Optimum Dietary Ca/P Ratio and Phytase for Growth and Bone Mineralization in Juvenile Clarias gariepinus Fed Soya Bean-Based Diet

The main source of plant protein in terrestrial and aquatic feed includes soybean meal, corn (gluten), groundnut meal, sunflower meal, canola/rapeseed meal, peas and lupins [1]. 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. Soybeans are the most plentiful of oilseed crops, with a worldwide production of 253.1 million metric tonnes in 2012 [2]. However, the major constraints that limit use of plant proteins in animal feed are the presence of anti-nutritional factors such as saponins [3], lectins (Santiago et al.), and phytate being one of importance [1]. They are also limiting in essential amino acids affecting growth and nutrient utilization in fish [4]. Apart from minerals, phytate also forms complexes with proteins and amino acids [5] at acidic pH leading to the formation of phytate-protein aggregates and precipitates, which results in a decreased accessibility for proteases, and consequently in inefficient protein and amino acid digestion [6]. Cao et al. reported that the growth and feed conversion efficiency in commonly cultured fish species, such as carps, tilapias, trout and salmons were negatively affected by the phytate in the diets [7]. Phytase in fish diet can breakdown a portion of the phytate, increasing availability of dietary phytate-phosphorus in diets for fish [1,8,9]. However, very low phytase activities have been reported for legume seeds, hence dietary supplementation is necessary in oil seed meals, particularly soy bean meal to improve protein and mineral digestibility and availability, which is very low compared to fish meal [10]. Although dietary phytase supplementation results in substantial reductions in total phosphorus excretion, a slight increase of soluble phosphorus in the excreta is possible [11]. A precise prescription of appropriate phytase inclusion in diets is needed to release the actually required phosphorus and other nutrients for the animals to avoid or minimize this side effect [12], hence there is need to investigate the intrinsic (native) phytase in fish diets and the optimum phytase supplementation needed to promote growth and nutrient utilization in fish. In dose–response studies, phytase addition of 250-1500 FTU/g is usually considered feasible in many fish species. The optimum dose changes along with many factors such as fish species, different phytase sources, diet formulation (amount of substrate for phytase) and selected response parameters [7]. Optimum phytase has been determined for Channel catfish [13,14], Nile tilapia [15], Korean rockfish [16], Pangasius pangasius [17], and Rainbow trout [18]. Van weerd et al. [19] and Nwanna et al. [20] reported 750 FTU/g (Natuphos 5000) and 8000 FTU/g (Ronozyme 5000) phytase, respectively, as optimum for growth of Clarias gariepinus, but did not consider consistently increasing levels of soya bean used in their studies. In the former study, increasing levels of soya bean depressed growth performance, which could be due to the inclusion of wheat in the study. Wheat has a high phytase (1193 FTU/g), according to Eeckhout and de Paepe (1994) [47], which can compromise responses to phytase and reduce its efficacy.


