Nutrient Digestibility in Labeo rohita Fingerlings Fed Citric Acid Acidified Phytase Sprayed Diet

Syed Zakir Hussain Shah1*, Muhammad Afzal2, Mahroze Fatima3, Syed Makhdoom Hussain4 and Tanveer Ahmed5 1Department of Zoology, University of Gujrat, Gujrat, Pakistan 2Fish Nutrition Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad 3Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore 4Department of Zoology, Government College University, Faisalabad 5Aquaculture Biotechnology Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad Article Information Received 23 September 2015 Revised 02 May 2019 Accepted 12 June 2019 Available online 22 June 2020


INTRODUCTION
F ishmeal is being used as a major protein source in aquafeeds (Hardy, 1995) as it has high protein content, balanced amino acid profile, utmost digestibility and presence of relatively less anti-nutritional factors. However, due to increasing demand, unstable supply and high price of fishmeal, other alternative protein sources have long been of interest (Lunger et al., 2007). Attempts are being made to use plant proteins in place of fishmeal in aquafeeds (Cain and Garling, 1995). However, these low cost plant proteins have many problems including the presence of anti-nutritional factors like phytate (myo-inositol-1, 2, 3, 4, 5, 6-hexaphosphates). Up to 80% of total phosphorus contents in plant ingredients are present in phytate form which are unavailable to agastric and mono-gastric fish O n l i n e

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to break the phytate is the use of organic acids. Among these organic acids, citric acid (CA) is considered as the most promising acidifier. Zyla et al. (1995) reported that CA dephosphorylate the phytate in vitro. Beside this, these organic acids also help to reduce the gastric pH, increase the activity of digestive enzymes, improve the nutrient digestibility and reduce the bacterial load (Hossain et al., 2007). Previous studies indicated that CA (3%) improve the P utilization in L. rohita (Baruah et al., 2005) while enhanced apparent absorption of P and Mg in Oncorhynchus mykiss was also observed in CA (5%) supplemented group (Sugiura et al., 2001). An important aspect of PHY is that it is a pH dependent enzyme and performs better at lower pH (2.5 and 5.0-5.5) (Simons et al., 1990). However, L. rohita is an agastric fish species having poor acid secretion in its gut, resulting in higher pH (above 6). Therefore, phytase performance can be enhanced by adding organic acids in the diet which may lower the dietary pH leading to decreased pH in fish gut. Present research was conducted to investigate the influence of CA acidification in PHY treated diet in improving the nutrient digestibility performance by L. rohita fingerlings. Information based on this research may prove useful to develop less expensive and less polluting feed for Labeo rohita.

MATERIALS AND METHODS
The experiment was carried out in Fish Nutrition Laboratory, Department of Zoology and Fisheries, University of Agriculture, Faisalabad, Pakistan. The experiment lasted for 60 days.

Experimental diets
Basal diet was formulated having 37.6% crude protein and 4.72 kcal/g gross energy. Fish meal and soybean meal was added as protein source in basal diet. Fish oil was used as lipids source. Composition of basal diet is shown in Table I. All ingredients were ground, sieved (0.5 mm) and were thoroughly mixed in electric mixer for 10-20 minutes with gradual addition of fish oil. In dry mixed ingredients citric acid was added at three concentrations 0%, 1.5% and 3% and partitioned into three test diets. Inert marker (1% Chromic oxide) was added for digestibility analysis. Dough for extrusion was made with addition of 10-15% of water. Pellets (2 mm) were made by using had pelletizer. These pellets were sprayed by following Jackson et al. (1996) with three concentrations of phytase; 0, 750 and 1000 FTU/kg diet. Hence, supplementation of citric acid and phytase resulted in nine test diets, ranging from T1 having 0% CA and 0 FTU/kg PHY to T9 containing 3% CA and 1000 FTU/kg PHY. Diets with 0 FTU/kg phytase concentration was sprayed with equal amount of water to make moisture contents constant. Pellets were dried up till 8-10% moisture contents remained and stored at 4 o C until initiation of feeding trial.

