https://orcid.org/0000-0002-1778-8776
Figures
Abstract
The objective of the current study was to assess the impact of dietary phytase supplementation on Labeo rohita fingerlings and to examine the effects on growth, nutrient digestibility and chemical characteristics of diets containing rice protein concentrate (RPC) as a major protein source. Six experimental diets were made, i.e., a positive control (fishmeal-based diet with no phytase), FM0; a negative control (RPC-based diet with no phytase), RPC0; and four supplemental phytase levels (250, 500, 1000, and 2000 FTU/kg). Fingerlings with an average weight of 9.42 ± 0.02 grams (mean ± SD) were randomly distributed into six experimental groups of three replicates, each containing 25 fish per tank (75 liters of water), provided with experimental diets at a rate equivalent to 5% of their body weight for 90 days, and uneaten feed was collected after 2 hours to determine feed consumption. The feces were collected before feeding to estimate digestibility. Phytase in combination with the RPC-based diet significantly (p < 0.05) enhanced phytate phosphorus in vitro hydrolysis; growth performance; nutrient (crude protein, crude fat, moisture and gross energy) and mineral (P, Ca, Mg, Na, K, Zn, Mn and Cu) digestibility; digestive enzyme (protease, lipase and amylase) activity; and mineral deposition up to 1000 FTU/kg phytase. However, the hepatosomatic and viscerosomatic indices and carcass composition were not influenced (p > 0.05) by phytase supplementation. Increasing phytase supplementation in the RPC-based diets led to a significant (p < 0.05) decrease in the serum biochemical parameters (alkaline phosphatase activity, aspartate aminotransferase, alanine aminotransferase), which resulted in improved liver health. In conclusion, phytase-supplemented RPC-based diets improved the growth, mineral/nutrient digestibility, digestive enzymes, serum biochemistry, and mineral deposition of L. rohita fingerlings up to 1000 FTU/kg. Broken line regression analysis revealed that the optimum phytase concentration in the RPC-based diet for L. rohita was 874.19 FTU/kg.
Citation: Khizar A, Fatima M, Khan N, Rashid MA (2024) Effects of phytase inclusion in diets containing rice protein concentrate (RPC) on the nutrient digestibility, growth and chemical characteristics of rohu (Labeo rohita). PLoS ONE 19(5): e0302859. https://doi.org/10.1371/journal.pone.0302859
Editor: Aziz ur Rahman Muhammad, University of Agriculture Faisalabad, PAKISTAN
Received: October 4, 2023; Accepted: April 11, 2024; Published: May 24, 2024
Copyright: © 2024 Khizar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its supporting information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Protein is a crucial component of aquafeeds, and fishmeal (FM) is widely acknowledged as the most suitable protein source due to its balanced amino acid profile [1]. However, the limited supply of FM and high cost have compelled aqua culturists to investigate alternative protein sources to fulfill the increasing demand for fish feed [2, 3]. Considering this situation, plant proteins (PPs) appear to be the most feasible choice, as they offer potential solutions to both cost and sustainability challenges in aquafeed production [4]. However, PP sources are associated with several problems, such as the presence of anti-nutritional factors (ANFs) [5].
Plant protein sources such as rice protein concentrate (RPC) are a byproduct of rice made from rice polish and contain ANF, specifically phytate. Some of the ANFs are denatured during the feed process; however, phytate is considered relatively heat stable and requires several enzymatic reactions for its breakdown [6, 7]. It forms a complex with proteins and affects the digestibility and intake of PP sources [6, 8]. RPC can be utilized as a protein source for fish feed. However, it contains remarkably high levels of phytate, which needs to be addressed [9].
To overcome this limitation, exogenous enzymes such as phytase have been proven to be effective in various PP meals [10–12]. Phytase supplementation in feed breaks down the phytate complex and releases bound phosphorus (P), leading to improved nutrient digestibility and availability from PP sources to fish. It also enhances P utilization, alleviates the excretion of P into the environment, and decreases water pollution [6, 7]. Moreover, it also enhances the absorption of chelated nutrients and minerals from the intestinal mucosa of fish [8, 13, 14]. Due to this potential, the utilization of phytase is currently being extensively assessed [15]. Various studies have been conducted on phytase supplementation in plant-based diets, which have shown enhanced growth performance and nutrient utilization in fish species [16–19]. However, no study on RPC-based diets has been reported.
Labeo rohita is one of the most widely cultured fish species in Asia due to its high nutritional value, delicious taste, low economic value, and high market value. Owing to its high demand, this species is cultured in intensive systems on formulated feeds. Therefore, this study aimed to evaluate the effects of phytase supplementation on the growth performance, digestibility, in vitro phytate hydrolysis, carcass composition, mineral content, serum biochemistry, and digestive enzyme activity of L. rohita fed RPC-based diets. Understanding the impact of phytase in this context can contribute to the development of more efficient and sustainable aquafeed formulations, fostering the growth of aquaculture to meet the demands of a growing global population.
Materials and methods
Ethics approval
Ethics approval (No. DR/677) for the experiment was obtained from the Ethical Review Committee of the University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan.
Study area and experimental fish
The study took place at the Fish Seed Rearing Unit, Ravi Campus, University of Veterinary and Animal Sciences, Pattoki, and L. rohita fingerlings with an initial weight of 9.42±0.02 g were used as the experimental fish.
Fish acclimatization
The fish fingerlings were procured from the ponds of the C block, Ravi campus, UVAS. To protect the fish from potential pathogens, the fingerlings were subjected to a bath solution containing 5 g/L KMnO4 to create an environment that would minimize the risk of infections and diseases. After this, the fingerlings underwent a fifteen-day acclimatization period under laboratory conditions. Throughout this period, the fingerlings were provided a basal (RPC0) diet containing 30% crude protein (CP) for feeding.