Introduction
The main source of plant protein in terrestrial and aquatic feed includes soybean meal, corn (gluten), groundnut meal, sunflower meal, canola/rapeseed meal, peas and lupins [1]. 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. Soybeans are the most plentiful of oilseed crops, with a worldwide production of 253.1 million metric tonnes in 2012 [2]. However, the major constraints that limit use of plant proteins in animal feed are the presence of anti-nutritional factors such as saponins [3], lectins (Santiago et al.), and phytate being one of importance [1]. They are also limiting in essential amino acids affecting growth and nutrient utilization in fish [4]. Apart from minerals, phytate also forms complexes with proteins and amino acids [5] at acidic pH leading to the formation of phytate-protein aggregates and precipitates, which results in a decreased accessibility for proteases, and consequently in inefficient protein and amino acid digestion [6]. Cao et al. reported that the growth and feed conversion efficiency in commonly cultured fish species, such as carps, tilapias, trout and salmons were negatively affected by the phytate in the diets [7]. Phytase in fish diet can breakdown a portion of the phytate, increasing availability of dietary phytate-phosphorus in diets for fish [1,8,9]. However, very low phytase activities have been reported for legume seeds, hence dietary supplementation is necessary in oil seed meals, particularly soy bean meal to improve protein and mineral digestibility and availability, which is very low compared to fish meal [10]. Although dietary phytase supplementation results in substantial reductions in total phosphorus excretion, a slight increase of soluble phosphorus in the excreta is possible [11]. A precise prescription of appropriate phytase inclusion in diets is needed to release the actually required phosphorus and other nutrients for the animals to avoid or minimize this side effect [12], hence there is need to investigate the intrinsic (native) phytase in fish diets and the optimum phytase supplementation needed to promote growth and nutrient utilization in fish. In dose-response studies, phytase addition of 250-1500 FTU/g is usually considered feasible in many fish species. The optimum dose changes along with many factors such as fish species, different phytase sources, diet formulation (amount of substrate for phytase) and selected response parameters [7]. Optimum phytase has been determined for Channel catfish [13,14], Nile tilapia [15], Korean rockfish [16], Pangasius pangasius [17], and Rainbow trout [18]. Van weerd et al. [19] and Nwanna et al. [20] reported 750 FTU/g (Natuphos 5000) and 8000 FTU/g (Ronozyme 5000) phytase, respectively, as optimum for growth of Clarias gariepinus, but did not consider consistently increasing levels of soya bean used in their studies. In the former study, increasing levels of soya bean depressed growth performance, which could be due to the inclusion of wheat in the study. Wheat has a high phytase (1193 FTU/g), according to Eeckhout and de Paepe (1994) [47], which can compromise responses to phytase and reduce its efficacy.
In diet, it is necessary to take into account the Ca/P ratio, because it has important consequences for bone development, which can be adversely affected when this ratio increases, resulting in aberrations in bone mineral homeostasis and bone metabolism [1]. Dietary levels of Ca:P ratios are crucial to phytase efficacy and activity [21]. Cao et al. [7] reported that data on this aspect in fish are limited. High dietary Ca or a high ratio of Ca: P interferes with phosphorus absorption and reduces the effectiveness of phytase activity [22,23]. High concentration of calcium in fish feed will chelate with phytate to become insoluble complex, or compete with phytase to change the phytase activity center site as an inhibitor, or increase the pH value to inhibit the activity of phytase. Increasing Ca:P ratio depressed E. coli derived phytase action in the pig diet and thus significantly depressed weight gain and feed efficiency. The aim of this research, therefore, is to define optimum dietary Ca/P ratio and effective phytase efficacy for optimum growth performance in soya bean diet of juvenile Clarias gariepinus.

Materials and Method Experimental design
The research investigated the interactive effect of five phytase supplemented at graded levels of 0 FTU/g (P0), 250 FTU/g (P1), 500 FTU/g (P2), 750 FTU/g (P3), 1000 FTU/g (P4) and substitution of four graded levels of fish meal with 25%, 50%, 75%, and 100% roasted, fullfat soya bean meal with hulls as S1, S2, S3 and, S4 and fed to duplicate group of juvenile Clarias gariepinus in a 4 × 5 factorial design so that there are 21 treatment combinations with treatment code S0P0-S4P4 (S for soya bean, P for phytase). A diet with 100% fish meal (S0), which served as control, was included in the experiment, but with no phytase addition (P0).
About 10kg of soya bean was purchased from a local grain market with good quality soya bean products. Soybean was subjected to heat treatment by roasting [20] to remove anti-nutritional factors affecting nutrient bioavailability in fish; roasted soybean were grinded, sundried, and packed into plastic bags, and stored at ambient temperature prior to inclusion with other feeding stuff for the fish. Compounded feeds were formulated using Pearson's method and mixed with cold, distilled water to a moisture content of 14%. Feed were cold pelleted and forced through a 2 mm hand mesh and sundried before phytase supplementation. Liquid Natuphos ® phytase (Natuphos 5000L, BASF, the Chemical Co., Germany) were prepared by serial dilution of the phytase concentrations using a dilution factor of 1:10 and bulk density of 1.2 g/cm 3 (1.15-1.25 g/cm 3 ) according to BASF to give 0.4917 ml, 0.9833 ml, 1.475 ml, and 1.967 ml/kg diets for 250 FTU/g, 500 FTU/g, 750 FTU/g, and 1000 FTU/g phytase, respectively, which were supplemented in the diets according to the design. Phytase was supplied by BASF, the Chemical Co., Ludwigshafen, Germany, while phytase activity of all experimental groups analysed by BASF SE, Lampertheim, Germany. Both phytase and non phytase-supplemented groups were analysed for total phosphorus, phytate phosphorus [26], calcium, and phytase activity. The control diet, S0 (100% fish meal), did not receive any phytase treatment. The gross and chemical composition of the basal diet is shown in Table 1 below.