Feeding and experimental units
Fingerlings of Labeo rohita (average weight 9 g) were taken from Government Fish Seed Hatchery, Faisalabad, Pakistan. Before feeding trial, fingerlings were treated with 5 g NaCl/L to make them ectoparasites free and acclimatized in V shaped tanks while feeding on basal diet (Allan and Rowland, 1992) with constant supply of oxygen. A group of 9 fish was stocked in each experimental V shaped tank of 70 L capacity (UA system), peculiarly designed for fecal collection from aquatic media. Triplicate tanks were allocated to each test diet. Water quality parameters (DO 5.8-7.3 mgL -1 , pH 7.4-8.6 and temp 24.9-28.7 o C) were monitored constantly among all tanks. Fish were fed 2% of live wet body weight twice a day and feeding was adjusted according to weekly weight measure of fish. After three hours of feeding all uneaten diet was removed with washing of tanks. After two hours of washing fecal material was collected via feaces collection tube of V shaped tanks equipped with two valves. Fecal material was collected by subsequent opening of valve I and II and was oven dried at 60oC and stored for further analysis.

Chemical analysis
At the end of feeding trial samples of feed ingredients, test diets and fecal material were homogenized with the help of pestle and mortar and analyzed following AOAC (1995). Dry matter contents were determined by drying in oven at 105 o C until constant weight. Crude protein (CP) was calculated by N×6.25 while N was determined by micro Kjeldahl apparatus while crude fat by ether extraction method through Soxtec HT2 1045 system. Gross energy was measured with the help of adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, USA). For estimation of minerals, test diets and feces samples were digested in boiling nitric acid and perchloric acid mixture (2:1). After recommended dilutions, minerals were estimated on atomic absorption spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan) except P, Na and K. Na and K were estimated on flame photometer (Jenway PFP-7, UK) while P was estimated calorimetrically (UV/VIS spectrophotometer Hitachi U-2001) at 720 nm absorbance. Chromic oxide contents were measured by using UV-VIS 2001 spectrophotometer at 350 nm absorbance after oxidation with molybdate reagent (Divakaran et al., 2002). Calculations and statistical analysis Apparent nutrient digestibility coefficients (ADC%) of test and reference diets were calculated following NRC (1993).

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Resulting data was subjected to two way analysis of variance (Steel et al., 1996). Significant differences among means were calculated by Tukey`s Honestly Significant Difference test at 5% significance level (Snedecor and Conhran, 1991). The CoStat computer package (Version 6.303, PMB 320, Monterey, CA, 93940 USA) was used for statistical analysis while interaction plots were made by using Minitab15. Table II. Both the supplements result in less excretion of dry matter, crude protein, crude fat and gross energy in feces in comparison with the control group (T1), however, minimum excretion was observed in T9 diet accompanied with 3% CA and 1000 FTU/kg phytase level. Similarly, addition of both the additives (CA and PHY) resulted in reduced amount of minerals in fish feces (Table II). Among all the tried levels of phytase, minimum excretion of minerals was recorded at 1000 FTU/kg level. Similarly, CA supplementation also resulted in less discharge of minerals in feces. Also, both the additives act synergistically (p<0.05) to decrease the mineral excretion in fish feces. However, their synergistic effect was not significant for Fe excretion in feces.

Effect of CA and PHY supplementations on nutrients content in feces is shown in
Nutrients digestibility results are presented in Table  III. Top spraying of phytase on extruded pellets showed improved absorption (digestibility) of dry matter, nutrients (crude protein and crude fat) and gross energy as compare to control group. However, different phytase supplementation levels showed statistically non-significant differences (p<0.05) among themselves for dry matter, crude protein and gross energy digestibility while crude fat was maximum digested by L. rohita at phytase level of 1000 FTU/kg. Similarly, dry matter, nutrients and gross energy digestibility was also enhanced by CA addition in the diet.
Main effect data of PHY addition indicated that top spraying of phytase to plant based diet improved the minerals (P, Ca, Mg, Na, K, Fe, Cu and Zn) digestibility significantly (p<0.05) except Mn. However, both the phytase levels showed same effect (p>0.05) for most of the minerals except Fe and Mn which showed highest digestibility at 750 FTU/kg and 1000 FTU/kg phytase levels respectively.
Dietary addition of CA also improved (p<0.05) mineral absorption as compare to control group. Ca, Na, Mn and Cu showed higher digestibility at 1.5% CA level while other minerals (Mg, K and Fe) showed utmost digestibility at 3% CA level.