Experimental diet preparation
Rice protein concentrate was purchased from Qurashi Brothers, Karachi, Pakistan, and its proximate composition was analyzed (Table 1) following the standard method of the AOAC [20]. The phytate content in the RPC ingredient and experimental diets were analyzed using a colorimetric method following Latta and Eskin [21]. In the feeding trial, the diets were made using RPC as the main ingredient, which contained 30% CP supplemented with different phytase (FTU/kg) concentrations, i.e., 0 (RPC0), 250 (RPC250), 500 (RPC500), 1000 (RPC1000), and 2000 (RPC2000). The FM-based diet was used as a positive control (FM0). For the preparation of the experimental diets, first, the dry feed ingredients (Table 1) were ground in an electric grinder (KENWOOD, AT284) to a fine powder, sieved (0.05 mm), and then mixed with the help of an electric mixer (KENWOOD, AT283). During the mixing process, the dry ingredients were supplemented with fish oil, a mineral mixture, vitamin premix, and chromic oxide (a marker for digestibility). Afterward, a dough was prepared by incorporating 15% water. The pellets were subsequently produced using a meat mincer (ANEX, AG 3060) and then shade-dried with up to 10% moisture [22]. Different dilutions of phytase (Microtech 10000 Plus granular, phytase 10,000 FTU/g, China, Pakistan) with distilled water (according to the experimental design) were sprayed on the pellets. Again, the pellets were dried and subsequently stored in airtight bags. The AOAC standard method [20] was used to determine the proximate composition of the RPC and experimental diets. The crude fat and CP contents were analyzed following the ether extraction method through a Soxhlet apparatus (behr Labor-Technik, Germany) and a Kjeldahl apparatus (FOSS Analytical A/S) after acid digestion, respectively. The ash and dry matter contents were determined on a muffle furnace (Eyela, TMF 3100) at 660°C and in a hot air oven at 105°C, respectively. Moreover, the gross energy content was quantified using an adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline. USA).
Rearing and feeding conditions
This experiment was conducted in a V-shaped steel tank placed indoors, and a natural photoperiod (8 h) was maintained throughout the experimental period. After acclimatization, the total fish fingerlings (n = 450) were stocked randomly into 18 V-shaped steel tanks (25 fish/tank) with 75 liters of water. The fish were fed experimental diets at 5% body weight for 90 days, and uneaten feed (feed waste) was collected after 2 hours to determine feed intake. For digestibility measurements, the sedimented feces were collected daily before feeding the fish by opening the valve at the bottom of each tank. Continuous aeration was given for the maintenance of dissolved oxygen (DO) within the optimum range (5.8–7.3 mg/L). The other water quality parameters, including temperature and pH, were monitored (Model 55, YSI, Inc., Yellow Springs, Ohio, USA), and average values were observed at 24.9–28.7°C and 7.4–8.6, respectively, throughout the experimental trial.
Sample collection
After the 90-day trial, the fish were subjected to a 24-hour fasting period, and the final weight of the fish in each tank was recorded for growth performance analysis. Subsequently, they were anesthetized using 150 mg/L tricane methanesulfate (MS 222, Sigma‒Aldrich) following the methods of Khan et al. [23]. Three fish from each tank were collected, pooled and placed in a hot air oven for carcass analysis, and another 3 fish were used for whole-body mineral analysis. For serum biochemical analysis, 5 fish were used, and blood was collected from the caudal vein in a 3 mL plain tube (BD vacutainers) and centrifuged for 15 minutes at 18,000 × g at 4°C. After centrifugation, the serum was collected in Eppendorf tubes for further analysis. Five additional fish were dissected, and their liver and viscera were measured for biological indices. The remaining 7 fish were dissected, and their intestine and bone samples were separated. The collected intestines were washed with distilled water and stored in sucrose solution at -20°C for further analysis of digestive enzyme activity. The bone samples were also stored at -20°C until analysis.
Growth performance and body indices
The growth performance was calculated in terms of average weight gain (AWG), weight gain (%), feed conversion ratio (FCR), specific growth rate (SGR), survival rate (SR), feed intake, and protein efficiency ratio (PER) using the following formulas:
The viscerosomatic index (VSI) and hepatosomatic index (HSI) were determined from the liver and viscera weights using the following formulas.
Carcass composition
The fish carcass composition (CP, crude fat, ash, and moisture) was determined following the standard method of the AOAC [20] as described in the experimental diet preparation section.
Mineral analysis
The dried whole-body and stored bone samples were subjected to mineral analysis. The collected bone samples were further processed, their soft tissues and muscles were separated, and only bones and spines were left after 2–3 min of boiling. After boiling, the collected bone samples were cleaned, rinsed, and dried in an oven and then subjected to ether extraction to eliminate fat. Finally, the bone samples from each replicate were dried, ground, and pooled together for mineral analysis. The samples (whole body and bones) were further subjected to wet digestion using a mixture of nitric acid and perchloric acid at a ratio of 3:1 following the AOAC [20] standard method, and the mineral [calcium (Ca), zinc (Zn), copper (Cu), magnesium (Mg), and manganese (Mn)] contents were determined through atomic absorption spectrophotometry (Hitachi Polarized Zeeman AAS, Z-8200, Japan); moreover, the P content in the samples was quantified using a UV visible spectrophotometer (U-2001, Hitachi), while the sodium (Na) and potassium (K) contents were determined via a flame photometer (Jenway, PFP 7, UK).
Nutrient digestibility and mineral absorption
The pooled fecal matter from each replicate and their respective test diets were dried in an oven at 60°C, ground, and pooled for analysis of nutrients and mineral digestibility. The apparent digestibility coefficient (ADC) of nutrients and mineral absorption was subsequently determined by the following formula.
Serum biochemical parameters
Serum biochemical parameters, such as alkaline phosphatase (ALP) (Cat no. BS:1/AP05.020.0100), aspartate aminotransferase (AST) (Cat no. BS:1/OT04.025.0100), and alanine aminotransferase (ALT) (Cat no. BS:1/PT04.0100) activity, were analyzed using specific kits from ARENA BioScien, Egypt, following Xu et al. [24].
Intestinal digestive enzyme analysis
The intestine samples from 7 fish were collected, pooled, and homogenized in a sucrose solution, after which the enzyme extract was separated via centrifugation for digestive enzyme analysis. The activity of amylase was measured by using a starch solution as a substrate at 2% (w/v) [25]. A spectrophotometric method was used to measure the activity of lipase using the substrate p-nitro phenyl palmitate (pNPP) [26]. The protease activity was calculated using Kunitz’s [27] method for casein digestion. The crude enzyme extract hydrolyzes casein and produces a color equivalent to that of 1 μmol/min tyrosine (pH = 7.5) at 37°C. The soluble protein concentration was measured in diluted homogenates following the Bradford method [28] using bovine serum albumin (BSA) as the standard. The enzymatic activity was measured as U/mg protein.