Experimental fish
To meet the requirement of the 4 × 5 factorial design in the first experimental group pf roasted , full fat soya bean diets, about 1160 Clarias gariepinus fish of average weight (4.5+0.2 g), were procured from a reliable fish farm with no trace of metal contamination and pollution. Fish were acclimated under laboratory conditions for 3 weeks prior to experimental feeding during which time they were fed commercial diet in 10 fish holding tanks supplied with clean water until they attained the juvenile status of average weight 11.55+0.2 g and length 11.79+1.03 cm before experimental feeding with phytase-treated diets. Water temperature, pH and oxygen maintained at optimum range between 25°C-32°C, 7.40-7.45, 4.80-5.0 mg/l. after which all fish were left unfed for 2-3 days prior to start of experiment. After acclimation, 1092 fish were randomly allocated to all 21 treatment combination groups of diets, including control diet, and fed to duplicate groups of fish stocked at 26 fish per tank ~300.30g. Each of the feed rations were shared so that experimental fish were fed twice daily (morning at 08:00 hrs) and evening (evening at 16:00 hrs) to obtain optimum growth with uneaten feed and faeces removed at each feeding period. Experimental fish were weighed biweekly throughout the experiments using an electronic compact balance S. Mettler scale with accuracy of 0.01 g (Model: K-BH). Water was changed once every two (2) days using static water renewal method and water quality monitored and measured for all treatment tanks at the end of the experiment. Fish (n=2) were sampled for bone mineralization after ashing to constant weight [27].

Determination of mineral composition
Duplicate sample of feeds were analyzed for phosphorus, calcium, sodium, copper, and manganese and determined using flame atomic absorption spectrophotometer model Buck 205, Buck Scientific, USA, while phosphorus (P) was estimated spectrophotometrically using molybdovanadate method [28].

Determination of phytate
Phytate in feeds was measured by alkaline picrate method of Oberleas [23]. Sample was extracted with 0.2 N HCl to give (3-30 µg ml -1 phytate solution). 0.5 ml of extract was pipetted into a test tube fitted with a ground glass stopper. 1 ml of ferric solution was added to the tube, which was covered with the stopper and fixed with a clip. The tube was heated in a boiling water bath for 30 minutes. Care was taken to ensure that for the first 5 minutes, the tube remained well stoppered. After cooling in ice water for 15 minutes, the tube was allowed to adjust to room temperature. Once the tube reached room temperature, the content of the tube was mixed and centrifuged for 30 minutes at 3000 g. 1 ml of the supernatant was transferred to another test tube and 1.5 ml of 2, 2'-Bipyridine solution added. The absorbance was measured at 519 nm against distilled water.

Determination of phytase activity
"Phytase activity in feed sample was determined by Engelen et al. [30] with slight modifications according to the relative method of BASF [29] using methods by Engelen et al. [30]. 100 g of each sample were incubated with sodium phytate as substrate, which was dissolved with acetic buffer. The incubation was stopped by adding acidic molybdate/ vanadate reagent to produce a colored complex with the phosphate formed. The enzyme phytase liberates inorganic phosphate from the substrate sodium phytate during incubation and the intensity of the yellow color of the vanadomolybdophosphorus complex is a measure of the amount of phosphate liberated and was measured at a wavelength of 415 nm.