DISCUSSION
Effects of different phytase inclusion methods in the fish diet have been studied including pre and posttreatments. Top spraying (post-treatment) of phytase on feed pellets can retain the phytase activity in the diet (Liu et al., 2013). Improved nutrients digestibility has been reported in carnivorous fish species by top spraying the phytase on dried pellets (Vielma et al., 2004;Wang et al., 2009;Tudkaew et al., 2008). However, there is scarcity O n l i n e

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Citric Acid and Phytase Supplementation in L. rohita Feed 5 of literature concerning the effect of phytase applications through top spraying on nutrients digestibility in the diets of non-carnivorous; agastric fish species. Present study demonstrated that phytase top spraying to plant based L. rohita diet significantly enhanced the apparent digestibility of dry matter, crude protein, crude fat, gross energy and minerals. Likewise, improved apparent nutrients and minerals digestibility was also observed in our previous study (Hussain et al., 2011a, b, c) in L. rohita fingerlings fed on plant based diets sprayed with graded levels of phytase. Baruah et al. (2007a, b) also observed improved nutrients and minerals absorption in response to phytase supplementation (through spraying) to L. rohita diet. Recently, Liu et al. (2013), while working on another agastric carp species, grass carp (Ctenopharyngodon idellus), reported enhanced ADC% of crude protein, crude lipid, dry matter and minerals in phytase sprayed group in comparison of fish fed on control diet. This increased nutrient digestibility will ultimately lead to improved growth performance of fish (Hussain et al., 2011b).
Inclusion of organic acids in the diet of agastric fish species is an increasing practice now days, as such fishes have no acid secretion mechanism in the gut. Results of present study showed that citric acid supplementation to L. rohita feed enhanced the crude protein, crude fat, gross energy and minerals digestibility. Increased nutrient absorption will not only improved the nutrient profile of fish, but would be helpful in reduction of nutrient loss in feces, which will decrease the need of nutrient supplementation in feed and aquatic pollution caused by nutrient loss in feces. The positive effects of diet acidification by CA supplementation on nutrients absorption were also reported by Baruah et al. (2007a, b) in L. rohita fingerlings fed on plant based diet. Rainbow trout also showed enhanced minerals absorption in response to CA addition to plant based diet (Sugiura et al., 2001). Citric acid increases the bioavailability of nutrients to fish in several different ways. It physically affects the chemical bonds between phytate and different nutrients resulting in release of these bounded nutrients (Atapattu and Nelligaswatta, 2005). Also, being a strong chelator of Ca and P, it removes these minerals from the phytate making it less stable and more susceptible to endogenous phytases (Khajepour and Hosseini, 2010). Furthermore, it helps in increasing the pepsin activity and in decreasing the intestinal microbial load by lowering the gastric pH (De Wet, 2005).
In the present study, CA and PHY synergistically enhanced the crude fat and gross energy digestibility. However, both the additives showed statistically nonsignificant interaction for the digestibility of dry matter and crude protein digestibility. So far, few studies have been conducted to investigate the nutrient digestibility in response to combined effect of CA and PHY. Similar to our study, Baruah et al. (2007b) also reported a non-significant interaction between CA and PHY on dry matter and crude protein digestibility in L. rohita fingerlings. In this study, a positive interaction between CA and PHY was observed on minerals absorption. CA may have provided optimum conditions for PHY to work by lowering the intestinal pH which lead to their synergitic effect on minerals digestibility. Furthermore, diet acidification lowers the rate of gastric emptying (Mayer, 1994) which also provide more time to the phytases for action. In agreement to our findings, improved minerals absorption was also reported by Baruah et al. (2007a) and Sugiura et al. (2001) in L. rohita and rainbow trout respectively fed on CA acidified phytase treated plant based diets.

CONCLUSION
Both the supplements (CA and PHY) act synergistically to improve the nutrient digestibility in L. rohita fingerlings which may lead to improved growth performance. Furthermore, more nutrient digestibility mean less excretion of these nutrients in the water bodies which may result in less water pollution. The information gathered in this study would contribute to develop the more nutritionally digestible feed for fish, however, more investigations on how increased nutrient digestibility improve the growth and nutrient retention and excretion in fish are needed. In addition, the effect of CA and PHY on digestive enzyme activities also require further evaluation

Statement of conflict of interest
The authors have declared no conflict of interest.

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Citric Acid and Phytase Supplementation in L. rohita Feed