In vitro phytate hydrolysis
In vitro phytate hydrolysis was measured following the methods of Baruah et al. [29]. A total of 0.5 g of RPC was mixed with an adequate amount of water, and then different dilutions of phytase were added (0, 250, 500, 1000, and 2000 FTU/kg). These mixtures were incubated at 37°C for 1 hour in triplicate. After incubation, the phytate P content of the samples was further determined [29]. Briefly, the P content was determined using the wet digestion method, and the P content was analyzed using a spectrophotometer.
Statistical analysis
The experiment was designed as a completely randomized design (CR Design), and the results were analyzed using one-way ANOVA. The linearity of the data was checked using the Levene test (homogeneity) and the Shapiro‒Wilk test (normality). Moreover, the different phytase levels were taken as fixed factors, while the allocation of fish to the treatment groups (tanks) was randomized (random factor). The mean values were compared using Tukey’s honestly significant difference (HSD) test if significant differences among the treatments were observed. Broken line regression analysis was used to evaluate the optimum requirement of phytase in the fish diet [30]. CoStat computer software (version 6.303) was used for one-way analysis, while broken-line regression analysis was performed in R Studio (version 2023.03.0). The results were considered significant at p<0.05.
Results
In vitro phytate hydrolysis
The in vitro hydrolysis of phytate P increased with increasing levels of phytase up to 1000 FTU/kg in the RPC diets (Table 2).
Growth performance and body indices
The highest WG% (390.47%), SGR (1.76%) and PER (3.02) and lowest FCR (1.10) were observed at the 1000 FTU/kg phytase level, which was not significantly different from those of the 500 FTU/kg diet (WG% (366.73%), SGR (1.71%), PER (2.81)) and FCR (1.18) compared to those of the other phytase-supplemented RPC-based diets. However, no significant effects of phytase supplementation were observed on the FI or SR (Table 3). Moreover, the optimum dietary level of phytase was calculated to be 874.19 FTU/kg according to the weight gain (%) data obtained via broken-line regression analysis (R2 = 0.87) (Fig 1).
The red dotted points indicate diets comprising FM0 (a fishmeal-based diet with no phytase supplementation), while others showed different levels of phytase in the RPC-based diet.
The body indices (HSI and VSI) did not significantly differ among the groups supplemented with phytase and those fed the RPC-based diet (Table 3).
Carcass composition
The addition of phytase to the RPC-based diets did not significantly impact the carcass composition in terms of moisture, CP, CF, or ash content of L. rohita. Moreover, the average carcass compositions of the RPC-based diets were determined to be moisture (74.60%), CP (16.80%), CF (2.67%), and ash (4.79%) (Table 4).
Mineral composition (whole body and bone)
The fish fed diets containing 1000 FTU/kg and 2000 FTU/kg phytase exhibited significant improvements in whole-body mineral content, such as Ca (14.82 mg g-1), P (14.85 mg g-1), Mg (1.83 mg g-1), Na (4.41 mg g-1), and K (8.19 mg g-1). Zn (83.50 μg g-1), Cu (7.49 μg g-1) and Mn (37.60 μg g-1) significantly improved in the 1000 FTU/kg treatment group compared to the control group; these included Ca (13.26 mg g-1), P (12.12 mg g-1), Mg (0.47 mg g-1), Na (3.02 mg g-1), K (6.73 mg g-1), Zn (74.30 μg g-1), Cu (5.26 μg g-1) and Mn (24.96 μg g-1). Furthermore, the highest bone mineral contents were also detected in the 1000FTU/kg phytase supplemented diet for Ca (89.39 mg g-1), P (64.81 mg g-1), Mg (1.87 mg g-1), Na (7.60 mg g-1), K (9.80 mg g-1), Zn (152.15 μg g-1), Cu (8.38 μg g-1) and Mn (47.94 μg g-1) compared to those in the control group, such as Ca (73.85 mg g-1), P (47.12 mg g-1), Mg (1.54 mg g-1), Na (3.86 mg g-1), K (6.95 mg g-1), Zn (139.11 μg g-1), Cu (4.02 μg g-1) and Mn (38.75 μg g-1) (Table 5).
Nutrient digestibility and mineral absorption
The supplemental level of phytase at 1000 FTU/kg resulted in the maximum values of ADC in terms of dry matter, CP, CF, and gross energy in L. rohita fingerlings fed RPC-based diets, and these values decreased thereafter (Table 6). However, the mineral absorption of Ca (64.83–68.79%), P (64.35–70.39%), Na (36.76–45.61%), Mg (49.66–69.55%), and K (62.98–73.69%) increased up to 1000 FTU/kg. Moreover, the Zn (40.68–60.24%), Cu (44.86–64.46%), and Mn (41.89–63.49%) contents increased with increasing supplemental phytase (2000 FTU/kg) in the RPC-based diet (p<0.01) (Table 7).
Serum biochemical parameters
The serum biochemical parameters (ALP, ALT, and AST activities) were significantly lower in L. rohita after up to 1000 FTU/kg phytase supplementation in RPC-based diets. However, there was no significant difference between the groups fed diets supplemented with phytase above 1000 FTU/kg and those fed the other RPC-based diets (Table 8).
Intestinal digestive enzyme analysis
The enzyme activities, i.e., protease, lipase, and amylase activities, were significantly greater in the 1000 FTU/kg and 2000 FTU/kg phytase supplementation groups than in the lower level group (Table 9).
Discussion
A plethora of studies have been conducted to replace the costly and scarcely available FM with other protein sources in aquaculture. Rice protein concentrate is considered the most feasible PP source and is used as a substitute for expensive FM, has a high nutrient content and is easily available. Nonetheless, the presence of ANF, such as phytate, hinders its utilization in fish [31, 32]. To maintain aquaculture sustainability, it is imperative to address and find solutions to this problem. Therefore, the current study aimed to overcome this deficiency by supplementing an RPC-based diet with phytase, an exogenous enzyme. The in vitro hydrolysis of phytate P in the RPC-based diet increased as the phytase supplementation level increased in the present study up to 1000 FTU/kg, which is in accordance with the findings reported by Baruah et al. [29] and Shah et al. [33]. This suggests that increasing the phytase levels in RPC-based diets increases P release, which might enhance P availability in fish [33]. However, there may be a maximum inclusion level of phytase, and the acceptability of this substitution concerning fish growth performance has exhibited considerable variation among various fish species [34–36].
