Phytase as a Feed Additive: Impact on Growth, Nutrient Digestibility and Carcass Composition of Catla catla Fingerlings Fed on Different Plant By-Products Based Diets

Ambreen Sabir1, Muhammad Zubair ul Hassan Arsalan1, Tanveer Ahmed1, Syed Makhdoom Hussain2*, Shafaqat Ali3,4**, Aumme Adeeba Bano2, Muhammad Wasim Aslam1, Huma Naz5, Sadia Tabassum2,

Muhammad Amjad2, Adan Naeem2 and Eman Naeem2

1Department of Life Sciences, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Pakistan.

2Fish Nutrition Laboratory, Department of Zoology, Government College University, Faisalabad, Pakistan

3Department of Environmental Sciences, Government College University, Faisalabad, Pakistan

4Department of Biological Sciences and Technology, China Medical University, Taichung 40402, Taiwan

5Department of Zoology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan

ABSTRACT

Owing to existence of anti-nutritional factors (ANFs) such as phytate in diets based on plant by-products, fish show poor growth and nutrition retention in their bodies because less minerals and nutrients are available to them. To overcome this problem, phytase was supplemented with different plant by-products based diets and impact was checked on digestibility of nutrients, growth performance and carcass composition of Catla catla fingerlings (average weight= 6.10±0.01 g), in a feeding trial lasting seventy days. The fingerlings were fed a variety of diets that were based on different plant by-products with phytase supplementation at levels of 0, 250 and 500 FTU Kg-1, in three-factorial configurations with a completely random design in triplicates. Chromium oxide was added to test diets at a concentration of 1% as an inert marker. Diets were served twice a day, @ 4% of body weight of fingerlings. Feces from every fish tank were gathered twice a day to measure nutrients digestion. Phytase supplementation into the test diets considerably (p<0.05) enhanced the growth rate, nutrient digestibility (including crude protein, crude fat, and gross energy), and carcass composition of C. catla. The findings showed that the test diet with 50% MOLM and 500 FTU Kg-1 phytase exhibited the best growth performance, nutrient digestibility, and carcass composition. Additionally, it was discovered that phytase supplementation lowered the nutrient discharge via feces, hence reducing the amount of contaminants in the water. In conclusion, adding phytase at a level of 500 FTU Kg-1 was favorable for using with different plant by-products to create an inexpensive and ecologically friendly fish diet.

Article Information

Received 30 July 2024

Revised 15 September 2024

Accepted 23 September 2024

Available online 16 January 2025

(early access)

Published 27 December 2025

Authors’ Contribution

Writing-original draft: AS. Conceptualization, data curation, methodology, supervision: MZHA. Investigation, formal analysis, data curation: TA, SMH, SA. Writing-review and editing, data curation: AAB, MWA, HN, ST, MA. Data curation, writing-review and editing: AN, EN. All authors read and approved the final manuscript.

Key words

Plant by-products, Phytase, Catla catla, Nutrients digestibility, Carcass composition, Growth performance

DOI: https://dx.doi.org/10.17582/journal.pjz/20240730064447

* Corresponding author: [email protected], [email protected]

0030-9923/2026/0001-0215 $ 9.00/0

Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

It is estimated that by 2030, there will be almost 8.5 billion people on the earth and global food security and sustainable development will be considerably supported by aquaculture, which is estimated to supply roughly 53% of the seafood consumed worldwide (FAO, 2022; UN DESA, 2023). By 2025, the world’s aquaculture output of fed species is expected to be 58.9 million tons, and the demand for aqua feed is expected to reach 69.5 million tons (Tacon et al., 2022). In order to facilitate this expansion, the sector must look at cutting-edge ways to get sustainable ingredients for raising aqua feed output (Hua et al., 2019). Since past two decades, reducing reliance on fishmeal (FM) by switching to plant based by-products has proved very challenging (Gatlin et al., 2007; Hussain et al., 2024). Plant-based proteins are beneficial to fish in terms of nutrition, nutrient utilization, growth, protein retention, digestibility, nutrient bioavailability, biochemical changes, meat quality, strength, and stress response (Shahzad et al., 2020).