Growth and nutrient utilization
Growth performance was monitored biweekly with the following parameters measured: Weight gain=Final weight-initial weight

Bone mineral analysis
At the beginning and end of each feeding trial, bones of fish sacrificed for each experiment were investigated for calcium, phosphorus, magnesium, iron and zinc analysis described as follows: Intact spinal, skull, pectoral, dorsal and caudal bones were removed from each group of fish and pooled for bone mineral analysis. Whole fish flesh was stripped off from the vertebrae (spinal) and the adhering flesh removed by light brushing and rinsing in distilled water. The vertebrae were oven-dried dried for 2 hours at 110°C and then pulverized, dried again, and weighed. The dried samples were ashed at 550°C for 6 h. Ash weight was calculated as a percentage of dry, fat-free bone weight. For mineral estimation, the ash was digested in a boiling nitric acid and perchloric acid mixture (2:1) according to the method by AOAC [28]. After appropriate dilution, calcium, magnesium, potassium, zinc content was estimated by atomic absorption spectrophotometer using flame atomic absorption spectrophotometer model Buck 205, Buck Scientific, USA, while phosphorus was estimated spectrophotometrically using molybdovanadate method [28].

Determination of phosphorus retention efficiency
Phosphorus retention efficiency was determined by method of Cao et al. [31] (10 mg

Statistical analysis
All data were subjected to one-way analysis of variance at significance level of 0.05. Individual differences in treatment means were determined by Duncan new multiple range test [32]. Interactions between dietary levels of phytase and experimental diets were determined using multiple regression analysis (factorial). Data was treated as mean+S.D. A dose-response data or growth model [33] of graded phytase inclusion was determined by the linear equation below.
Y=a 0 +a 1 X

Mineral composition of the diet
Phytase addition to fish diet has been reported to increase dietary mineral retention in fish [8,[34][35][36]. In this experiment, significant effect of phytase on mineral availability was observed. Although phosphorus in 25% soya bean and 50% soya bean (full fat) were sufficient for fish in meeting the requirement for the mineral (0.67-0.82%) as determined by Nwanna et al. [37], the significant increase in dietary phosphorus by phytase in the former could be related to phytate hydrolysis and   Mean values with the same alphabet superscript in the same column are not significantly different (P>0.05) (2-tailed). n=2 Table 6: Growth and nutrient utilization in juvenile Clarias gariepinus suboptimal phytase diet based on soyabean meal (full fat).
breakdown, which made phosphorus available (Table 2) in these diet [5,6,8] in excess of requirement [14]. While dietary phosphorus declined in diet with 50% full fat soya bean with increasing phytase addition from 250 FTU/g to 1000 FTU/g ( Table 2), values of available phosphorus increased with 1000 FTU/g phytase. Increase in available phosphorus in both diets could be due to high phytate (Table 1) in 25% (0.52%) and 50% soya bean (0.57%) without phytase, compared to phytate levels in 75% (0.43%) and 100% (0.40%) full fat soya bean diet. The magnitude of response to phytase was greater in ingredients containing high level of phytate for basal diet with 25% and 50% soya bean with phytase supplementation compared to 75% and 100% full fat soya bean diet [6,38]. Available phosphorus levels (Table 2) in 75% full fat soya bean with 250 FTU/g (S3P1) was highest compared to values in other diets (S3P0, S3P2, S3P3, S3P4), which indicated that phytase at that level significantly increased phytate hydrolysis [6] and thus increasing the level of available phosphorus from 0.33 ± 0.01% in control S3P0 to 0.42 ± 0.02%, which was above the requirement (0.33%-0.40%) for catfish [13,39]. Values of available phosphorus in 100% soya bean diet (full fat) with phytase showed a significant increase and could be due increased phytate utilization even at lowest level of dietary phosphorus compared to other dietary soya bean levels [6,40]. Although, levels of calcium were above the requirement of 0.45% for catfish [24], calcium increased with phytase in all diet except in 25% soya bean. Increase in calcium could be due to phytase effect, which released calcium from calcium-phytate complex and the presence of high phytate in these diets [6,38], thus making it available in the diet [23]. Phytase has little effect in diet when phytate content is less than 2.0 g/kg diet [6]. Increase in dietary zinc is associated with phytaseinduced breakdown of phytate-zinc complex [6], thus improving utilization of phytate-bound minerals, especially phosphorus and zinc.