The present study demonstrated that dietary supplementation with phytase supplemented with RPC, a substitute for FM, had positive effects on growth performance up to 1000 FTU/kg. Similar findings have been reported for Nile tilapia [37–39], African catfish [8], channel catfish [40], rohu [41], and Pacific white shrimp [42]. Phytase decreases phytate contents in diets, which might release bound nutrients, especially P, and improve the digestion of protein-bound phytate. Thus, phytase supplementation improved protein utilization and overall nutritional efficiency in fish [19, 43–45]. Nevertheless, supplementation above the required level reduced the utilization of feed and growth performance, possibly due to the degradation of nutrients present in the feed, e.g., proteins and amino acids, from the overdose of phytase [18, 38, 39, 40]. Therefore, the broken line regression analysis of the weight gain % revealed that the recommended level of phytase supplementation for L. rohita fingerlings is estimated to be 874.19 FTU/kg. The HSI and VSI are indicators used to evaluate the effects of environmental or dietary manipulations on fish. In the present study, no changes in the HSI or VSI were observed, which indicates that there was no nutritional deficiency, disease, or environmental stressor in the fish; these findings are consistent with previous reports on various fish species [11, 24].
The carcass composition, such as moisture content, CP, CF, and ash content, is a reliable parameter for estimating the nutrient profile of fish and is crucial for consumers [46]. Phytate complex formation is reported to hinder the availability of nutrients such as CP and CF to fish species, preventing their utilization [47, 48]. No alteration in body composition in the present study indicated no negative effect on the nutrient profile, which is consistent with the findings of previous reports examining gibel carp and tilapia [49, 50] and might be due to the hydrolysis of phytate P. The P release from phytate enhances the utilization of P at the highest supplementation level of a PP-based diet [51]. In contrast to our study, a significant alteration was observed, which might be due to the presence of NADPH in the PP sources, which ultimately affects the lipogenic pathway and the production of ATP [42, 48, 52].
Minerals are vital for facilitating the growth, development, and overall well-being of fish and play vital roles in various physiological processes. Phytate is a strong chelator that can bind to minerals (Fe, Ca, and Mg), forming complexes that are insoluble and unavailable for absorption by fish [17]. The present study revealed that L. rohita fingerlings supplemented with phytase had higher mineral contents (P, Ca, Mg, Na, K, Mn, Cu, and Zn) in the whole body and bones, up to 1000 FTU/kg phytase, than did the fish fed diets with no phytase enzyme, which could be attributed to the efficacy of the enzyme in hydrolyzing the phytate content, leading to the liberation of bound minerals that were subsequently utilized and retained by the fish [48]. Similarly, Olugbenga et al. [53] reported that the addition of phytase at 750–1000 FTU/kg enhanced the mineral content of African catfish. Similar results were observed in various fish species, e.g., crucian carp, African catfish, and grass carp [53–56].
The digestibility of alternative protein sources as ingredients plays a crucial role in assessing their suitability in the formulation of fish feed [57]. Phytate binds with other enzymes and components present in the diet, ultimately lowering nutrient digestibility and activating specific enzymes as well as potentially affecting culture conditions [16, 43, 58, 59]. The present study revealed that 1000 FTU/kg phytase supplementation significantly improved the ADC, which is consistent with the findings of previous studies [58, 60]. However, no effect on the ADC of nutrients was shown for some fish species when dietary phytase was added to their feeds [61]. In the present study, the increase in phytase supplementation in an RPC-based diet increased the mineral digestibility of Ca, Mg, P, Na, K, Zn, Mn, and Cu. Similarly, previous findings have indicated that phytase addition enhances mineral absorption in many fish species under different cultures and environmental conditions [54, 59, 62–65]. The observed phenomenon can be attributed to phytate hydrolysis facilitated by phytase supplementation, as is evident in in vitro phytate hydrolysis. In contrast to the current study, the tiger puffer Takifu gurubripes [66] showed greater mineral digestibility at the 2000 FTU/kg phytase level. Moreover, Nwanna and Olusola [67] suggested that phytase addition to PP-based diets had no effect on the ADC of minerals in Oreochromis niloticus.
Amino acid transaminases, namely, AST and ALT, are integral components of metabolic processes and are found primarily within hepatocytes. In aquatic animals, the activities of these enzymes in serum may serve as markers of the overall health of the liver [68, 69]. The increased concentrations of ALT and AST are discharged into the serum, which may indicate damage to liver cells [70]. ALP is crucial for membrane transport and mineralization of the skeleton in aquatic species [11]. In the present study, the activities of ALT, AST, and ALP decreased with phytase supplementation in response to RPC-based diets, which was inconsistent with the findings of previous studies by Liu et al. [71] on Ctenopharyngdon idella and Yang et al. [11] on red swamp crayfish (Procambarus clarkia). In contrast, no effect was observed for different fish species when phytase was supplemented in PP-based diets [36, 37, 72].
Digestive enzymes such as protease, lipase, and amylase mostly represent the ability to digest and absorb nutrients, which helps in reproduction and enhanced growth performance in many fish species [73, 74]. Phytate binds to digestive enzymes or their cofactors and affects their efficacy [75]. The current study demonstrated that dietary phytase addition increased the intestinal digestive enzyme activity of L. rohita. The addition of phytase dissolved the phytate by breaking down the complexes and discharging the bound minerals, hence playing a crucial role in enhancing enzyme activity [11]. Similar results have been reported in other fish species, e.g., rohu, red swamp crayfish, and Nile tilapia [11, 70, 76].
Conclusion
In conclusion, RPC in combination with phytase is a viable and efficient substitute protein source for promoting sustainable aquaculture practices and maintaining the health and productivity of L. rohita without causing any significant negative effects on growth performance, digestibility, mineral status, digestive enzymes, or serum biochemistry. The optimum fish performance was observed with the inclusion of 1000 FTU/kg phytase containing 32% RPC. However, the phytase level in the diet of L. rohita fingerlings determined via broken line regression analysis of weight gain (%) data was calculated to be 874.19 FTU/kg when 6.8 g/kg phytate was present in the RPC-based diet.
Supporting information
S1 Raw data. This is the link of S1 Raw data.
https://figshare.com/s/63b4fea61c3438a187c3, (doi: 10.6084/m9.figshare.25688556).
https://doi.org/10.1371/journal.pone.0302859.s001
(XLSX)
References
- 1. Luthada-Raswiswi R, Mukaratirwa S, O’Brien G. Animal protein sources as a substitute for fishmeal in aquaculture diets: A systematic review and meta-analysis. Applied Sciences. 2021 Apr 24;11(9):3854. https://doi.org/10.3390/app11093854.