Moringa oleifera is a fast-growing plant that is abundant in macro and micronutrients, including proteins, minerals (iron, potassium, calcium, phosphorus), carbohydrates, vitamins, and many other bioactive compounds (Sahay et al., 2017). Its leaves contain 260 g of crude protein per kilogram of leaf, which is significantly more than the amount found in many other legumes i.e. soybean seeds (Ferreira et al., 2008; Hussain et al., 2024). Sunflower meal (SFM) is another promising substitute of FM to be used in aqua feed due to its high protein amount, palatability and low content of anti-nutritional factors (ANFs) (Saleh et al., 2021). It is utilized in formulations of feed because it contains natural proteolytic enzymes that aid in protein digestion in fish (Kocher et al., 2000; Soltan et al., 2023). After soybean meal, canola meal (CM) is rich source of protein and has higher concentrations of important amino acids like cysteine and methionine. CM is often utilized instead to FM in aquaculture feed (Hussain et al., 2022). Guar meal (GM) has rich protein value with better amino acid composition and is less expensive per unit of generated protein. Additionally, because guar gum is used less frequently as a binder, guar meal has improved digestibility and higher pellet quality (Hardy, 1999; Brinker and Friedrich, 2012).

In fish nutrition, plant proteins are a feasible sources of protein. However, presence of a number of ANFs in plants decrease their use in aqua feed. In addition to other significant ANFs such as fiber, tannins, glucosinolates, protease inhibitors, or saponins, one of the most common ANFs in plant feed sources is phytic acid or phytate (inositol hexakisphosphate, IP6) (Francis et al., 2001; Serra et al., 2024). One of the main issues with phytate in aqua feed is its detrimental impacts on growth, energy and nutrient utilization, and minerals uptake. The enzyme phytase have a critical role in increasing the discharge of minerals, such as phosphorus, that are bound to phytic acid, increasing their bioavailability for animal assimilation. The aqua feed sector has been using phytase for the past ten years to improve fish growth and nutrient utilization as well as to lower P pollution in the aquatic environment (Kumar et al., 2015; Negm et al., 2024).

Catla catla is the sixth-most recognized fish species in all known fish species of the world, making up roughly 5.6% of total output. C. catla, often called Thaila, is a surface feeder. The best source of human protein among all fish species is C. catla, which is also the cheapest. It is characterized by a high concentration of excellent quality nutrients, including vitamins, minerals, fatty acids and a balanced amino acid content (Khan et al., 2012; FAO, 2020). Thus, the goal of present research was to ascertain the impacts of phytase on growth, nutrient digestibility and carcass composition of C. catla fingerlings given different plant by-products based diets.

MATERIALS AND METHODS

This study was designed to check the impact of supplemented phytase on growth, nutrient digestibility and carcass composition of C. catla fingerlings fed on different plant by products based test diets. The trial was held in Fish Nutrition Lab, Department of Life Sciences, KFUEIT, Rahim Yar Khan.

Conditions of trial and fish

C. catla fish (average weight= 6.10±0.01 g) was taken from Fish Seed Hatchery, Rahim Yar Khan and looked after in glass aquarium (especially designed to collect feces) for 15 days to acclimatize them to trial conditions. The fingerlings were treated with NaCl (5 g L-1) solution before start of trial in order to avoid any fungal infections. The fingerlings were fed on basal diet throughout this acclimation stage. Water quality indicators i.e. dissolved O2 (4-6 mg L-1), temperature (26-28°C) and pH (6.7-7.5) were checked on daily basis.

Test diets formulation

Four different plant by-products including MOLM, SFM, CM and GM were used to replace FM in test diets, in which phytase was added at the level of 250 and 500 FTU Kg-1, except control diet (FM based) with 0 FTU Kg-1 phytase supplementation. All feed ingredients (Table I) were sourced from a commercial feed mill, and recognized methods were used to analyze their chemical composition (AOAC, 2005), before formulation of diets. To get the required particle size to pass through a 0.5 mm filter, all ingredients were ground and sieved.

The fish oil was slowly mixed with the mixture by blending in the blender for 5 min. About 10 to 15% water was included to produce a suitable paste. Then, granulator turned the already prepared dough into food granules. To assess digestibility, 1% chromium oxide was included in the test meals as an inert marker. Two grams of phytase were dissolved in 1000ml of purified water. According to

 

Table I. Ingredients composition (%) of test diets.

