Efficacy of acidified phytase supplemented cottonseed meal based diets on growth performance and proximate composition of Labeo rohita fingerlings

Abstract The growth of aquaculture sector is strongly dependent upon the continuous supply of inexpensive fish feed with balanced nutritional profile. However, fish meal (FM) is unable to satisfy this demand due to its scarce supply and high cost. In order to test the potential of cottonseed meal (CSM) as a fish meal replacer, a feeding trial of 12 weeks was conducted to check growth performance and proximate composition of Labeo rohita fingerlings. The protein ration of the test feed was satisfied by replacing FM with CSM at 0, 25, 50 and 75%. Sixteen test diets viz., TD1 (control), TD2, TD3, TD4, TD5, TD6, TD7, TD8, TD9, TD10, TD11, TD12, TD13, TD14, TD15 and TD16 were supplemented with citric acid (CA; 0 and 2.5%) and phytase (PHY; 0 and 750 FTU/kg) in a completely randomized design with 3×3 factorial arrangement. The highest weight gain (11.03g), weight gain% (249.21%), specific growth rate (1.39) and best feed conversion ratio (1.20) were recorded by fish fed with TD12. Furthermore, the same level increased the crude protein (59.26%) and fat (16.04%) being significantly different (p<0.05) than that of control. Conclusively, the addition of acidified phytase (CA; 2.5%, PHY; 750 FTU/kg) in TD12 (CSM=50%) led to the improved growth and proximate composition of L. rohita fingerlings.

Efficacy of acidified phytase supplemented cottonseed meal based diets on growth performance and proximate composition of Labeo rohita fingerlings for commercially important specie i.e., L. rohita, to improve its growth performance and proximate composition.

Fish and experimental conditions
This study was conducted in the Fish Nutrient Laboratory, Department of Zoology, Government College University, Faisalabad, Pakistan (Latitude 31.4166°N and Longitude 73.0707°E). L. rohita fingerlings of average 4.51g initial weight were purchased from Government Seed Hatchery, Satiana road, Faisalabad and kept in V-shaped tanks for acclimation to laboratory conditions. Initially, fingerlings were fed with basal diet @5% body weight once daily to the apparent satiation (Allan and Rowland, 1992). Before the start of feeding trial, fingerlings were given a dip prophylactically in NaCl solution (5g/L), so to get rid of fungal and bacterial pathogens (Rowland and Ingram, 1991). Parameters such as temperature, pH and dissolved oxygen were checked daily that should be between 25.9-29.5 °C, 7.3-8.5 and 5.4-7.5mg/L, respectively.

Experimental design
Fish rearing system was comprised of 48 V-shaped tanks representing 16 groups; in triplicate fashion. These tanks were supplied with fresh water and air supply, so, to maintain dissolved oxygen level. A total of 720 fingerlings of initial weight (4.51 g) were stocked in each tank with 15 fish stocking density. Sixteen test diets (TD 1, TD 2 , TD 3 , TD 4 , TD 5 , TD 6 , TD 7 , TD 8 , TD 9 , TD 10 , TD 11 , TD 12 , TD 13 , TD 14 , TD 15 and TD 16 ) in 3×3 factorial arrangement was prepared by replacing FM with CSM at levels of 0%, 25%, 50% and 75% supplemented with CA (0 and 2.5%) and PHY (0 and 750 FTU/kg). Each test diet was allocated to triplicate tanks by using Completely Randomized Design (CRD).