- 2. Olsen RL, Hasan MR. A limited supply of fishmeal: Impact on future increases in global aquaculture production. Trends in Food Science & Technology. 2012 Oct 1;27(2):120–128. https://doi.org/10.1016/j.tifs.2012.06.003.
- 3. Siddiqui SA, Alvi T, Sameen A, Khan S, Blinov AV, Nagdalian AA, et al. Consumer acceptance of alternative proteins: a systematic review of current alternative protein sources and interventions adapted to increase their acceptability. Sustainability. 2022 Nov 18;14(22):15370. https://doi.org/10.3390/su142215370
- 4. Chakraborty P, Mallik A, Sarang N, Lingam SS. A review on alternative plant protein sources available for future sustainable aqua feed production. International Journal of Chemical Studies. 2019;7(3):1399–1404.
- 5. Bandara T. Alternative feed ingredients in aquaculture: Opportunities and challenges. Journal of Entomology and Zoological Studies. 2018;6(2):3087–3094.
- 6. Kumar V, Sinha AK, Makkar HP, De Boeck G, Becker K. Phytate and phytase in fish nutrition. Journal of Animal Physiology and Animal Nutrition. 2012 Jun;96(3):335–364. pmid:21692871
- 7. Hussain SM, Afzal M, Javid A, Hussain AI, Ali Q, Mustafa I, et al. Efficacy of Phytase Supplementation on Growth Performance and Mineral Digestibility of Labeo rohita Fingerlings Fed on Cottonseed Meal Based Diet. Pakistan Journal of Zoology. 2015 May 1;47(3): 699709.
- 8. Adeshina I, Akpoilih BU, Tiamiyu LO, Badmos AA, Emikpe BO, Abdel‐Tawwab M. Effects of dietary supplementation with microbial phytase on the growth, bone minerals, antioxidant status, innate immunity and disease resistance of African catfish fed on high soybean meal‐based diets. Journal of Animal Physiology and Animal Nutrition. 2023 Mar;107(2):733–745. pmid:35979610
- 9. Makkar HP, Francis G, Becker K. Protein concentrate from Jatropha curcas screw‐pressed seed cake and toxic and antinutritional factors in protein concentrate. Journal of the Science of Food and Agriculture. 2008 Jul;88(9):1542–8. https://doi.org/10.1002/jsfa.3248
- 10. Manikandan K, Felix N, Prabu E, Bharathi S, Tamilarasu A. An essential review on the challenges with plant based ingredients with respect to phytic acid and phytase activities in the aquafeeds. Journal of Aquaculture in the Tropics. 2020;35(1–4):93–105.
- 11. Yang W, Gu Z, Chen X, Gao W, Wen H, Wu F, et al. Effects of phytase supplementation of high-plant-protein diets on growth, phosphorus utilization, antioxidant, and digestion in red swamp crayfish (Procambarus clarkii). Fish & Shellfish Immunology. 2022 Aug 1;127:797–803.Doi: https://doi.org/https://doi.org/10.1016/j.fsi.2022.07.034.
- 12. Sarfraz Q, Hussain SM, Sharif A, Selamoglu Z, Bashir F, Ahsan S. Potential of phytase supplemented Moringa oleifera leaf meal based diet on mineral digestibility of Oreochromis niloticus fingerlings. Journal of Survey in Fisheries Sciences. 2023 Jan 24:65–77. http://www.sifisheriessciences.com/index.php/journal/article/view/199.
- 13. Shahzad MM, Rafique T, Hussain SM, Hussain Z, Zahoor MY, Hussain M, et al. Effect of phytase supplemented Moringa byproducts based diets on the performance of Oreochromis niloticus fingerlings. JAPS: Journal of Animal & Plant Sciences. 2021 Feb 1;31(1). http://dx.doi.org/10.36899/japs.2021.1.0216.
- 14. Priya , Virmani I, Pragya , Goswami RK, Singh B, Sharma JG, et al. Role of microbial phytases in improving fish health. Reviews in Aquaculture. 2023 Jan 27. https://doi.org/10.1111/raq.12790.
- 15. Hlophe‐Ginindza SN, Moyo NA, Ngambi JW, Ncube I. The effect of exogenous enzyme supplementation on growth performance and digestive enzyme activities in Oreochromis mossambicus fed kikuyu‐based diets. Aquaculture Research. 2016 Dec;47(12):3777–3787. https://doi.org/10.1111/are.12828
- 16. Marjan S, Afzal M, Shah SZH, Hussain SM, Mubarik MS. Role of phytase supplementation in improving mineral digestibility of dry bread meal based diet fed to Labeo rohita fingerlings. Pakistan Journal of Life and Social Sciences. 2014; 12: 150–154.
- 17. Hussain SM, Afzal M, Nasir S, Jabeen F, Javid A, Asrar M, et al. Effect of phytase supplementation on growth performance and mineral digestibility in Labeo rohita (Hamilton, 1822) fingerlings fed on sunflower meal based diet. Indian Journal of Fisheries. 2015 Jan 1;62(4):71–79.
- 18. Hussain SM, Shahzad MM, Aslam N, Javid A, Hussain AI, Hussain M, et al. Use of phytase at graded levels for improving nutrient digestibility, growth, and hematology of Catla catla fingerlings fed Moringa oleifera seed meal (MOSM) based diet. Indian Journal of Fisheries. 2017 Jan 1;64:48–57.
- 19. Shahzad MM, Hussain SM, Javid A, Hussain M. Role of phytase supplementation in improving growth parameters and mineral digestibility of Catla catla fingerlings fed moringa byproducts based test diet. Turkish Journal of Fisheries and Aquatic Sciences. 2018 Apr 1;18(4):557–66.
- 20.
AOAC (Association of Official Analytical Chemists). Official Methods of Analysis of AOAC International. 20th Ed. George W. Latimer, Jr. 2016.
- 21. Latta M, Eskin M. A simple and rapid colorimetric method for phytate determination. Journal of Agricultural and Food Chemistry. 1980 Nov;28(6):1313–1315.
