOLA Michael for his technical support during the research.
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