Growth and nutrient utilization
Phytase has been extensively used in animal nutrition to enhance growth and feed and nutrient utilization [19,37,38,41]. Phytase enhanced apparent digestibility of minerals and their deposition in the fish [20]. It increased mineral utilization through the breakdown of phytate-mineral complex [23], which resulted in increased growth and feed efficiency of the fish [7,37] through improvement in phosphorus bioavailability [23]. Improved growth performance and nutrient Mean values with the same alphabet superscript in the same column are not significantly different at the 0.05 level (2-tailed)  utilization were observed in this research investigating the possibility of substituting entirely or almost entirely level of fish meal in fish diet [7]. Phytase addition at 250, 500, 750, and 1000 FTU/g to 25% full fat soya bean meal recorded positive weight gain (Table 3) compared to diet without phytase (25% soya bean, no phytase). When fish meal was substituted with 50% soya bean (full fat), positive effect was observed with 750 FTU/g compared to other diets (S2P1, S2P2, S2P4), and was comparable to weight gain of fish fed control diet (S0P0). At higher level of fish substitution by soya bean (full fat), phytase inclusion at 250 FTU/g was better than at 0, 500, 750, and 1000 FTU/g.
Weight gain at 250 FTU/g in 75% soya bean (full fat) was better than at lower fish meal substitution (25% and 50%) by soya bean (full fat) with phytase addition at any level, and performed as fish fed control (100% fish meal). This could be explained by fact that phytase is effective in hydrolyzing phytate at low dietary phosphorus and low available phosphorus level [6,7,23]. The total and available phosphorus in 75% soya bean diet (full fat) without phytase are much lower (0.75 ± 0.01% and 0.33 ± 0.01%) than at 25% (1.15 ± 0.01% and 0.63 ± 0.01%) and 50% (1.01 ± 0.01% and 0.44 ± 0.01%). Denbow et al. [42] tried seven levels of supplemental phytase at 0, 200, 400, 600, 800, 1000, and 1200 FTU/g in the semi purified diet of broilers containing 0.20, 0.27 or 0.34% available phosphorus. They observed improved body weight gain and feed intake (P<0.01) at all available phosphorus levels, but the magnitude of response was maximum at low available phosphorus level. Lim et al. [40] supplemented microbial phytase at 500 FTU/g in a corn-soya bean meal based broiler diet (0.25%, 0.35% and 0.45% available phosphorus) and observed significant (P<0.01) improvements in body weight gain. They reported enhanced benefits of phytase in low available phosphorus diet, but no significant (P<0.01) effect in diets containing higher level of phytate phosphorus. Johnston and Southern (2000) used eight levels of phytase supplementation at 0, 200, 400, 600, 800, 1000, and 1200 FTU/g) in low phytate phosphorus broiler diet and reported improvements in body weight gain, and that increased levels of phytase supplementation had little effect on growth performance (P<0.01). Phytase supplementation at 500 FTU/g to low available phosphorus (0.30%) diets significantly (P<0.05) improved the growth performance of broilers. This was also observed in improved growth performance in fish fed with phytase (250 FTU/g) soya bean diet (75%) with low basal available phosphorus (0.33 ± 0.01 g) compared with fish fed 25% and 50% soya bean with available phosphorus of 0.63 ± 0.01% and 0.44 ± 0.01%, respectively (Table 2). Additionally, fish fed 75% soya bean with 250 FTU/g showed the best growth performance and nutrient utilization, which was reported to be best by Castro et al. who reported 75% soya bean with phytase (4,000 FTU/g, Ronozyme) as best for Rainbow trout. In the study of Li and Robinson, fish fed the diets containing 250 FTU/g phytase or above consumed more feed, gained more weight, and had a lower feed conversion ratio in comparison to fish fed the basal diet containing no microbial phytase. In Li et al., phytase supplementation at 250 FTU/g in catfish diet increased feed