- 22. Akram Z, Fatima M, Shah SZ, Afzal M, Hussain SM, Hussain M, et al. Dietary zinc requirement of Labeo rohita juveniles fed practical diets. Journal of Applied Animal Research. 2019 Jan 1;47(1):223–229. https://doi.org/10.1080/09712119.2019.1613238.
- 23. Khan T, Fatima M, Shah SZ, Khan N, Ali W, Sabir A, et al. Quercetin supplementation in the diet of Labeo rohita: Effects on growth, proximate composition, antioxidative indices, and immunity. Animal Feed Science and Technology. 2023 Jun 8:115699. https://doi.org/10.1016/j.anifeedsci.2023.115699.
- 24. Xu GL, Xing W, Li TL, Ma ZH, Jiang N, Xu SD, et al. The effects of different fishmeal level diets with or without phytase supplementation on growth performance, body composition, digestibility, immunological and biochemical parameters of juvenile hybrid sturgeon (Acipenser baeri Brandt♀× A. schrenckii Brandt♂). Aquaculture Nutrition. 2020 Apr;26(2):261–74. https://doi.org/10.1111/anu.12987.
- 25.
Rick W, Stegbauer HP. α-Amylase measurement of reducing groups. In Methods of enzymatic analysis 1974 Jan 1 (pp. 885–890). Academic Press. https://doi.org/10.1016/B978-0-12-091302-2.50074-8.
- 26. Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV. Production of acidic lipase by Aspergillus niger in solid-state fermentation. Process Biochemistry. 2002 Dec 31;38(5):715–721. https://doi.org/10.1016/S0032-9592(02)00194-2.
- 27. Kunitz M. Crystalline soybean trypsin inhibitor: II. General properties. The Journal of General Physiology. 1947 Mar 20;30(4):291–310. pmid:19873496
- 28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976 May 7;72(1–2):248–254. pmid:942051
- 29. Baruah K, Sahu NP, Pal AK, Jain KK, Debnath D, Mukherjee SC. Dietary microbial phytase and citric acid synergistically enhances nutrient digestibility and growth performance of Labeo rohita (Hamilton) juveniles at sub‐optimal protein level. Aquaculture Research. 2007 Feb;38(2):109–20. https://doi.org/10.1111/j.1365-2109.2006.01624.x.
- 30.
Steel RG, Torrie JH. Principles and procedures of statistics. Principles and procedures of statistics, 2nd Ed. McGraw Hill Book Co. Singapore. 1960.
- 31. Palmegiano GB, Costanzo MT, Dapra F, Gai F, Galletta MG, Maricchiolo G, et al. Rice protein concentrate meal is a potential dietary ingredient in practical diets for blackspot seabream (Pagellus bogaraveo). Journal of Animal Physiology and Animal Nutrition. 2007; 91(56): 235 239. https://doi.org/10.1111/j.1439-0396.2007.00697.x.
- 32. Oujifard A, Seyfabadi J, Kenari AA, Rezaei M. Growth and apparent digestibility of nutrients, fatty acids, and amino acids in Pacific white shrimp, Litopenaeus vannamei, fed diets with rice protein concentrate as total and partial replacement of fish meal. Aquaculture. 2012 Apr 15;342:56–61. https://doi.org/10.1016/j.aquaculture.2011.12.038.
- 33. Shah SZ, Fatima M, Afzal M, Bilal M. Interactive effect of citric acid, phytase, and chelated mineral on growth performance, nutrient digestibility and whole‐body composition of Labeo rohita fingerlings. Aquaculture Research. 2021 Feb;52(2):842–58. https://doi.org/10.1111/are.14939.
- 34. Bian F, Zhou H, He G, Wang C, Peng H, Pu X, et al. Effects of replacing fishmeal with different cottonseed meals on growth, feed utilization, hematological indices, intestinal and liver morphology of juvenile turbot (Scophthalmus maximus L.). Aquaculture Nutrition. 2017 Dec;23(6):1429–39. https://doi.org/10.1111/anu.12518.
- 35. Ren X, Wang Y, Chen JM, Wu YB, Huang D, Jiang DL, et al. Replacement of fishmeal with a blend of poultry byproduct meal and soybean meal in diets for largemouth bass, Micropterus salmoides. Journal of the World Aquaculture Society. 2018 Feb;49(1):155–64. https://doi.org/10.1111/jwas.12415.
- 36. Cao SP, Zou T, Zhang PY, Han D, Jin JY, Liu HK, et al. Effects of dietary fishmeal replacement with Spirulina platensis on the growth, feed utilization, digestion, and physiological parameters in juvenile gibel carp (Carassis auratus gibelio var. CAS III). Aquaculture Research. 2018 Mar;49(3):1320–1328. https://doi.org/10.1111/are.13590.
- 37. Norag MA, El-Shenawy AM, Fadl SE, Abdo WS, Gad DM, Rashed MA, et al. Effect of phytase enzyme on growth performance, serum biochemical alteration, immune response and gene expression in Nile tilapia. Fish & Shellfish Immunology. 2018 Sep 1;80:97–108. https://doi.org/10.1016/j.fsi.2018.05.051.
- 38. Hassaan MS, El-Sayed AI, Soltan MA, Iraqi MM, Goda AM, Davies SJ, et al. Partial dietary fish meal replacement with cotton seed meal and supplementation with exogenous protease alters growth, feed performance, hematological indices, and associated gene expression markers (GH, IGF-I) for Nile tilapia, Oreochromis niloticus. Aquaculture. 2019 Mar 30;503:282–92. https://doi.org/10.1016/j.aquaculture.2019.01.009.
- 39. Adeshina I, Akpoilih BU, Udom BF, Adeniyi OV, Abdel-Tawwab M. Interactive effects of dietary phosphorus and microbial phytase on growth performance, intestinal morphometry, and welfare of Nile tilapia (Oreochromis niloticus) fed on low-fishmeal diets. Aquaculture. 2023 Jan 30;563:738995. https://doi.org/10.1016/j.aquaculture.2022.738995.
- 40. Chen A, Liu X, Cui C, Yang C, Wang Y, Bu X, et al. An evaluation of phytase for Channel catfish (Ictalurus punctatus) fed all plant‐protein diet: Growth performance, nutrient utilization, and P equivalency value. Aquaculture Nutrition. 2019 Feb;25(1):215–24. https://doi.org/10.1111/anu.12845.