Ingredients	T1 (control)	T2 (MOLM)	T3 (MOLM)	T4 (SFM)	T5 (SFM)	T6 (CM)	T7 (CM)	T8 (GM)	T9 (GM)
Fishmeal	50	35	50	35	50	35	50	35	50
		15		15		15		15
Soybean meal	15	15	15	15	15	15	15	15	15
Wheat flour	17	17	17	17	17	17	17	17	17
Rice polish	8	8	8	8	8	8	8	8	8
Fish oil	6	6	6	6	6	6	6	6	6
Vitamin premix	1	1	1	1	1	1	1	1	1
Mineral premix	1	1	1	1	1	1	1	1	1
Ascorbic acid	1	1	1	1	1	1	1	1	1
Chromic oxide	1	1	1	1	1	1	1	1	1
Phytase (FTU Kg-1)	0	250	500	250	500	250	500	250	500

 

MOLM, M. oleifera leaf meal; SFM, Sunflower meal; CM, Canola meal; GM, Guar meal; Phytase inclusion was by the substitution of wheat flour.

 

Robinson et al. (2002) test diets were sprayed with the recommended concentrations of phytase. One test diet without phytase (T1) and eight test diets (T2, T3, T4, T5, T6, T7, T8, and T9) with varying levels of phytase (250 and 500 FTU Kg-1) were formulated.

Feeding protocol and sampling

In a completely randomized design, nine fish groups (N = 405) were created in triplicates having 15 fingerlings in each group. Test diets were fed to fish about 5 % of their body mass twice per day. Following the feeding period, unused feed was taken out of fish tanks in order to gauge feed intake. Fecal matter was collected after 2 h of feeding via the tanks’ fecal collection tubes. After being dried, the feces were preserved for chemical examination.

Growth analysis

The fingerlings of each tank were bulk weighed at the beginning and end the trial to check growth performance. Growth features including weight gain percentage (WG%), feed conversion ratio (FCR), and specific growth rate (SGR%) were determined by following standard formulae:

Feed, feces and carcass chemical analysis

The feed, feces and carcass samples were homogenized separately by using pestle and motor and then chemically checked by using conventional methods (AOAC, 2005). Moisture content was evaluated by oven drying at 105℃ for about 12 h. Crude protein (N×6.25) was measured with a micro-Kjeldahl apparatus. A Soxtec HT2 1045 system was used to extract crude fat by ether extraction method; calcination at 650°C for about 12 h. at constant weight was used to determine crude ash content. Bomb calorimeter was utilized to measure the gross energy of samples.

Chromic oxide estimation

The content of chromic oxide (Cr2O3) was gauged by acid digestion method as described by Divakaran et al. (2002) following oxidation of ash samples of feed and feces by using reagent HCLO4.

Nutrient digestibility calculations

The nutrient digestibility of experimental diets was determined by calculating apparent digestibility coefficients (ADC%) by utilizing following standard formula:

Statistical assay

Utilizing one-way ANOVA, the data of feed digestibility, growth, mineral absorption, and overall body composition was analyzed (Steel and Torrie, 1996). Using Tukey’s Honesty Significant Difference Test, differences between means were contrasted. According to Snedecor and Cochran (1989), when comparing variances across means, a significance threshold of p<0.05 was taken into account. For this aim, the Co-stat computer program was employed.

RESULTS

This study explored the effects of phytase as a supplement on the growth performance, carcass composition, and nutrient digestibility of C. catla

 

Table III. Analyzed nutrient compositions in diets based on different plant by-products supplemented with phytase.