intake, body weight gain, feed efficiency, and bone ash and phosphorus concentration. In the present research, 250 FTU/g at 75% soya bean and at all levels of soya bean (irrespective of soya bean levels, Table 4), had the highest specific growth rate, weight gain, and lowest feed conversion compared with others levels of soya bean diet (full fat). Feed conversion declined with increasing phytase as seen from the regression graph between FCR and analyzed phytase activity (Figure 3), which means weight gain is optimum or highest with low phytase. In Li and Robinson, channel catfish fed the diets containing 250 FTU/g or above consumed more feed, gained more weight, and had a lower feed conversion ratio in comparison to fish fed the basal diet containing no microbial phytase. Robinson et al. reported that phytase at 250 FTU/g could effectively replace dicalcium phosphate supplement in the diet of channel catfish without affecting growth, feed efficiency or bone P deposition. This was also observed in this experiment in which diets with 250 FTU/g at 75% soya bean (S3P1) had better growth performance (Table 2) compared to positive control, S 3 D (Table 6), suggesting phytase at this level can effectively replace inorganic phosphorus in Clarias gariepinus diet. Fish fed suboptimal phytase diet (Table 6) showed poor growth performance compared to diet with optimal level of phytase at 250 FTU/g (Table 3), which could be due to low phosphorus availability and high Ca/P ratios in the diet ( Table 6) compared to S3P1 and S0P0 (Table 3).
Fish fed 100% soya bean diet (full fat) showed the poorest growth response compared to other soya bean levels, although there were significant improvement in weight gain compared to control (S4P0). The low growth in 100% soya bean fish could be related to unbalanced amino acid profile of soya bean compared to fish meal [43] limiting its total substitution of fish meal as well as the high level of fat, which was beyond the maximum level that can be used in fish feed [24]. Fish fed 100% soya bean also showed the lowest feed intake, specific growth rate, protein efficiency, and the highest feed conversion. 100% soya bean diet (full fat) also showed a significantly higher calcium/phosphorus ratio, which depresses phytate hydrolysis and reduce growth [23]. The Ca:P of 75% soya bean meal supplemented with 250 FTU/g (1.41:1) is comparable to that of 100% fish meal diet (1.41:1), suggesting optimum level in soya bean diet (full fat) for Clarias gariepinus fish diet. In Table 4, phytase at 250 FTU/g gave the highest growth response with optimum Ca/P of 1.46 compared to phytase at 0 FTU/g (Ca/P=1.40), 500 FTU/g (Ca/P=1.71), 750FTU/g (Ca/P=1.59) and 1000 FTU/g (Ca/ P=1.54) ( Table 6). In Kim et al. [44], growth performance and nutrient utilization in weaning pig fed phytase-supplemented-canola-soya bean diet with high phytate and Ca/P of 1.46 was significantly higher than diets without phytase. Cao et al. [7] recommended a range of Ca:P between 1.1:1 and 1.4:1 for optimum phytase activity in fish feed. The high Ca:P in S2P1 (1.54), S2P2 (2.00) and S2P4 (1.54) as well as the high level in 100% soya bean with phytase, in the diet could be responsible for the poor weight gain of fish fed these diets compared to S0P0, S2P0, S2P3, S3P1 and S3P4. Ca:P ratios are crucial to phytase efficacy as reviewed by Lei and Stahl [22], since high level chelate with phytate and compete with active site of phytase, which reduces phytase efficacy and phosphorus availability [1]. Qian et al. reported that reducing Ca:P ratios from 2.0 to 1.2:1 in phytase supplemented diets (700 and 1050 FTU/kg) of pig, increased phytase efficacy by the order of 16%. Liu et al. observed that reducing Ca:P ratios from 1.5 to 1.0:1 in phytase (500 FTU/kg) supplemented low phosphorus diets increased P absorption in the small intestine of pigs. Similarly, Qian et al. found that increasing Calcium (5.61-10.20 g/kg) and Ca:P ratios (1.1-2.0:1) depressed weight gain of broilers.