- 41. Bano N, Afzal M. Synchronized effect of citric acid and phytase supplementation on growth performance and nutrient digestibility of Labeo rohita. Aquaculture Nutrition. 2018 Apr;24(2):786–92. https://doi.org/10.1111/anu.12607.
- 42. Pakravan S, Akbarzadeh A, Sajjadi MM, Hajimoradloo A, Noori F. Partial and total replacement of fish meal by marine microalga Spirulina platensis in the diet of Pacific white shrimp Litopenaeus vannamei: Growth, digestive enzyme activities, fatty acid composition and responses to ammonia and hypoxia stress. Aquaculture Research. 2017 Nov;48(11):5576–86. https://doi.org/10.1111/are.13379.
- 43. Hussain SM, Hameed T, Afzal M, Javid A, Aslam N, Shah SZ, et al. Growth performance and nutrient digestibility of Cirrhinus mrigala fingerlings fed phytase supplemented sunflower meal based diet. Pakistan Journal of Zoology. 2017 Oct 1;49(5). http://dx.doi.org/10.17582/journal.pjz/2017.49.5.1713.1724.
- 44. Farrokhi H, Abdullahpour R, Rezaeipour V. Influence of dietary phytase and protease, individually or in combination, on growth performance, intestinal morphology, microbiota composition and nutrient utilization in broiler chickens fed sesame meal-based diets. Italian Journal of Animal Science. 2021 Jan 1; 20(1), 2122–2130. https://doi.org/10.1080/1828051X.2021.2001698.
- 45. Roofchaei A, Rezaeipour V, Vatandour S, Zaefarian F. Influence of dietary carbohydrases, individually or in combination with phytase or an acidifier, on performance, gut morphology and microbial population in broiler chickens fed a wheat-based diet. Animal Nutrition. 2019 Mar 1;5(1):63–67. pmid:30899811
- 46. Akter T, Hossain A, Rabiul Islam M, Hossain MA, Das M, Rahman MM, et al. Effects of spirulina (Arthrospira platensis) as a fishmeal replacer in practical diets on growth performance, proximate composition, and amino acids profile of pabda catfish (Ompok pabda). Journal of Applied Aquaculture. 2023 Jan 2;35(1):69–82. https://doi.org/10.1080/10454438.2021.1936740.
- 47. Cao L, Wang W, Yang C, Yang Y, Diana J, Yakupitiyage A, et al. Application of microbial phytase in fish feed. Enzyme and Microbial Technology. 2007 Mar 5;40(4):497–507. https://doi.org/10.1016/j. enzmictec.2007.01.007.
- 48. Naseem N, Abdullah S, Aziz S. Effect of phytase supplemented diet on whole body proximate composition of Labeo rohita fingerlings. Pakistan Journal of Zoology. 2021;54(3):1479–82. https://dx.doi.org/10.17582/journal.pjz/20200928150918.
- 49. Liu LW, Su J, Luo Y. Effect of partial replacement of dietary monocalcium phosphate with neutral phytase on growth performance and phosphorus digestibility in gibel carp, Carassius auratus gibelio (Bloch). Aquaculture Research. 2012 Aug;43(9):1404–13. https://doi.org/10.1111/j.1365-2109.2011.02944.x.
- 50. Adeoye AA, Jaramillo-Torres A, Fox SW, Merrifield DL, Davies SJ. Supplementation of formulated diets for tilapia (Oreochromis niloticus) with selected exogenous enzymes: Overall performance and effects on intestinal histology and microbiota. Animal Feed Science and Technology. 2016 May 1;215:133–43. https://doi.org/10.1016/j.anifeedsci.2016.03.002.
- 51. Lewandowski V, Feiden A, Signor A, Bittencourt F, Moro EB, Pessini JE, et al. Digestibility of vegetal energetic ingredients supplemented with phytase for silver catfish (Rhamdia voulezi). Aquaculture. 2017 Jan 20;467:71–5. https://doi.org/10.1016/j.aquaculture.2016.05.022.
- 52. Ahmad B, Hussain SM, Ali S, Arsalan M, Tabassum S, Sharif A. Efficacy of acidified phytase supplemented cottonseed meal based diets on growth performance and proximate composition of Labeo rohita fingerlings. Brazilian Journal of Biology. 2021 Aug 20;83. https://doi.org/10.1590/1519-6984.247791.
- 53. Olugbenga O, Falaye AE, Kareem OK. Effect of phytase supplementation on the growth, mineral composition, and phosphorus digestibility of African catfish (Clarias gariepinus) juveniles. Animal Research International. 2017;14(2):2741–50. https://www.ajol.info/index.php/ari/article/view/186900.
- 54. Liu L, Zhou Y, Wu J, Zhang W, Abbas K, Xu Fang L, et al. Supplemental graded levels of neutral phytase using pretreatment and spraying methods in the diet of grass carp, Ctenopharyngodon idellus. Aquaculture Research. 2014 Nov;45(12):1932–41. https://doi.org/10.1111/are.12145.
- 55. Nie XZ, Chen S, Zhang XX, Dai BY, Qian LC. Effects of neutral phytase on growth performance and phosphorus utilization in crucian carp (Carassius auratus). Journal of Zhejiang University. Science. B. 2017 Oct;18(10):886. https://doi.org/10.1631%2Fjzus.B1600280.
- 56. Akpoilih BU, Omitoyin BO, Ajani EK. Phosphorus utilization in juvenile Clarias gariepinus fed phytase-supplemented diets based on soya bean (oil-extracted) and full-fat (roasted): A comparison. Journal of Applied Aquaculture. 2017 Apr 3;29(2):126–51. https://doi.org/10.1080/10454438.2016.1276000.
- 57. Kalhoro H, Zhou J, Hua Y, Ng WK, Ye L, Zhang J, et al. Soy protein concentrate as a substitute for fish meal in diets for juvenile Acanthopagrus schlegelii: effects on growth, phosphorus discharge, and digestive enzyme activity. Aquaculture Research. 2018 May;49(5):1896–906. https://doi.org/10.1111/are.13645.
- 58. Rodrigues EJ, Ito PI, Ribeiro LF, de Carvalho PL, Xavier WD, Guimaraes MG, et al. Phytase Supplementation under Commercially Intensive Rearing Conditions: Impacts on Nile Tilapia Growth Performance and Nutrient Digestibility. Animals. 2022 Dec 29;13(1):136. pmid:36611745
- 59. Hussain SM, Afzal M, Javid A, Aslam N, Hussain M, Shah SZ, et al. Role of phytase supplementation in improving nutrient digestibility in Labeo rohita (Hamilton, 1822) fingerlings fed on cottonseed meal based diet. Indian Journal of Fisheries. 2015 Jan 1;62(1):78–84.