Test diets	Fishmeal protein replacement levels (%)	Plant by products	Plant by-products replacement levels (%)	Phytase (FTU Kg-1)	Crude protein	Crude fat	Gross energy
T1	50	MOLM	0	0	30.63±0.02	8.22±0.01	3.53±0.02
T2	35		15	250	30.55±0.04	8.23±0.02	3.53±0.01
T3	0		50	500	30.67±0.03	8.24±0.01	3.54±0.02
T4	35	SFM	15	250	30.53±0.03	8.24±0.02	3.52±0.01
T5	0		50	500	30.53±0.02	8.25±0.02	3.53±0.02
T6	35	CM	15	250	30.66±0.02	8.24±0.03	3.53±0.02
T7	0		50	500	30.65±0.02	8.24±0.03	3.53±0.02
T8	35	GM	15	250	30.56±0.02	8.21±0.01	3.53±0.02
T9	0		50	500	30.45±0.02	8.22±0.01	3.53±0.02

 

Data is mean ± standard deviation (N=3). In columns, means with distinct superscripts differ significantly (p<0.05).

 

fingerlings given different plant by product based diets. MOLM, SFM, CM, and GM plant by-products were the basic ingredients to replace FM. The current study’s findings are as follows:

Growth performance

The growth indices of C. catla fingerlings fed phytase supplemented diets based on different plant by-products are shown in Table II. It was recorded that T3 diet; having 50% MOLM with 500 FTU Kg-1 phytase showed maximum WG% (271.1%), SGR (1.87%), and lowest FCR (1.46). The second best values were observed when fish were given diet T7; in which 50% CM with 500 FTU Kg-1 phytase resulted in the WG% (236.7%) and SGR (1.73%) with FCR (1.39), respectively. The minimum values of WG% (57.1%), SGR (0.65%) and highest FCR (6.22) were resulted from T1 test diet with 0 FTU Kg-1 phytase. The results of growth performance showed that MOLM and CM with phytase inclusion are better alternatives of protein for fish diets in place of costly FM.

Nutrient digestibility

Tables III and IV show the nutrients percentage in test diets and feces of C. catla, while Table V shows their digestibility values. In different plant by-product based diets, the phytase as a supplement improved the nutrients digestibility by decomposing phytate. The test diet T3 (50% MOLM with 500 FTU Kg-1 phytase) resulted in the highest ADCs (CP: 73.45%, CF: 77.94%, and GE: 75.79%), while the control test diet T1 gave the lowest values of ADCs (CP: 40.46%, CF: 43.74%, and GE: 31.64%). Second highest ADC% of CP, CF, and GE were resulted by T7 (50% CM with 500 FTU Kg-1 phytase) with values 68.22%, 75.93%, and 72.95%, correspondingly.

 

Table IV. Analyzed nutrient compositions in feces of C. catla fed diets based on different plant by products supplemented with phytase.

Test diets	Fishmeal protein replacement levels (%)	Plant by products	Plant by-products replacement levels (%)	Phytase (FTU Kg-1)	Crude protein	Crude fat	Gross energy
T1	50	MOLM	0	0	24.73±0.26i	6.27±0.16i	2.95±0.03i
T2	35		15	250	14.50±0.51c	2.94±0.04c	1.38±0.09c
T3	0		50	500	10.18±0.08a	2.27±0.08a	1.04±0.01a
T4	35	SFM	15	250	19.07±0.05e	4.86±0.03e	1.94±0.02e
T5	0		50	500	20.54±0.33f	4.12±0.01f	1.66±0.03f
T6	35	CM	15	250	16.22±0.32d	3.86±0.03d	1.56±0.05d
T7	0		50	500	12.54±0.33b	2.56±0.03b	1.16±0.04b
T8	35	GM	15	250	23.22±0.32h	5.96±0.03h	2.25±0.03h
T9	0		50	500	21.54±0.33g	5.36±0.03g	2.06±0.04g

 

Data is mean ± standard deviation (N=3). In columns, means with distinct superscripts differ significantly (p<0.05).

 

Table V. Apparent digestibility coefficients (ADC%) of nutrients for C. catla fingerlings fed diets based on different plant by- products supplemented with phytase.