Bone mineralization
The effect of phytase supplementation in diet fish has been shown improve bone mineralization in several studies [1,7,9] Channel catfish fed phytase-supplemented diets had higher concentrations of ash, calcium, phosphorus and manganese in their bones than the fish fed on a control diet. Bone mineralization in Common carp was improved by phytase addition in the diet [37,41].
In this study, phytase supplementation improved bone parameters, depending on the level of soya bean in the diet. Bone phosphorus, calcium, iron, and zinc (Table 7) were improved in fish fed 25 and 50% soya bean (full fat) with phytase supplementation at 250-750 FTU/g and 250-500 FTU/g, respectively, compared to basal controls in these diets [5,45]. Increased mobilization of phosphorus store in soft tissue such as the flesh from fish fed these diets improved bone mineralization in these fish without depleting their store in the flesh, suggesting adequate phosphorus intake, contrary to who reported that insufficient phosphorus intake leads to the mobilization of phosphorus from the bone and transfer to soft tissues and metabolic processes [46]. According to Baruah et al. [5], dietary supplementation of microbial phytase at 500 FTU/g in Labeo rohita (Hamilton) juveniles' diet significantly increased bone sodium, calcium, potassium, manganese and iron content by 15, 12.1, 17.4, 20.4 and 40.7%, respectively. Robinson et al. [14] reported that phytase of 250 FTU/g could effectively replace dicalcium phosphate supplement in the diet of channel catfish without affecting growth, feed efficiency or bone phosphorus deposition. In this study, fish fed 75% soya bean (full fat) with 250 FTU showed significant reduction in bone phosphorus and calcium, which could be due to inadequate supply [36,46] fat in the diet. Reduction in bone phosphorus and calcium in 75% soya bean with 250 FTU/g could be due to inhibition of their absorption by fat in the diet for whole body retention and bone mineralization. However, in other diets (25%, 50% and 100% soya bean), phytase at 250 FTU/g improved significantly bone phosphorus compared to controls without phytase. Reqz et al. reported that soyabean oil affected bone characteristics (bone length and volume) in animals. There was, however, significant increase in iron and zinc, indicating phytase effectively released zinc from zinc-phytate complex [23] for retention in whole body and bone mineralization. Improvement of zinc in diet and bone by supplementing phytase at 250 FTU/g could be the reason why it produced the greatest growth response in fish fed 75% soya bean meal diet compared to other phytase supplemented soya bean diet, and could effectively replace fish meal at that level of soya bean in the diet of juvenile Clarias gariepinus. Zinc exhibits a greater effect on growth than growth hormone and the nutrition of zinc for monogastric animals is similar physiologically to that of man. Most zinc deficiency is attributable to the abundance of dietary phytate, a natural compound found in all plant seeds and some roots and tubers. Fish fed 100% soya bean showed the greatest positive and consistent response to increasing phytase with the highest phosphorus at 250 FTU/g as observed in 25% and 50% soya bean, which could explained by low phosphorus level that enhances phytase response [40], and improving bone parameters. Irrespective of soyabean levels, fish fed diets with 250 FTU/g phytase showed the highest bone phosphorus and calcium compared to 500, 750, and 1000 FTU/g (Table 8).
In conclusion, the research has demonstrated that phytase at 250 FTU/g was optimum for juvenile Clarias gariepinus as observed by improvement in growth (Figure 1), bone mineralization (Table  8) and phosphorus retention efficiency (Table 7) compared to other levels, and can replace dicalcium phosphate [14] however, the effect of Ca/P, from the research, has also shown the negative effect its negative effects at high levels as seen from the relationships between weight gain and total weight. Hence, it is recommended that, since the optimum phytase diet, which produced the best growth at 75% soya bean diet at 250 FTU/g (Ca/P for S3P1=1.41), which also gave the highest growth response (Ca/P=1.46) compared to phytase at 0 FTU/g (Ca/P=1.40), 500 FTU/g (Ca/P=1.71), 750 FTU/g (Ca/P=1.59) and 1000 FTU/g (Ca/ P=1.54), Ca/P in the diet of juvenile Clarias gariepinus should range between 1.41-1.46 for optimum phytase activity. Available phosphorus should be as low as possible (<0.33%).