- 60. Maas RM, Verdegem MC, Dersjant-Li Y, Schrama JW. The effect of phytase, xylanase and their combination on growth performance and nutrient utilization in Nile tilapia. Aquaculture. 2018 Feb 25;487:7–14. https://doi.org/10.1016/j.aquaculture.2017.12.040.
- 61. Maas RM, Verdegem MC, Stevens TL, Schrama JW. Effect of exogenous enzymes (phytase and xylanase) supplementation on nutrient digestibility and growth performance of Nile tilapia (Oreochromis niloticus) fed different quality diets. Aquaculture. 2020 Dec 15;529:735723. https://doi.org/10.1016/j.aquaculture.2020.735723.
- 62. Shahzad MM, Hussain SM, Jabeen F, Hussain AI, Javid A, Asrar M, et al. Improvement in Mineral Digestibility and Whole Body Composition of Catla catla Fingerlings Fed Phytase Supplemented MOSM Based Diet. Pakistan Journal of Zoology. 2018 Oct 1;50(5). http://dx.doi.org/10.17582/journal.pjz/2018.50.5.1909.1920.
- 63. Zhu Y, Qiu X, Ding Q, Duan M, Wang C. Combined effects of dietary phytase and organic acid on growth and phosphorus utilization of juvenile yellow catfish Pelteobagrus fulvidraco. Aquaculture. 2014 Jun 20;430:1–8. https://doi.org/10.1016/j.aquaculture.2014.03.023.
- 64. Cheng N, Chen P, Lei W, Feng M, Wang C. The sparing effect of phytase in plant‐protein‐based diets with decreasing supplementation of dietary NaH2PO4 for juvenile yellow catfish Pelteobagrus fulvidraco. Aquaculture Research. 2016 Dec;47(12):3952–63. https://doi.org/10.1111/are.12845.
- 65. Hussain SM, Afzal M, Nasir S, Javid A, Azmat H, Mamoona Makhdoom S, et al. Role of phytase supplementation in improving nutrient digestibility and growth performance for Labeo rohita fingerlings fed on canola meal-based diet. Journal of Applied Animal Research. 2017 Jan 1;45(1):15–21. https://doi.org/10.1080/09712119.2015.1091331.
- 66. Laining A, Ishikawa M, Kyaw K, Gao J, Binh NT, Koshio S, et al. Dietary calcium/phosphorus ratio influences the efficacy of microbial phytase on growth, mineral digestibility, and vertebral mineralization in juvenile tiger puffer, Takifugu rubripes. Aquaculture Nutrition. 2011 Jun;17(3):267–77. https://doi.org/10.1111/j.1365-2095.2009.00749.x.
- 67. Nwanna LC, Olusola SE. Effect of supplemental phytase on phosphorus digestibility and mineral composition in Nile tilapia (Oreochromis niloticus). International Journal of Aquaculture. 2014 Jun 2;4.
- 68. Ceriotti F, Henny J, Queralto J, Ziyu S, Ozarda Y, Chen B, et al. Common reference intervals for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transferase (GGT) in serum: results from an IFCC multicenter study. Clinical Chemistry and Laboratory Medicine. 2010 Nov 1;48(11):1593–601. https://doi.org/10.1515/CCLM.2010.315.
- 69. Fawole FJ, Sahu NP, Shamna N, Phulia V, Emikpe BO, Adeoye AA, et al. Effects of detoxified Jatropha curcas protein isolate on growth performance, nutrient digestibility, and physio‐metabolic response of Labeo rohita fingerlings. Aquaculture Nutrition. 2018 Aug;24(4):1223–33. https://doi.org/10.1111/anu.12660.
- 70. Kumar V, Makkar HP, Devappa RK, Becker K. Isolation of phytate from Jatropha curcas kernel meal and effects of isolated phytate on growth, digestive physiology and metabolic changes in Nile tilapia (Oreochromis niloticus L.). Food and Chemical Toxicology. 2011 Sep 1;49(9):2144–56. https://doi.org/10.1016/j.fct.2011.05.029.
- 71. Liu LW, Su JM, Zhang T, Liang XF, Luo YL. Apparent digestibility of nutrients in grass carp (Ctenopharyngodon idellus) diet supplemented with graded levels of neutral phytase using pretreatment and spraying methods. Aquaculture Nutrition. 2013 Feb;19(1):91–9. https://doi.org/10.1111/j.1365-2095.2012.00942.x.
- 72. Cantas IB, Yildirim O. Supplementation of microbial phytase with safflower meal in rainbow trout (Oncorhynchus mykiss): the effects on growth, digestibility, environmental, and serum biological parameters. Journal of Chemistry. 2020 Jul 8;2020. https://doi.org/10.1155/2020/4634796.
- 73. Furne M, Hidalgo MC, Lopez A, Garcia-Gallego M, Morales AE, Domezain A, et al. Digestive enzyme activities in Adriatic sturgeon Acipenser naccarii and rainbow trout Oncorhynchus mykiss. A comparative study. Aquaculture. 2005 Nov 14;250(1–2):391–8. https://doi.org/10.1016/j.aquaculture.2005.05.017.
- 74. Ribeiro L, Couto A, Olmedo M, Alvarez‐Blazquez B, Linares F, Valente LM. Digestive enzyme activity at different developmental stages of blackspot seabream, Pagellus bogaraveo (Brunnich 1768). Aquaculture Research. 2008 Mar;39(4):339–46. https://doi.org/10.1111/j.1365-2109.2007.01684.x.
- 75. Li JS, Li JL, Wu TT. Effects of non‐starch polysaccharides enzyme, phytase, and citric acid on activities of endogenous digestive enzymes of tilapia (Oreochromis niloticus× Oreochromis aureus). Aquaculture Nutrition. 2009 Aug;15(4):415–20. https://doi.org/10.1111/j.1365-2095.2008.00606.x
- 76. Shah SZ, Afzal MU, Hussain SM, Fatima M, Bilal M, Ahmed T, et al. Supplementation of citric acid and phytase improves the digestive enzyme activities in Labeo rohita fingerlings. Biologia. 2015;61:63–8. PKISSN 0006–3096.