Test diets	Fishmeal protein replacement levels (%)	Plant by-products	Plant by-products replacement levels (%)	Phytase (FTU Kg-1)	Crude protein	Crude fat	Gross energy
T1	50	MOLM	0	0	40.46±0.58i	43.74±2.45i	31.64±2.06i
T2	35		15	250	60.65±2.06d	66.31±0.64d	63.34±2.46d
T3	0		50	500	73.45±0.92a	77.94±1.43a	75.79±0.38a
T4	35	SFM	15	250	47.27±2.23f	51.01±1.56f	54.23±1.63f
T5	0		50	500	54.16±0.70e	65.98±0.84e	61.89±0.36e
T6	35	CM	15	250	61.89±1.59c	70.84±0.05c	68.02±3.19c
T7	0		50	500	68.22±1.20b	75.93±0.30b	72.95±1.06b
T8	35	GM	15	250	46.41±2.35g	49.40±0.76g	52.17±2.34g
T9	0		50	500	45.07±1.58h	47.73±0.98h	47.20±1.27h

 

Data is mean ± standard deviation (N=3). In columns, means with distinct superscripts differ significantly (p<0.05).

 

Table VI. Analyzed body composition of C. catla fingerlings fed diets based on different plant by-products supplemented with phytase.

Test diets	Fishmeal protein replace-ment levels (%)	Plant by products	Plant by-products replacement levels (%)	Phytase (FTU Kg-1)	Protein	Fat	Ash	Moisture
T1	50		0	0	14.75±0.03i	12.81±0.04i	1.00±0.01i	71.44±0.02a
T2	35	MOLM	15	250	17.18±0.01c	5.22±0.02c	2.33±0.03c	75.27±0.01g
T3	0		50	500	18.05±0.03a	2.97±0.01a	2.49±0.02a	76.50±0.00h
T4	35	SFM	15	250	16.03±0.02f	8.75±0.04f	1.48±0.01f	73.74±0.02d
T5	0		50	500	16.27±0.02e	7.98±0.04e	1.68±0.01e	74.07±0.01e
T6	35	CM	15	250	16.87±0.02d	6.13±0.04d	2.03±0.03d	74.97±0.01f
T7	0		50	500	17.84±0.05b	3.03±0.02b	2.47±0.00b	76.67±0.06i
T8	35	GM	15	250	15.38±0.55h	10.82±0.54h	1.05±0.02h	72.74±0.02b
T9	0		50	500	15.78±0.55g	10.10±0.55g	1.08±0.01g	73.04±0.02c

 

Data is mean ± standard deviation (N=3). In columns, means with distinct superscripts differ significantly (p<0.05).

 

Carcass composition

The body composition of C. catla given diets based on different plant by-products supplemented with phytase is displayed in Table VI. The body composition of fingerlings of different groups varied significantly (p<0.05). The T3 diet resulted in the highest level of crude protein (18.05%), ash (2.49%) and lowest value of crude fat (2.97%). In contrast, the T7 diet, had a second higher value of crude protein (17.84%), ash (2.47%) and second lowest value of crude fat (3.03%). Highest value (76.67%) of moisture was recorded at T7 diet and the second higher value (76.50%) was observed at T3 diet. The lowest value (71.44%) of moisture was observed from T1 diet.

DISCUSSION

The substitution of FM, a protein source, in fish feed is needed in these days, owing to its uncertain availability and high price. Since FM is the primary source of protein, replacing another protein source for FM must not reduce the feed’s nutritional value. One of the best substitutes is plant by-products. But plant ingredients utilization is hampered by the existence of ANFs such phytic acid, having a number of negative impacts including reduced fish growth rates (Punia and Singh, 2024). To reduce these negative impacts, a number of strategies are being used. In a current study, phytase was supplemented with plant by-products based diets and efficacy was observed on growth, nutrients digestibility and carcass composition of C. catla.

In this study, it was recorded that phytase supplementation improves growth performance. Phytase efficacy was maximum at T3 diet (50% MOLM with 500 FTU Kg-1 phytase), in contrast to T1 diet (50% FM with 0 FTU Kg-1 phytase). Same growth improvements were recorded for fishes fed phytase supplemented diets; i.e. phytase inclusion at 1500 to 2000 UF kg-1 for Oreochromis niloticus (Rodrigues et al., 2022), at 300 to 1500 FTU Kg-1 with MOSM for C. catla (Hussain et al., 2017) and up to 1000 FTU Kg-1 with rice protein concentrate for Labeo rohita (Khizar et al., 2024) enhanced the growth parameters including WG and SGR, increased the feed intake and reduced the FCR. This improvement can be attributed to the phytase, which effectively broke down the complex phytate and released the nutrients, resulting in an increase in feed palatability (Maas et al., 2020; Rodrigues et al., 2022). Similarly, Adeshina et al. (2023) recorded that dietary phytase improved the growth of African catfish fed SBM based diet and stimulated feed intake and Maas et al. (2019) noticed an increase in the growth rate of Nile tilapia given SFM based diets having phytase level 1000 FTU Kg-1. In contrast, Adeoye et al. (2016), found that adding phytase to juvenile tilapia had no statistically significant influence on growth parameters.

