Effects of X-xylanase Incorporated in the Diet on the Growth Performance of Red tilapia (Oreochromis sp.)

The aim of this study was to evaluate the feasibility of using X-xylanase in the diet to improve tilapia growth. Four experimental diets containing X-xylanase (Hostazym®, Huvepharma, Antwerp, Belgium) at levels of 0 (control), 1050, 1500, and 1950 endopentosanase units (EPU) were used in this study. The growth performance trial was carried out for 56 days. At the end of the study, there were no significant differences between the groups fed by control and the experimental diets in terms of survival rate, condition factor and weight gain. However, the highest weight gain was recorded in fish fed a diet containing 1500 EPU of X-xylanase. Fish fed diets containing 1500 and 1950 EPU X-xylanase for 56 days had significant better feed efficiency compared to those of fish fed the commercial diet and other experimental diets. It was therefore concluded that the addition of X-xylanase to the tilapia diet was able to improve the feed utilization and consequently enhance fish growth.


Introduction
Tilapia (Oreochromis sp.) farming is a widespread industry in the world because of its high growth performance and market value, and it has become one of the most important aquaculture species in the world. The production of tilapia is increasing annually, and 5,576,800 metric tons were produced worldwide in 2015. As tilapia aquaculture has dramatically grown over the past decade, feed quality and utilization have become major issues standing in the way of the sustainable development of tilapia aquaculture.
Formulated diets are the primary food source used in tilapia aquaculture. Plant-derived proteins, such as soybean meal, rice gluten meal, rapeseed meal, rice gluten meal, and wheat middling's, are widely used as ingredients in tilapia feed. However, the low nutrient utilization of plant-derived proteins by fish might result in lower growth performances and pollution of pond environment (Leenhouwer et al., 2007). The limited nutrient availability in aquafeed is mainly caused by non-starch polysaccharide (NSP) components (Sinha et al., 2011) and/or other anti-nutritional factors in plant-derived proteins (Cheng et al., 2013). Therefore, issues of anti-nutritional factors in plant-derived proteins must be dealt with to improve nutrient utilization and animal growth to bring down consequent costs of aquafeed.
Microbial enzymes are widely used in feed to minimize the problem of anti-nutritional factors, thereby improving the nutritive value (Forster et al., 1999), nutrient utilization (Cheng and Hardy, 2003;Cheng et al., 2013), animal growth (Sajjadi and Carter, 2004;Cheng et al., 2013;Saputra et al., 2016), and so on. Xylanases are a class of enzymes that degrade linear polysaccharides in plant cell walls into xylose, thus breaking down hemicellulose. They are found in numerous bacteria, yeasts, and fungi and play a role in those organisms' ability to thrive on plant sources by degrading plant matter into usable nutrition. Because of xylanase's capability of enzymatic digestion, it is considered a potential candidate for improving the efficiency of feed utilization and degrading NSPs of plant ingredients in animal feed. Xylanase has been widely used in the poultry (Yang et al., 2008;Pirgozliev et al., 2015) and livestock (Prandini et al., 2014;Passos et al., 2015) industries to enhance energy and nutrient availability of plant ingredients, but little is known about its application in aquaculture. Xylanase-producing yeasts were found in some freshwater fish species, such as Indian major carp (Cirrhinus mrigala) and Nile tilapia (Oreochromis niloticus), and xylanases released from yeast strains are thus available in the gut of fish and might promote NSP digestibility and utilization by fish (Gatesoupe, 2007;Banerjee and Ghosh, 2014). Purified xylanase was used in a plant protein-enriched diet of Jian carp (Cyprinus carpio var. Jian), which resulted in improvements in growth, in intestinal enzyme activities, and the microflora (Jiang et al., 2014); similar results were also recorded in Japanese seabass (Lateolabrax japonicas) (Ai et al., 2007).
It is well known that xylanases have great potential, as they are widely applied in the feed of terrestrial animals (Prandini et  analysis. The level of significance was accepted at p<0.05.

Proximate analysis of the experimental diets
Proximate analysis of the experimental diets showed no significant differences in the moisture (2.34%-2.50%), ash (8.85%-9.2%), crude protein (24.31%-24.56%), or crude lipid (8.24%-9.12%) levels among all experimental diets. Compared to the proximate analysis of the commercial diet (crude protein 23.92%, moisture 6.33%, crude lipids 8.03% and ash 7.8%), the experimental diets seemed to have similar nutrient values, which should not have influenced the growth performance of tilapia compared to fish fed the commercial diet.

Growth performance
During the experiment, the water quality did not significantly differ among the treatments. The water temperature ranged 25-26.3°C, pH 7.3-8.3, DO >5 mg/L, ammonia-N 0.002-0.22 mg/L, and nitrite-N 0.013-0.23 mg/L. Figure 1 shows the growth performance of tilapia fed the different experimental diets for 56 days. Growth performance results indicated that no significant difference was seen in the growth of tilapia fed the experimental diets and commercial diet, which may have been caused by individual growth differences among fish. However, fish fed the diet containing X-xylanase at a level of 1500 EPU seemed to have the highest weight compared to those of fish fed the other experimental diets and the commercial diet, but the growth rate of tilapia was reduced when an excessive dose of X-xylanase in the diet was offered to fish for 56 days in this study (Figure 1). Fish fed xylanase-supplemented diets having better growth performances was also found in juvenile Jian weeks during the growing-out phase. At the end of the experiment fish were individually weighted. Condition factor, survival rate, weight gain and feed efficiency were determined using following equations: Condition factor=body weight/(body length) 3 × 100%, Survival rate (%)=(final number of tilapia/initial number of tilapia) × 100%, Percentage weight gain (%)=[(final weight -initial weight)/ initial weight] × 100%, and Feed efficiency=weight gain/total feed intake.
The cost per kg fish production (NT$) was calculated as following formula: Cost per kg fish production=(Feed cost per kg × total feed intake (kg))/ weight gain (kg).