Formulation of pellets and feeding technique
All feed ingredients were purchased from commercial feed mill and analyzed for chemical composition following standard procedures of AOAC (2005) before formulation of experimental diets. Diet composition of nutrients showed that there was an equal amount of nutrients in all the CSM based test diets including the control diet as shown in Table 1. Feed components were grinded to pass through 0.5 mm sieve size. Fish oil was poured in mixture and mixed for 5 minutes. To prepare suitable dough, 10-15% distilled water was added (Lovell, 1989). To get feed pellets, previously made dough was processed by pelleting machine (Model SYSLG30-5 experimental extruder). Phytase was later on sprayed on test diets as defined by Robinson et al. (2002). After drying, pellets diameter was adjusted according to fish size and stored at 4 °C to keep it oxidation-free. The actual feeding trial prolonged for 12 weeks (90 days). Fish were hand fed at the rate of the 5%/day of their total biomass. The total amount of feed was divided into two halves and offered to the fish in morning (8:00 am) and afternoon (12:00 pm). 2020). Since last few years, the quest for cost effective and nutrient rich ingredients is high on the agenda by a number of fish nutritionists; so, to culture aquatic animals in an economical way (Froehlich et al., 2018). Now-a-days, the forefront trend is to replace fish meal by the most viable feed stuff i.e., plant meal (Daniel, 2018). It has become an inevitable issue to find cost-effective and highly nutritious plant protein sources for fish feed formulation. Growth promoting potential of various plant meals like soybean meal (Chu et al., 2016), Moringa oleifera by-products meal  and corn gluten meal (Hussain et al., 2018) has been verified by different researchers. CSM is one of the important protein sources (23-53%) and thought as highly valuable for its cheaper cost and local availability (Sun et al., 2015). After soybean and canola meal, it is the third largest oilseed meal product in the world. It is a rich source of essential amino acid arginine, which is even higher than that of fishmeal and soybean meal (EI-Saidy and Gaber, 2004). Several studies have confirmed its inclusion in the diets of Carassius auratus gibelio (Gui and Zhou, 2010), Oplegnathus fasciatus (Lim and Lee, 2009) and Oreochromis niloticus (Yue and Zhou, 2008).
In plants, most of the phosphorus is found in the form of phytate and phytic acid which is hardly assimilated by agastric or monogastric fishes (NRC, 1993). This phytate complex can only be broken down by the addition of exogenous enzymes like phytase which improves digestibility in gut, nutrient availability to fish and hematology of different fish species (Shahzad et al., 2017). Furthermore, phytase maximizes the absorption of chelated minerals and nutrients present in plant by-products meal . Having this potential, exogenous enzymes are being evaluated on a wider scale (Hlophe-Ginindza et al., 2016). Due to the differences in feeding habits and digestive potential, the activity of digestive enzymes varies in different fish species (Iqbal et al., 2016). Furthermore, organic acids are proving to be a promising dietary additive to improve intestinal health and growth performance (Hassan et al., 2015). Citric acid (CA) has become the one of the world's most usable organic acid (Sauer et al., 2008). Due to its unique flavor and high buffering capacity, it is being used in food, pharmaceutical and other industries (Yin et al., 2015). Recent studies have found positive effects of organic acids on the activity of digestive enzymes which led to improved growth performance (Castillo et al., 2014;Ebrahimi et al., 2017). Supplementation of CA decreases the pH in digestive tract of fish (Baruah et al., 2005). Both CA and PHY not only improve intestinal physiology but also enhance growth, immune response and mineralization in fish body (Emami et al., 2013).
Indian major carps are known as highly cultured fish species around the globe (FAO, 2015). Amidst these, L. rohita is the most popular fish species and considered as the key species in fresh water aquaculture due to its fast growth rate, great economic value, delicious flesh quality, balanced amino acids profile and better disease resistance (Bharathi et al., 2013). So, the aim of the current study was to find out the best level of CA and PHY treated CSM diets The unconsumed diet was siphoned out from every tank, dried and weighed for determination of daily feed intake.

Growth study
To determine growth performance of fish, weight measurement was undertaken every fortnightly. Fish from each tank was caught up smoothly and then dried using tissue. Finally, its length and weight were noted down. Standard formulae (NRC, 1993) were used to determine growth performance (Equations 1, 2 and 3).

Determination of whole-body composition
After 90 days of feeding experiment, 5 unstressed fish of L. rohita were taken from each tank and sacrificed for determination of proximate composition following standard procedures of AOAC (2005). Then fish whole body samples were air dried and analyzed for determination of body composition (Korhonen et al., 1990). By a mortar and pestle, whole fish body was homogenized and examined by standard techniques; crude fat by petroleum ether extraction through Soxhlet system, ash by ignition at 650°C for 12 hours in an electric furnace to constant weight, crude protein (N × 6.25) contents by micro Kjeldahl apparatus, moisture by oven-drying at 105°C for 12h; crude fiber as loss on ignition of desiccated residues that are lipid free after breakdown with 1.25% NAOH and 1.25% H 2 SO 4 . Total carbohydrates were calculated by following formula i.e., Total carbohydrates (%) =100-(EE % + CP % + Ash % + CF%).

Statistical analysis
Data of growth performance and proximate composition was subjected to three-way Analysis of Variance (Steel et al., 1996). By Tukey's Honesty Significant Difference Test, the differences among treatments were compared and determined significant at p<0.05 (Snedecor and Cochran, 1991). For statistical analysis, the Co-Stat computer software (Version 6.303, PMB 320, Monterey, CA, 93940 USA) was used. Table 2 shows the growth performance of L. rohita fingerlings fed on acidified phytase supplemented CSM based diets. The results demonstrated that growth were noted in fingerlings fed on TD 1 (no supplementation) having more inclusion of FM. High FCR value (2.16) also predicted that least amount of nutrients were made part of fish body. Conclusively, the growth of L. rohita was improved with the increase in CSM protein up to 50% and subsequent decrease in FM, along with supplementation of CA (2.5%) and PHY (750 FTU/kg) and started decrease with any further inclusion of CSM.