The results of current study demonstrated that phytase inclusion to plant by-products based diets significantly raised the nutrients digestion when compared to diet without phytase and these findings are consistent with previous findings. The maximum ADC% of CP, CF and GE values were recorded by T3 diet (50% MOLM with 500 FTU Kg-1 phytase). Hussain et al. (2017), using MOSM based diet having 900 FTU Kg-1 phytase, observed maximum increase in digestibility in C. catla fingerlings. Phytase-fed fish groups showed improved digestibility, which could potentially be related to increased protease production, which is linked to protein digestibility (Morales et al., 2016). The results demonstrated that, phytase inclusion to plant based diets decreased the amount of nutrients excreted into the water through feces, thereby improving nutrient digestibility in contrast to a diet with 0 FTU Kg-1 phytase. Reducing pollution in the aquatic environment is greatly helped by fish bodies by using maximum nutrients of diet and releasing less pollutants into the water (Kumar et al., 2015). Hussain et al. (2017) observed that feeding Cirrhinus mrigala fingerlings a test diet based on SFM supplemented with phytase resulted increase in CP, CF and GE digestibility. On the other hand, adding dietary phytase to the diets of several fish species did not demonstrate any impact on the ADC of nutrients (Maas et al., 2020). Several factors, including the type of fish, feed composition (especially basic plant ingredients having different levels of phytate), enzyme source, inclusion method and other response characteristics, might affect the activity of phytase (Debnath et al., 2005).

According to current findings, plant by-products based diets with phytase as a supplement improved the nutritional composition of body of C. catla fingerlings. The best carcass composition was observed by T3 (50% MOLM with 500 FTU Kg-1 phytase) in comparison to T1 diet (50% FM without phytase). Consistent with these findings, Shahzad et al. (2021) found improvement in carcass composition of Nile tilapia fed on moringa based diets with phytase inclusion in a range 500 to 1250 FTU Kg-1. In contrast, Akpoilih (2015) found no significant improvement in crude protein by phytase when compared to non phytase diets fed to C. gariepinus. In line with current findings, Naseem et al. (2021) observed increase in body protein, ash and fat of L. rohita fingerlings by phytase supplementation.

CONCLUSION

In this study, effects of phytase were checked systematically on the growth, nutrients digestibility and carcass composition of C. catla fingerlings fed different plant by-products based diets. The findings showed that dietary supplementation of phytase boosted the growth rate of C. catla fingerlings. These positive outcomes might be related to improved nutrient availability, digestibility and nutrient retention. Best results were recorded for diet having 50% MOLM with 500 FTU Kg-1 phytase, in contrast to diet with 50% FM and without phytase. Conclusively, phytase supplementation with different plant by-products based diets is beneficial to produce low cost, highly nutritive and environmentally friendly fish diets. Future research must focus on more economical and eco-friendly aqua-feeds.

Declarations

Acknowledgment

The authors are thankful to HEC Pakistan for funding Project No. 20-4892/NRPU/RandD/HEC/14/1145 at Department of Zoology, Government College University Faisalabad for provision of facilities for this research.

Funding

HEC Islamabad, Pakistan provided funds for this study under Project # 5649/Punjab/NRPU/R and D/HEC/2016 and Project # 20-4892/NRPU/R and D/HEC/14/1145.

IRB approval

All applicable institutional, national and international guidelines for the care and use of animals were followed.

Ethical statement

All the procedures and methods used in this study followed the ethical guidelines provided by Government College University, Faisalabad.

Statement of conflict of interest

The authors have declared no conflict of interest.

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