Whole-body composition analysis
After the experiment, six fish from each treatment were selected for a whole-body composition analysis. All fish samples were analyzed in duplicate for proximate composition following the standard methods (AOAC, 1984). Moisture was determined by a moisture analyzer (MX-50, A & DJapan). Crude protein was measured by the Kjeldahl procedure using the Kjeltec System (Tecator, Sweden) after acid digestion. Crude lipids were analyzed by the method of Folch et al., 1957. Ash was determined after burning at 550 o C in a muffle furnace.

Statistical Analysis
Experimental data were statistically analyzed by a one-way analysis of variance (ANOVA) and Duncan's multiple-range test in SAS software (Cary, NC, USA) to determine variations and the significance of differences between treatments. All percentage data were square-root arcsine-transformed before the statistical carp (Jiang et al., 2014) and Japanese seabass (Ai et al., 2007). In addition, animals with poor weight gain after consuming a diet with an excessive amount of supplemented xylanase were also recorded in Jian carp (Jiang et al., 2014) and broilers (Mendoes et al., 2013), which might have been due to the overproduction of xylooligosaccharides and xylose (Schutte, 1999;Xu et al., 2009).
Xylanase applied in animal feed to improve feed utilization was reviewed (Goswami and Pathak, 2013), and previous studies showed that xylanase was able to improve the growth performance and feed utilization in poultry and pigs (Goswami and Pathak, 2013;Woyengo et al., 2013). In aquaculture, a case of using multiple exogenous enzymes (including xylanase) in the diet of African catfish (Clarias gariepinus) resulted in enhanced fish growth performance and feed utilization (Yildirim and Turan, 2010). In Nile tilapia, fish fed a diet containing the xylanase-expressing probiotic, Bacillus amyloliquefaciens R8, which expresses the xylanase gene from rumen fungi, also had a significantly better growth performance and health status compared to fish fed a control diet or wild B. amyloliquefaciens (Saputra et al., 2016). Similarity, X-xylanase incorporated in the diet at a level of 1500 EPU was able to improve the feed efficiency, a good indicator for evaluating feed quality ( Table 2). The improved feed efficiency of fish fed the X-xylanase-supplemented diet at a level of 150 EPU indicated that the product really would be beneficial to tilapia aquaculture by improving feed utilization. At the end of the experiment, mean weights of fish in the commercial diet, control, 1050 EPU, 1500 EPU, and 1950 EPU groups were 9.36, 10.27 ± 0.7, 9.96 ± 1.15, 11.07 ± 0.48, and 9.01 ± 0.57 g, respectively (Table 2).
This study found no significant difference in the body composition of red tilapia, including moisture, protein, lipid, and ash contents among fish fed the commercial, control, and experimental diets. However, fish fed X-xylanase-supplemented diets seemed to have higher protein contents compared to the control and commercial diet groups (Table 3), and the increased tendency of protein content in the fish body was similar to the increase in feed efficiency which was highest at the optimal level (1500 EPU) of xylanase in the diet, and then decreased in the group supplemented with an excessive amount (1950 EPU) of X-xylanase in the diet. Net nutrient retention is considered an accurate and important indicator for evaluating the feed efficiency in fish (Belal, 2005). Results of increased body protein contents and feed efficiency in red tilapia fed X-xylanase-supplemented diets agree with results of Jiang et al. (2014). Furthermore, data on the body composition of tilapia in this study were similar to those of tilapia in a previous study (Gao et al., 2015).

Cost analysis
The cost of the experimental diet was estimated to be NT $31.38/kg. (The average exchange rate is US $1≈New Taiwan (NT) $30) (Table 4). No difference in the cost was calculated among the experimental diets because the inactive enzyme was added with a corresponding decrease in the amount of X-xylanase (the cost of X-xylanase was NT $600 /kg). The cost of feed for 1 kg of tilapia production in the control, and 1050, 1500, and 1950 EPU groups were NT $44.46 ± 0.30, NT $43.68 ± 2.07, NT $38.78 ± 0.46, and NT $41.62 ± 1.06, respectively. Therefore, producing 1 kg of tilapia using the X-xylanase-supplemented diets at the levels of 1050, 1500, and 1950 EPU could save NT $0.78, NT $5.68, and NT $2.84, respectively, compared to the control. It was thus concluded that using X-xylanase in the diet for tilapia aquaculture would be profitable, and the recommended level is 1500 EPU. Body weight (g)

Rearing days
Commercial feed Control 1050 EPU 1500 EPU 1950 EPU Figure 1: Growth of tilapia during the 56 days of the feeding experiment. EPU-Endopentosanase units.