Proximate composition
The effect of acidified phytase supplemented CSM based diet on proximate composition of L. rohita fingerlings is illustrated in Table 3 and Figure 3. Our results made it clear that nutrient composition of fish was optimally improved by feeding TD 12 having acidified phytase (750 FTU/kg, 2.5% CA) CSM (50%) based diet as compared to those fed on control diet. Significant differences in terms of crude protein (59.26%) and crude fat (16.04%) were prevalent in fish body when fed at TD 12 . It confers the idea that phytate complex was degraded maximally at this level and released   phosphorus to be a part of fish body. Second best values (58.54%, 15.89%) were obtained by using TD 8 having FM protein replaced by CSM up to 25% supplemented with CA and PHY at their highest concentration i.e., 2.5% and 750 FTU/kg, respectively. However, lowest values of protein (49.30%) and fat (8.73%) were observed in those fed on TD 1 . The highest values of moisture (7.25%), crude fiber (2.46%), crude ash (13.25%) and carbohydrates (19%) were also recorded on the same level.

Discussion
In present study, L. rohita fingerlings fed on CSM based diet supplemented with CA (2.5%) and PHY (750 FTU/kg) led to improved body parameters like WG% (249.21%) and FCR (1.20). Similarly, Khaled (2019) reported a significant effect of 1000 FTU/kg phytase in improving WG (%), specific growth rate and FCR of Nile tilapia. In line with our studies, Baruah et al. (2007) and Bano andAfzal (2018) in L. rohita andWang et al. (2009) in Oncorhynchus mykiss found growth promoting results. They proved that supplementation of acidified phytase trigger the phytate hydrolysis by reducing stomach pH and improved growth was resulted. Luckstadt, (2008) found that citric acid inclusion to phytase deposit H + ions in stomach and enhance nutrient bioavailability. A number of researchers came to the point that supplementation of exogenous enzymes in plant meal based diets helps to mitigate the effects of anti-nutritional factors (Hlophe-Ginindza et al., 2016) and maximum quantity of amino acids become part of fish body (Hussain et al., 2015b). Supporting our results, Singh et al. (2020) found that citric acid (1.5%) and phytase (1200FTU/kg) positively affected the growth parameters of O. mykiss. Meanwhile, improved growth performance in many fish species by addition of phytase in citric acid was observed such as tilapia (Tahoun et al., 2009),  catfish (Hung et al., 2015) and grass carp (Liu et al., 2014). Hussain et al. (2015a) found positive results in L. rohita fingerlings fed on CSM based diet supplemented with 750 FTU/kg level; just similar to our values and reported best growth performance. On the other hand, Lim andLee (2009) andWang et al. (2009) did not find any significant effect of phytase supplemented plant based diets on fingerlings' growth performance. The possible reason can be the thermal inactivation of phytase enzymes during feed formulation, diet composition, storage conditions of feed, plant meal replacement level and fish species (Hassaan et al., 2019). Supplementation of 750 FTU/kg phytase with 2.5% CA resulted in an interaction and enhanced the whole body composition like crude protein (59.26%) and crude fat (16.04%). Similar results were proposed by other researchers following the dietary addition of CA (Hossain et al., 2007;Khajepour and Hosseini, 2012). Recent studies have shown that poor fish carcass composition is due to phytate complex, which usually bounds important nutrients such as CP, EE and gross energy making them unavailable to fish (Cao et al., 2007). Thus, in present research, the acidified phytase supplemented diet enhanced the proximate composition of whole body by acting on chelated complex and increased the availability of nutrients to fingerlings. Similarly, Khajepour and Hosseini (2012) noticed increased crude protein contents in muscles of Huso huso while adding CA (30g/kg) in the diet. Whereas, Liu et al. (2014) and Cao et al. (2008) reported that acidified phytase supplementation decreased body lipids of grass carp and Nile tilapia, respectively.

Conclusions
Based upon the obtained results, it can be concluded that TD 12 having CSM protein up to 50% supplemented with 2.5% CA and 750 FTU/kg PHY proved to be the most efficient diet in improving growth performance of L. rohita fingerlings. Furthermore, the proximate composition of fish body was also improved by the synergistic effect of CA and PHY. Hence, it assisted in the formulation of cost-effective and eco-friendly feed for L. rohita.